Reviewer #3 (Public review):

Summary:

Kern et al. critically assess the sensitivity of temporally delayed linear modelling (TDLM), a relatively new method used to detect memory replay in humans via MEG. While TDLM has recently gained traction and been used to report many exciting links between replay and behavior in humans, Kern et al. were unable to detect replay during a post-learning rest period. To determine whether this null result reflected an actual absence of replay or sensitivity of the method, the authors ran a simulation: synthetic replay events were inserted into a control dataset, and TDLM was used to decode them, varying both replay density and its correlation with behavior. The results revealed that TDLM could only reliably detect replay at unrealistically (not-physiological) high replay densities, and the authors were unable to induce strong behavior correlations. These findings highlight important limitations of TDLM, particularly for detecting replay over extended, minutes-long time periods.

Strengths:

Overall, I think this is an extremely important paper, given the growing use of TDLM to report exciting relationships between replay and behavior in humans. I found the text clear, the results compelling, and the critique of TDLM quite fair: it is not that this method can never be applied, but just that it has limits in its sensitivity to detect replay during minutes-long periods. Further, I greatly appreciated the authors' efforts to describe ways to improve TDLM: developing better decoders and applying them to smaller time windows.

The power of this paper comes from the simulation, whereby the authors inserted replay events and attempted to detect them using TDLM. Regarding their first study, there are many alternative explanations or possible analysis strategies that the authors do not discuss; however, none of these are relevant if, under conditions where it is synthetically inserted, replay cannot be detected.

Additionally, the authors are relatively clear about which parameters they chose, why they chose them, and how well they match previous literature (they seem well matched).

Finally, I found the application of TDLM to a baseline period particularly important, as it demonstrated that there are fluctuations in sequenceness in control conditions (where no replay would be expected); it is important to contrast/calculate the difference between control (pre-resting state) and target (post-resting state) sequenceness values.

Weaknesses:

While I found this paper compelling, I was left with a series of questions.

(1) I am still left wondering why other studies were able to detect replay using this method. My takeaway from this paper is that large time windows lead to high significance thresholds/required replay density, making it extremely challenging to detect replay at physiological levels during resting periods. While it is true that some previous studies applying TDLM used smaller time windows (e.g., Kern's previous paper detected replay in 1500ms windows), others, including Liu et al. (2019), successfully detected replay during a 5-minute resting period. Why do the authors believe others have nevertheless been able to detect replay during multi-minute time windows?

For example, some studies using TDLM report evidence of sequenceness as a contrast between evidence of forwards (f) versus backwards (b) sequenceness; sequenceness was defined as ZfΔt - ZbΔt (where Z refers to the sequence alignment coefficient for a transition matrix at a specific time lag). This use case is not discussed in the present paper, despite its prevalence in the literature. If the same logic were applied to the data in this study, would significant sequenceness have been uncovered? Whether it would or not, I believe this point is important for understanding methodological differences between this paper and others.

(2) Relatedly, while the authors note that smaller time windows are necessary for TDLM to succeed, a more precise description of the appropriate window size would greatly improve the utility of this paper. As it stands, the discussion feels incomplete without this information, as providing explicit guidance on optimal window sizes would help future researchers apply TDLM effectively. Under what window size range can physiological levels of replay actually be detected using TDLM? Or, is there some scaling factor that should be considered, in terms of window size and significance threshold/replay density? If the authors are unable to provide a concrete recommendation, they could add information about time windows used in previous studies (perhaps, is 1500ms as used in their previous paper a good recommendation?).

(3) In their simulation, the authors define a replay event as a single transition from one item to another (example: A to B). However, in rodents, replay often traverses more than a single transition (example: A to B to C, even to D and E). Observing multistep sequences increases confidence that true replay is present. How does sequence length impact the authors' conclusions? Similarly, can the authors comment on how the length of the inserted events impacts TDLM sensitivity, if at all?

For example, regarding sequence length, is it possible that TDLM would detect multiple parts of a longer sequence independently, meaning that the high density needed to detect replay is actually not quite so dense? (example: if 20 four-step sequences (A to B to C to D to E) were sampled by TDLM such that it recorded each transition separately, that would lead to a density of 80 events/min).

Reviewer #3 (Public review):

Summary:

Borghi and colleagues present results from 4 experiments aimed at investigating the effects of dual γtACS and iTBS stimulation of the precuneus on behavioral and neural markers of memory formation. In their first experiment (n = 20), they find that a 3-minute offline (i.e., prior to task completion) stimulation that combines both techniques leads to superior memory recall performance in an associative memory task immediately after learning associations between pictures of faces, names, and occupation, as well as after a 15-minute delay, compared to iTBS alone (+ tACS sham) or no stimulation (sham for both iTBS and tACS). Performance in a second task probing short-term memory was unaffected by the stimulation condition. In a second experiment (n = 10), they show that these effects persist over 24 hours and up to a full week after initial stimulation. A third (n = 14) and fourth (n = 16) experiment were conducted to investigate neural effects of the stimulation protocol. The authors report that, once again, only combined iTBS and γtACS increases gamma oscillatory activity and neural excitability (as measured by concurrent TMS-EEG) specific to the stimulated area at the precuneus compared to a control region, as well as precuneus-hippocampus functional connectivity (measured by resting state MRI), which seemed to be associated with structural white matter integrity of the bilateral middle longitudinal fasciculus (measured by DTI).

Strengths:

Combining non-invasive brain stimulation techniques is a novel, potentially very powerful method to maximize the effects of these kinds of interventions that are usually well-tolerated and thus accepted by patients and healthy participants. It is also very impressive that the stimulation-induced improvements in memory performance resulted from a short (3 min) intervention protocol. If the effects reported here turn out to be as clinically meaningful and generalizable across populations as implied, this approach could represent a promising avenue for treatment of impaired memory functions in many conditions.

Methodologically, this study is expertly done! I don't see any serious issues with the technical setup in any of the experiments. It is also very commendable that the authors conceptually replicated the behavioral effects of experiment 1 in experiment 2 and then conducted two additional experiments to probe the neural mechanisms associated with these effects. This certainly increases the value of the study and the confidence in the results considerably.

The authors used a within-subject approach in their experiments, which increases statistical power and allows for stronger inferences about the tested effects. They also used to individualize stimulation locations and intensities, which should further optimize the signal-to-noise ratio.

Reviewer #3 (Public review):

Summary:

In the manuscript by Shen, Yeung, and colleagues, the authors generate an improved and expanded Mosaic analysis by gRNA-induced crossing-over (MAGIC) toolkit for use in making mosaic clones in Drosophila. This is a clever method by which mitotic clones can be induced in dividing cells by using CRISPR/Cas9 to generate double-strand breaks at specific locations that induce crossing over at those locations. This is conceptually similar to previous mosaic methods in flies that utilized FRT sites that had been inserted near centromeres along with heat-shock inducible FLPase. The advantage of the MAGIC system is that it can be used along with chromosomes lacking FRT sites already introduced, such as those found in many deficiency collections or in EMS mutant lines. It may also be simpler to implement than FRT-based mosaic systems. There are two flavors of the MAGIC system: nMAGIC and pMAGIC. In nMAGIC, the main constituents are a transgene insertion that contains gRNAs that target DNA near the centromere, along with a fluorescent marker. In pMAGIC, the main constituents are a transgenic insertion that contains gRNAs that target DNA near the centromere, along with ubiquitous expression of GAL80. As such, nMAGIC can be used to generate clones that are not labelled, whereas pMAGIC (along with a GAL4 line and UAS-marker) can be used much like MARCM to positively label a clone of cells. This manuscript introduces MAGIC transgenic reagents that allow all 4 chromosomes to be targeted. They demonstrate its use in a variety of tissues, including with mutants not compatible with current FLP/FRT methods, and also show it works well in tissues that prove challenging for FLP/FRT mosaic analyses (such as motor neurons). They further demonstrate that it can be used to generate mosaic clones in non-melanogaster hybrid tissues. Overall, this work represents a valuable improvement to the MAGIC method that should promote even more widespread adoption of this powerful genetic technique.

Strengths:

(1) Improves the design of the gRNA-marker by updating the gRNA backbone and also the markers used. GAL80 now includes a DE region that reduces the perdurance of the protein and thus better labeling of pMAGIC clones. The data presented to demonstrate these improvements is rigorous and of high quality.

(2) Introduces a toolkit that now covers all chromosome arms in Drosophila. In addition, the efficiency of 3 target different sites is characterized for each chromosome arm (e.g., 3 different gRNA-Marker combinations), which demonstrate differences in efficiency. This could be useful to titrate how many clones an experimenter might want (e.g., lower efficiency combinations might prove advantageous).

(3) The manuscript is well written and easy to follow. The authors achieved their aims of creating and demonstrating MAGIC reagents suitable for mosaic analysis of any Drosophila chromosome arm.

(4) The MAGIC method is a valuable addition to the Drosophila genetics toolkit, and the new reagents described in this manuscript should allow it to become more widely adopted.

Weaknesses:

(1) The MAGIC method might not be well known to most readers, and the manuscript could have benefited from schematics introducing the technique.

(2) Traditional mosaic analyses using the FLP/FRT system have strongly utilized heat-shock FLPase for inducible temporal control over mitotic clones, as well as a way to titrate how many clones are induced (e.g., shorter heat shocks will induce fewer clones). This has proven highly valuable, especially for developmental studies. A heat-shock Cas9 is available, and it would have been beneficial to determine the efficiency of inducing MAGIC clones using this Cas9 source.

Reviewer #3 (Public review):

Leshem et al have generated a transcriptional cell atlas of the human outflow tract at two developmental timepoints and its adult valvular derivatives. This carefully performed study provides a useful resource for the study of known genes implicated in outflow tract defects and potentially also for discovering new disease genes. The authors reveal neural crest and mesodermal contributions to different outflow tract components and show that GATA6, known to play a role in arterial valve development, controls a set of genes expressed in endocardium-derived cells during valve development. Interestingly, the results suggest lineage persistence of expression of certain genes through to the adult timepoint, a main new finding of this study.

The following points should be addressed to reinforce the conclusions and emphasize the novel features of this study.

(1) It would be helpful to clarify how these new findings confirm or diverge from what is known from analysis of neural crest and mesodermal lineage contributions to different cell populations in the mouse heart. Did the authors identify any human-specific populations of cells, such as the LGR5 population reported by Sahara et al?

(2) The authors should clarify in the introduction and results that they consider the endocardium to be on the SHF trajectory as indicated in Figure S4C. Please add a reference for this point.

(3) The GATA6 results are interesting and support this experimental approach. The paper would be reinforced if the authors could provide any functional validation (in addition to their GATA6 genomic occupancy data) that the designated target genes are regulated by GATA6. This might involve looking at mutant mouse embryos or cultured cells. Do the authors consider that GATA6 may regulate the endocardial to mesenchymal transition during the early stages of valve development? Or the valve interstitial cell versus fibroblast fate choice?

(4) Do the new findings reveal whether human valves have a direct SHF to VIC trajectory (ie, without transiting through endocardium) as has been recently shown in the murine non-coronary valve leaflet? Relevant to this point, Figure 5E appears to show contributions to a single adult aortic valve leaflet - this should be explained, or corrected.

Reviewer #3 (Public review):

Summary:

In this manuscript, Sumegi et al. use calcium imaging in head-fixed mice to test whether new place fields tend to emerge due to events that resemble behavioral time scale plasticity (BTSP) or other mechanisms. An impressive dataset was amassed (163 sessions from 45 mice with 500-1000 neurons per sample) to study spontaneous emergence of new place fields in area CA1 that had the signature of BTSP. The authors observed that place fields could emerge due to BTSP and non-BTSP-like mechanisms. Interestingly, when non-BTSP mechanisms seemed to generate a place field, this tended to occur on a trial with a spontaneous reset in neural coding (a remapping event). Novelty seemed to upregulate non-BTSP events relative to BTSP events. Finally, large calcium transients (presumed plateau potentials) were not sufficient to generate a place field.

Strengths:

I found this manuscript to be exceptionally well written, well powered, and timely given the outstanding debate and confusion surrounding whether all place fields must arise from BTSP event. Working at the same institute, Albert Lee (e.g. Epszstein et al., 2011 - which should be cited) and Jeff Magee (e.g. Bittner et al., 2017) showed contradictory results for how place fields arise. These accounts have not fully been put toe-to-toe and reconciled in the literature. This manuscript addresses this gap and shows that both accounts are correct - place fields can emerge due to a pre-existing map and due to BTSP.

Weaknesses:

I find only three significant areas for improvement in the present study:

First, can it be concluded that non-BTSP events occur exclusively due to a global remapping event, as stated in the manuscript "these PFF surges included a high fraction of both non-BTSP- and BTSP-like PFF events, and were associated with global remapping of the CA1 representation"? Global remapping has a precise definition that involves quantifying the stability of all place fields recorded. Without a color scale bar in Figure 3D (which should be added), we cannot know whether the overall representations were independent before and after the spontaneous reset. It would be good to know if some neurons are able to maintain place coding (more often than expected by chance), suggestive of a partial-remapping phenomenon.

Second, BTSP has a flip side that involves weakening of existing place fields when a novel field emerges. Was this observed in the present study? Presumably place fields can disappear due to this bidirectional-BTSP or due to global remapping. For a full comparison of the two phenomena, the disappearance of place fields must also be assessed.

Finally, it would be good to know if place fields differ according to how they are born. For example, are there differences in reliability, width, peak rate, out of field firing, etc for those that arise due BTSP vs non-BTSP.

Comments on revisions:

The authors have mostly addressed my feedback. Compelling evidence for a fundamental observation.

Reviewer #3 (Public review):

Summary:

The authors tackle an important problem: defining the topological changes that occur during tumorigenesis. To study this, they use an established stepwise cell model of breast cancer. A strength of their study is a careful, robust differential analysis of topological features across each cell state, which is presented clearly and rigorously. They define changes in compartmentalization, TAD structure, and chromatin looping. Intriguingly, when the authors integrate differential gene expression with chromatin looping, they see that most differentially regulated genes are not involved in loop changes, suggesting that changes in promoter or enhancer chromatin marks may play a bigger role in regulating transcription than differential loops. The differential topology analysis and its integration with transcription is very well done- one of the best versions of this I have read in the 3D genome field! However, the paper is framed largely as a cancer biology study, and it teaches us much less about this. I am worried that some of the trends for each topologic feature are not going to be consistent across the pre-malignant-malignant-metastatic spectrum and would like the authors to soften some of their claims a bit regarding how this clarifies our understanding of cancer evolution.

Weaknesses:

Major Concerns:

(1) The integration of gene expression and chromatin loops is intriguing. The authors' differential analysis, however, omits consideration of genes that are on and simply further upregulated versus genes that transition on/off or off/on. It would be nice to see the authors break out looping patterns for these two different patterns of regulation, as it may be instructive regarding the rules for how EP loops govern transcription.

(2) Given the paucity of differential loops at the majority of genes whose expression changes, the authors should examine chromatin subcompartments, as these may associate more with differential transcription.

(3) The authors could push their TAD analysis further by integrating it with transcription. Can they look at genes and their enhancers that span these altered boundaries to see if these shifts impact transcription?

(4) The progression of cancer critically goes from a benign -> pre-malignant -> malignant -> metastatic series of steps. The AT1 line is described as 'premalignant' and thus the authors' series omits a malignant line. While I think adding such a sample is an unreasonable request at this point (as it would have had to have been studied in 'batch' with these other samples), the authors should acknowledge that they omit this step and spend some time discussing the genetic, morphologic, and phenotypic features for their 3 conditions. The images in Figure 1S aren't particularly useful- they don't tell the reader that these cells are malignant/benign. The karyotypic data are intriguing but not fully analyzed, so it is hard to know what true phenotype these cells represent. For example, malignant means DCIS/invasive carcinoma - so then what does this pre-malignant cell model represent? The described alteration in the AT1 line is a Ras oncogene, so in some sense, the transition to this line really is just +/- Ras. The authors could spend some time thinking about the effects of Ras specifically on the 3D genome.

Reviewer #3 (Public review):

Summary:

This paper seeks to identify underlying mechanisms contributing to memory deficits observed in Alzheimer's disease (AD) mouse models. By understanding these mechanisms, they hope to uncover insights into subtle cognitive changes early in AD to inform interventions for early-stage decline.

Strengths:

The paper provides a comprehensive exploration of memory deficits in an AD mouse model, covering early and late stages of the disease. The experimental design was robust, confirming age-dependent increases in Aβ plaque accumulation in the AD model mice and using multiple behavior tasks that collectively highlighted difficulties in maintaining multiple competing memory cues, with deficits most pronounced in older mice.

In the fear acquisition, extinction, and reinstatement task, AD model mice exhibited a significantly higher fear response after acquisition compared to controls, as well as a greater drop in fear response during reinstatement. These findings suggest that AD mice struggle to retain the fear memory associated with the conditioned stimulus, with the group differences being more pronounced in the older mice.

In the reversal Barnes maze task, the AD model mice displayed a tendency to explore the maze perimeter rather than the two potential target holes, indicating a failure to integrate multiple memory cues into their strategy. This contrasted with the control mice, which used the more confirmatory strategy of focusing on the two target holes. Despite this, the AD mice were quicker to reach the target hole, suggesting that their impairments were specific to memory retrieval rather than basic task performance.

The authors strengthened their findings by analyzing their data with a leading computational model, which describes how animals balance competing memories. They found that AD mice showed somewhat of a contradiction: a tendency to both treat trials as more alike than they are (lower α) and similar stimuli as more distinct than they are (lower σx) compared to controls.

Weaknesses:

While conceptually solid, the model struggles to fit the data and to support the key hypothesis about AD mice's inability to retain competing memories. These issues are evident in Figure 3:

(1) The model misses trends in the data, including the gradual learning of fear in all groups during acquisition, the absence of a fear response at the start of the experiment, and the faster return of fear during reinstatement compared to the gradual learning of fear during acquisition. It also underestimates the increase in fear at the start of day 2 of extinction, particularly in controls.

(2) The model explains the higher fear response in controls during reinstatement largely through a stronger association to the context formed during the unsignaled shock phase, rather than to any memory of the conditioned stimulus from acquisition (as seen in Figure 3C). In the experiment, however, this memory does seem to be important for explaining the higher fear response in controls during reinstatement (as seen in Author Response Figure 3). The model does show a necessary condition for memory retrieval, which is that controls rely more on the latent causes from acquisition. But this alone is not sufficient, since the associations within that cause may have been overwritten during extinction. The Rescorla-Wagner model illustrates this point: it too uses the latent cause from acquisition (as it only ever uses a single cause across phases) but does not retain the original stimulus-shock memory, updating and overwriting it continuously. Similarly, the latent cause model may reuse a cause from acquisition without preserving its original stimulus-shock association.

These issues lead to potential overinterpretation of the model parameters. The differences in α and σx are being used to make claims about cognitive processes (e.g., overgeneralization vs. over differentiation), but the model itself does not appear to capture these processes accurately.

The authors could benefit from a model that better matches the data and captures the retention and retrieval of fear memories across phases. While they explored alternatives, including the Rescorla-Wagner model and a latent state model, these showed no meaningful improvement in fit. This highlights a broader issue: these models are well-motivated but may not fully capture observed behavior.

Conclusion:

Overall, the data support the authors' hypothesis that AD model mice struggle to retain competing memories, with the effect becoming more pronounced with age. While I believe the right computational model could highlight these differences, the current models fall short in doing so.

Reviewer #3 (Public review):

Summary:

Kamal L. Nahas et al. demonstrated that pUL16, pUL21, pUL34, VP16, and pUS3 are involved in the egress of the capsids from the nucleous, since mutant viruses ΔpUL16, ΔpUL21, ΔUL34, ΔVP16, and ΔUS3 HSV-1 show nuclear egress attenuation determined by measuring the nuclear:cytoplasmic ratio of the capsids, the dfParental, or the mutants. Then, they showed that gM-mCherry+ endomembrane association and capsid clustering were different in pUL11, pUL51, gE, gK, and VP16 mutants. Furthermore, the 3D view of cytoplasmic budding events suggests an envelopment mechanism where capsid budding into spherical/ellipsoidal vesicles drives the envelopment.

Strengths:

The authors employed both structured illumination microscopy and cellular ultrastructure analysis to examine the same infected cells, using cryo-soft-X-ray tomography to capture images. This combination, set here for the first time, enabled the authors to obtain holistic data regarding a biological process, as a viral assembly. Using this approach, the researchers studied various stages of HSV-1 assembly. For this, they constructed a dual-fluorescently labelled recombinant virus, consisting of eYFP-tagged capsids and mCherry-tagged envelopes, allowing for the independent identification of both unenveloped and enveloped particles. They then constructed nine mutants, each targeting a single viral protein known to be involved in nuclear egress and envelopment in the cytoplasm, using this dual-fluorescent as the parental one. The experimental setting, both the microscopic and the virological, is robust and well-controlled. The manuscript is well-written, and the data generated is robust and consistent with previous observations made in the field.

I congratulate the authors. The work is robust, and I personally highlight the way they managed to include others' results merged among their own, providing a complete view of the story.

Comments on the latest version:

I reviewed the responses and the updated manuscript, and I agree with the reviewer's #1 words: "The manuscript was already strong, but with the addition of the summary table and the separated images, it is now excellent."

Reviewer #3 (Public review):

Summary:

The authors seek to determine the underlying traits that support the exceptional capacity of Aspergillus oryzae to secrete enzymes and heterologous proteins. To do so, they leverage the availability of multiple domesticated isolates of A. oryzae along with other Aspergillus species to perform comparative imaging and genomic analysis.

Strengths:

The strength of this study lies in the use of multifaceted approaches to identify significant differences in hyphal morphology that correlate with enzyme secretion, which is then followed by the use of genomics to identify candidate functions that underlie these differences.

Weaknesses:

The authors addressed all suggestions satisfactorily.

Reviewer #3 (Public review):

The authors have satisfactorily addressed my inquiries. However, I had to look quite hard to find where they responded to my final comment regarding the potential role of Arpc2 post-fusion during myofiber growth and/or maintenance, which I eventually located on page 7. I would appreciate it if the authors could state this point more explicitly, perhaps by adding a sentence such as "However, we cannot rule out the possibility that Arpc2 may also play a role in....." to improve clarity of communication.

While I understood from the original version that this issue falls beyond the immediate scope of the study, I believe it is important to adopt a more cautious and rigorous interpretative framework, especially given the widespread use of this experimental approach. In particular, when a gene could potentially have additional roles in myofibers, it may be helpful to explicitly acknowledge that possibility. Even if Arpc2 may not necessarily be one of them, such roles cannot be fully excluded without direct testing.

Reviewer #3 (Public review):

Mondal et al. use computational modeling to investigate how activity-dependent shifts in voltage-dependent (in)activation curves can complement changes in ion channel conductance to support homeostatic plasticity. While it is well established that the voltage-dependent properties of ion channels influence neuronal excitability, their potential role in homeostatic regulation, alongside conductance changes, has remained largely unexplored. The results presented here demonstrate that activity-dependent regulation of voltage dependence can interact with conductance plasticity to enable neurons to attain and maintain target activity patterns, in this case, intrinsic bursting. Notably, the timescale of these voltage-dependent shifts influences the final steady-state configuration of the model, shaping both channel parameters and activity features such as burst period and duration. A major conclusion of the study is that altering this timescale can seamlessly modulate a neuron's intrinsic properties, which the authors suggest may be a mechanism for adaptation to perturbations.

While this conclusion is largely well-supported, additional analyses could help clarify its scope. For instance, the effects of timescale alterations are clearly demonstrated when the model transitions from an initial state that does not meet the target activity pattern to a new stable state. However, Fig. 6 and the accompanying discussion appear to suggest that changing the timescale alone is sufficient to shift neuronal activity more generally. It would be helpful to clarify that this effect primarily applies during periods of adaptation, such as neurodevelopment or in response to perturbations, and not necessarily once the system has reached a stable, steady state. As currently presented, the simulations do not test whether modifying the timescale can influence activity after the model has stabilized. In such conditions, changes in timescale are unlikely to affect network dynamics unless they somehow alter the stability of the solution, which is not shown here. That said, it seems plausible that real neurons experience ongoing small perturbations which, in conjunction with changes in timescale, could allow gradual shifts toward new solutions. This possibility is not discussed but could be a fruitful direction for future work.

Editor's note: The authors have adequately addressed the concerns raised in the public reviews above, as well as the previous recommendations, and revised the manuscript where necessary.

Reviewer #3 (Public review):

Summary:

Submitted to the Tools and Resources series, this study reports on the use of a single-domain antibody targeting the nucleoporin Nup84 to probe and track NPCs in budding yeast. The authors demonstrate their ability to rapidly label or pull down NPCs by inducing the expression of a tagged version of the nanobody (Figure 1).

Strengths:

This tool's main strength is its versatility as an inexpensive, easy-to-set-up alternative to metabolic labelling or optical switching. This same rationale could, in principle, be applied to the study of other multiprotein complexes using similar strategies, provided that single-chain antibodies are available.

Weaknesses:

This approach has no inherent weaknesses, but it would be useful for the authors to verify that their pulse labelling strategy can also be used to detect assembly intermediates, structural variants, or damaged NPCs.

Overall, the data clearly show that Nup84 nanobodies are a valuable tool for imaging NPC dynamics and investigating their interactomes through affinity purification.

Reviewer #4 (Public review):

The manuscript reports on a large-scale study correlating genomic architecture with splicing complexity over almost 1,500 species. We still know relatively little about alternative splicing functional consequences and evolution, and thus, the study is relevant and timely. The methodology relies on annotations from NCBI for high-quality genomes and a main metric proposed by the authors and named Alternative Splicing Ratio (ASR). It quantifies the level of redundancy of each coding nucleotide in the annotated isoforms.

According to the authors' response to the first reviewers' comments, the present version of the manuscript seems to be a profoundly revised version compared to the original submission. I did not have access to the reviewers' comments.

Although the study addresses an important question and the authors have visibly made an important effort to make their claims more statistically robust, I have a number of major concerns regarding the methodology and its presentation.

(1) A large part of the manuscript is speculative and vague. For instance, the Discussion is very long (almost longer than the Results section) and the items discussed are sometimes not in direct connection with the present work. I would suggest merging the last 2 paragraphs, for instance, since the before last paragraph is essentially a review of the literature without direct connection to the present work.

(2) The Methods section lacks clarity and precision. A large part is devoted to explaining the biases in the data without any reference or quantification. The definition of ASR is very confusing. It is first defined in equation 2, with a different name, and then again in the next subsection from a different perspective on lines 512-518. Why build matrices of co-occurrences if these are, in practice, never used? It seems the authors exploit only the trace. A major revision, if I understood correctly, was the correction/normalisation of the ASR metric. This normalisation is not explained. The authors argue that they will write another paper about it, I do not think this is acceptable for the publication of the present manuscript. Furthermore, there is no information about the technical details of the implementation: which packages did the authors use?

(3) Could the authors motivate why they do not directly focus on the MC permutation test? They motivate the use of permutations because the data contains extreme outliers and are non normal in most cases. Hence, it seems the Welch's ANOVA is not adapted. "To further validate our findings, we also conducted<br /> 148 a Monte Carlo permutation test, which supported the conclusions (see Methods)." Where is the comparison shown? I did not see any report of the results for the non-permuted version of the Welch's ANOVA.

(4) What are the assumptions for the Phylogenetic Generalized Least Squares? Which evolution model was chosen and why? What is the impact of changing the model? Could the authors define more precisely (e.g. with equations) what is lambda? Is it estimated or fixed?

(5) I think the authors could improve their account of recent literature on the topic. For instance, the paper https://doi.org/10.7554/eLife.93629.3, published in the same journal last year, should be discussed. It perfectly fits in the scope of the subsection "Evidence for the adaptive role of alternative splicing". Methods and findings reported in https://doi.org/10.1186/s13059-021-02441-9 and https://www.genome.org/cgi/doi/10.1101/gr.274696.120 directly concern the assessment of AS evolutionary conservation across long evolutionary times and/or across many species. These aspects are mentioned in the introduction on p.3. but without pointing to such works. Can we really qualify a work published in 2011 as "recent" (line 348-350)?

The generated data and codes are available on Zenodo, which is a good point for reproducibility and knowledge sharing with the community.

Reviewer #3 (Public review):

Summary:

Su et al. sought to understand how the opportunistic pathogen Staphylococcus aureus responds to multiple selection pressures during infection. Specifically, the authors were interested in how the host environment and antibiotic exposure impact the evolution of both virulence and antibiotic resistance in S. aureus. To accomplish this, the authors performed an evolution experiment where S. aureus was fed to Caenorhabditis elegans as a model system to study the host environment and then either subjected to the antibiotic oxacillin or not. Additionally, the authors investigated the difference in evolution between an antibiotic-resistant strain, MRSA, and an isogenic susceptible strain, MSSA. They found that MRSA strains evolved in both antibiotic and host conditions became more virulent, and that strains evolved outside these conditions lost virulence. Looking at the strains evolved in just antibiotic conditions, the authors found that S. aureus maintained its ability to lyse blood cells. Mutations in codY, gdpP, and pbpA were found to be associated with increased virulence. Additionally, these mutations identified in these experiments were found in S. aureus strains isolated from human infections.

Strengths:

The data are well-presented, thorough, and are an important addition to the understanding of how certain pathogens might adapt to different selective pressures in complex environments.

Weaknesses:

There are a few clarifications that could be made to better understand and contextualize the results. Primarily, when comparing the number of mutations and selection across conditions in an evolution experiment, information about population sizes is important to be able to calculate the mutation supply and number of generations throughout the experiment. These calculations can be difficult in vivo, but since several steps in the methodology require plating and regrowth, those population sizes could be determined. There was also no mention of how the authors controlled the inoculation density of bacteria introduced to each host. This would need to be known to calculate the generation time within the host. These caveats should be addressed in the manuscript.

Another concern is the number of generations the populations of S. aureus spent either with relaxed selection in rich media or under antibiotic pressure in between the host exposure periods. It is probable then that the majority of mutations were selected for in these intervening periods between host infection. Again, a more detailed understanding of population sizes would contribute to the understanding of which phase of the experiment contributed to the mutation profile observed.

Reviewer #3 (Public review):

Summary:

This study aimed to investigate pseudouridylation across various RNA species in multiple bacterial strains using an optimized BID-seq approach. It examined both conserved and divergent modification patterns, the potential functional roles of pseudouridylation, and its dynamic regulation across different growth conditions.

Strengths:

The authors optimized the BID-seq method and applied this important technique to bacterial systems, identifying multiple pseudouridylation sites across different species. They investigated the distribution of these modifications, associated sequence motifs, their dynamics across growth phases, and potential functional roles. These data are of great interest to researchers focused on understanding the significance of RNA modifications, particularly mRNA modifications, in bacteria.

Weaknesses:

(1) The reliability of BID-seq data is questionable due to a lack of experimental validations.

(2) The manuscript is not well-written, and the presented work shows a major lack of scientific rigor, as several key pieces of information are missing.

(3) The manuscript's organization requires significant improvement, and numerous instances of missing or inconsistent information make it difficult to understand the key objectives and conclusions of the study.

(4) The rationale for selecting specific bacterial species is not clearly explained, and the manuscript lacks a systematic comparison of pseudouridylation among these species.

Reviewer #3 (Public review):

Summary:

This is a well-done study. It shows, in a comprehensive manner, that Sp5 and Sp8 play essential roles in maintaining the complicated positive feedback circuitry needed for specification of neuromesodermal competent progenitors (NMCs) in caudal mesodermal development in murine embryos.

Strengths:

The developmental genetics, transcriptomic, and genomic survey of TF binding are all satisfactory and make a compelling story. The CRISPR deletion of the Wnt3a downstream enhancer clearly demonstrates that it plays an important role in the positive feedback circuit.

Weaknesses:

My only concerns are some of the language surrounding the mechanistic interpretation of the Wnt3a downstream enhancer and the relationship between TCF and TLE binding.

Reviewer #3 (Public review):

Summary:

It has been a long time since I enjoyed reviewing a paper as much as this one. In it, the authors generate an unprecedented view of the aboral organ of a 5-day-old ctenophore. They proceed to derive numerous insights by reconstructing the populations and connections of cell types, with up to 150 connections from the main Q1-4 neuron.

Strengths:

The strengths of the analysis are the sophisticated imaging methods used, the labor-intensive reconstruction of individual neurons and organelles, and especially the mapping of synapses. The synaptic connections to and from the main coordinating neurons allow the authors to create a polarized network diagram for these components of the aboral organ. These connections give insight into the potential functions of the major neurons. This also gives some unexpected results, particularly the lack of connections from the balancer system to the coordinating system.

Weaknesses:

There were no significant weaknesses in the paper - only a slate of interesting unanswered questions to motivate future studies.

Reviewer #3 (Public review):

Summary:

Zhu et al. set out to elucidate how the moral emotions of guilt and shame emerge from specific cognitive antecedents - harm and responsibility - and how these emotions subsequently drive compensatory behavior. Consistent with their prediction derived from functionalist theories of emotion, their behavioral findings indicate that guilt is more influenced by harm, whereas shame is more influenced by responsibility. In line with previous research, their results also demonstrate that guilt has a stronger facilitating effect on compensatory behavior than shame. Furthermore, computational modeling and neuroimaging results suggest that individuals integrate harm and responsibility information into a composite representation of the individual's share of the harm caused. Brain areas such as the striatum, insula, temporoparietal junction, lateral prefrontal cortex, and cingulate cortex were implicated in distinct stages of the processing of guilt and/or shame. In general, this work makes an important contribution to the field of moral emotions. Its impact could be further enhanced by clarifying methodological details, offering a more nuanced interpretation of the findings, and discussing their potential practical implications in greater depth.

Strengths:

First, this work conceptualizes guilt and shame as processes unfolding across distinct stages (cognitive appraisal, emotional experience, and behavioral response) and investigates the psychological and neural characteristics associated with their transitions from one stage to the next.

Second, the well-designed experiment effectively manipulates harm and responsibility - two critical antecedents of guilt and shame.

Third, the findings deepen our understanding of the mechanisms underlying guilt and shame beyond what has been established in previous research.

Weaknesses:

(1) Over the course of the task, participants may gradually become aware of their high error rate in the dot estimation task. This could lead them to discount their own judgments and become inclined to rely on the choices of other deciders. It is unclear whether participants in the experiment had the opportunity to observe or inquire about others' choices. This point is important, as the compensatory decision-making process may differ depending on whether choices are made independently or influenced by external input.

(2) Given the inherent complexity of human decision-making, it is crucial to acknowledge that, although the authors compared eight candidate models, other plausible alternatives may exist. As such, caution is warranted when interpreting the computational modeling results.

(3) I do not agree with the authors' claim that "computational modeling results indicated that individuals integrate harm and responsibility in the form of a quotient" (i.e., harm/responsibility). Rather, the findings appear to suggest that individuals may form a composite representation of the harm attributable to each individual (i.e., harm/the number of people involved). The explanation of the modeling results ought to be precise.

(4) Many studies have reported positive associations between trait gratitude, social value orientation, and altruistic behavior. It would be helpful if the authors could provide an explanation about why this study failed to replicate these associations.

(5) As the authors noted, guilt and shame are closely linked to various psychiatric disorders. It would be valuable to discuss whether this study has any implications for understanding or even informing the treatment of these disorders.

Reviewer #3 (Public review):

This study is a follow-up to a recent study of synaptic development based on a powerful data set that combines anterograde labeling, immunofluorescence labeling of synaptic proteins, and STORM imaging (Cell Reports, 2023). Specifically, they use anti-Vglut2 label to determine the size of the presynaptic structure (which they describe as the vesicle pool size), anti-Bassoon to label active zones with the resolution to count them, and anti-Homer to identify postsynaptic densities. Their previous study compared the detailed synaptic structure across the development of synapses made with contra-projecting vs. ipsi-projecting RGCs and compared this developmental profile with a mouse model with reduced retinal waves. In this study, they produce a new detailed analysis on the same data set in which they classify synapses into "multi-active zone" vs. "single-active zone" synapses and assess the number and spacing of these synapses. The authors use measurements to make conclusions about the role of retinal waves in the generation of same-eye synaptic clusters. The authors interpret these results as providing insight into how neural activity drives synapse maturation, the strength of their conclusions is not directly tested by their analysis.

Strengths:

This is a fantastic data set for describing the structural details of synapse development in a part of the brain undergoing activity-dependent synaptic rearrangements. The fact that they can differentiate the eye of origin is what makes this data set unique over previous structural work. The addition of example images from the EM dataset provides confidence in their categorization scheme.

Weaknesses:

Though the descriptions of single vs multi-active zone synapses are important and represent a significant advance, the authors continue to make unsupported conclusions regarding the biological processes driving these changes. Although this revision includes additional information about the populations tested and the tests conducted, the authors do not address the issue raised by previous reviews. Specifically, they provide no assessment of what effect size represents a biologically meaningful result. For example, a more appropriate title is "The distribution of eye-specific single vs multi-active zone is altered in mice with reduced spontaneous activity" rather than concluding that this difference in clustering is somehow related to synaptic competition. Of course, the authors are free to speculate, but many of the conclusions of the paper are not supported by their results.

Reviewer #3 (Public review):

Summary:

The present manuscript investigates and proposes different mechanisms for the effects of two therapeutic approaches - cognitive distancing technique and use of antidepressants - on subjective ratings of happiness, confidence, and task engagement, and on the influence of such subjective experiences on choice behavior. Both approaches were found to link to changes in affective state dynamics in a choice task, specifically reduced drift (cognitive distancing) and increased baseline (antidepressant use). Results also suggest that cognitive distancing may reduce the weighing of recent expected values in the happiness model, while antidepressant use may reduce forgetting of choices and outcomes.

Strengths:

This is a timely topic and a significant contribution to ongoing efforts to improve our mechanistic understanding of psychopathology and devise effective novel interventions. The relevance of the manuscript's central question is clear, and the links to previous literature and the broader field of computational psychiatry are well established. The modelling approaches are thoughtful and rigorously tested, with appropriate model checks and persuasive evidence that modelling complements the theoretical argument and empirical findings.

Weaknesses:

Some vagueness and lack of clarity in theoretical mechanisms and interpretation of results leave outstanding questions regarding (a) the specific links drawn between affective biases, therapies aimed at mitigating them, and mental health function, and (b) the structure and assumptions of the modelling, and how they support the manuscript's central claims. Broadly, I do not fully understand the distinction between how choice behavior vs. affect are impacted separately or together by cognitive distancing. Clarification on this point is needed, possibly through a more explicit proposal of a mechanism (or several alternative mechanisms?) in the introduction and more explicit interpretation of the modelling results in the context of the cyclical choice-affect mechanism.

(1) Theoretical framework and proposed mechanisms

The link between affective biases and negative thinking patterns is a bit unclear. The authors seem to make a causal claim that "affective biases are precipitated and maintained by negative thinking patterns", but it is unclear what precisely these negative patterns are; earlier in the same paragraph, they state that affective biases "cause low mood" and possibly shift choices toward those that maintain low mood. So the directionality of the mechanism here is unclear - possibly explaining a bit more of the cyclic nature of this mechanism, and maybe clarifying what "negative thinking patterns" refer to will be helpful.

More generally, this link between affect and choices, especially given the modelling results later on, should be clarified further. What is the mechanism by which these two impact each other? How do the models of choice and affect ratings in the RL task test this mechanism? I'm not quite sure the paper answers these questions clearly right now.

The authors also seem to implicitly make the claim that symptoms of mental ill-health are at least in part related to choice behavior. I find this a persuasive claim generally; however, it is understated and undersupported in the introduction, to the point where a reader may need to rely on significant prior knowledge to understand why mitigating the impact of affective biases on choice behavior would make sense as the target of therapeutic interventions. This is a core tenet of the paper, and it would be beneficial to clarify this earlier on.

It would be helpful to interpret a bit more clearly the findings from 3.4. on decreased drift in all three subjective assessments in the cognitive distancing group. What is the proposed mechanism for this? The discussion mentions that "attenuated declines [...] over time, [add] to our previously reported findings that this psychotherapeutic technique alters aspects of reward learning" - but this is vague and I do not understand, if an explanation for how this happens is offered, what that explanation is. Given the strong correlation of the drift with fatigue, is the explanation that cognitive distancing mitigates affect drift under fatigue? Or is this merely reporting the result without an interpretation around potential mechanisms?

(Relatedly, aside from possibly explaining the drift parameter, do the fatigue ratings link with choice behavior in any way? Is it possible that the cognitive distancing was helping participants improve choices under fatigue?)

(2) Task Structure and Modelling

It is unclear what counted as a "rewarding" vs. "unrewarding" trial in the model. From my understanding of the task description, participants obtained positive or no reward (no losses), and verbal feedback, Correct/Incorrect. But given the probabilistic nature of the task, it follows that even some correct choices likely had unrewarding results. Was the verbal feedback still "Correct" in those cases, but with no points shown? I did not see any discussion on whether it is the #points earned or the verbal feedback that is considered a reward in the model. I am assuming the former, but based on previous literature, likely both play a role; so it would be interesting - and possibly necessary to strengthen the paper's argument - to see a model that assigns value to positive/negative feedback and earned points separately.

From a theory perspective, it's interesting that the authors chose to assume separate learning rates for rewarding and non-rewarding trials. Why not, for example, separate reward sensitivity parameters? E.g., rather than a scaling parameter on the PE, a parameter modifying the r term inside the PE equation to, perhaps, assign different values to positive and zero points? (While I think overall the math works out similarly at the fitting time, this type of model should be less flexible on scaling the expected value and more flexible on scaling the actual #points / the subjective experience of the obtained verbal feedback, which seems more in line with the theoretical argument made in the introduction). The introduction explicitly states that negative biases "may cause low mood by making outcomes appear less rewarding" - which in modelling equations seems more likely to translate to different reward-perception biases, and not different learning rates. Alternatively, one might incorporate a perseveration parameter (e.g., similar to Collins et al. 2014) that would also accomplish a negative bias. Either of these two mechanisms seems perhaps worth testing out in a model - especially in a model that defines more clearly what rewarding vs. unrewarding may mean to the participant.

If I understand correctly, the affect ratings models assume that the Q-value and the PE independently impact rating (so they have different weights, w2 and w3), but there is no parameter allowing for different impact for perceived rewarding and unrewarding outcomes? (I may be misreading equations 4-5, but if not, Q-value and PE impact the model via static rather than dynamic parameters.) Given the joint RL-affect fit, this seems to carry the assumption that any perceptual processing differences leading to different subjective perceptions of reward associated with each outcome only impact choice behavior, but not affect? (whereas affect is more broadly impacted, if I'm understanding this correctly, just by the magnitude of the values and PEs?) This is an interesting assumption, and the authors seem to have tested it a bit more in the Supplementary material, as shown in Figure S4. I'm wondering why this was excluded from the main text - it seems like the more flexible model found some potentially interesting differences which may be worth including, especially as they might shed additional insight into the influence of cognitive distancing on the cyclical choice-affect mechanisms proposed.

Minor comments:

If fatigue ratings were strongly associated with drift in the best-fitting model (as per page 13), I wonder if it would make sense to use those fatigue ratings as a proxy rather than allow the parameter to vary freely? (This does not in any way detract from the winning model's explanatory power, but if a parameter seems to be strongly explained by a variable we have empirical data for, it's not clear what extra benefit is earned by having that parameter in the model).

Reviewer #3 (Public review):

Summary:

This report describes the development and initial applications of the ARM (Automated Reproducible Mechano-stimulator), a programmable tool that delivers various mechanical stimuli to a select target (most frequently, a rodent hindpaw). Comparisons to traditional testing methods (e.g., experimenter application of stimuli) reveal that the ARM reduces variability in the anatomical targeting, height, velocity, and total time of stimulus application. Given that the ARM can be controlled remotely, this device was also used to assess effects of experimenter presence on reflexive responses to mechanical stimulation. Although not every experimenter had notable sex-dependent effects on animal behavior, use of the ARM never had this effect (for obvious reasons!). Lastly, the ARM was used to stimulate rodent hindpaws while measuring neuronal activity in the basolateral nucleus of the amygdala (BLA), a brain region that is associated with the negative affect of pain. This device, and similar automated devices, will undoubtedly reduce experimenter-related variability in reflexive mechanical behavior tests; this may increase experimental reproducibility between laboratories who are able to invest in this type of technology.

Strengths:

Clear examples of variability in experimenter stimulus application are provided and then contrasted with uniform stimulus application that is inherent to the ARM.

The ARM is able to quickly oscillate between delivery of various mechanical stimuli; this is advantageous for experimental efficiency.

New additions to the ARM and PAWS platforms have been methodically tested to ensure reproducibility and reliability.

Reviewer #3 (Public review):

Summary:

The authors have identified novel dRTA causing SLC4A1 mutations and studied the resulting kAE1 proteins to determine how they cause dRTA. Based on a previous study on mice expressing the dRTA kAE1 R607H variant, the authors hypothesize that kAE1 variants cause an increase in intracellular pH, which disrupts autophagic and degradative flux pathways. The authors clone these new kAE1 variants and study their transport function and subcellular localization in mIMCD cells. The authors show increased abundance of LC3B II in mIMCD cells expressing some of the kAE1 variants, as well as reduced autophagic flux using eGFP-RFP-LC3. These data, as well as the abundance of autophagosomes, serve as the key evidence that these kAE1 mutants disrupt autophagy. Furthermore, the authors demonstrate that decreasing the intracellular pH abrogates the expression of LC3B II in mIMCD cells expressing mutant SLC4A1. Lastly, the authors argue that mitochondrial function, and specifically ATP synthesis, is suppressed in mIMCD cells expressing dRTA variants and that mitochondria are less abundant in AICs from the kidney of R607H kAE1 mice. While the manuscript does reveal some interesting new results about novel dRTA causing kAE1 mutations, the quality of the data to support the hypothesis that these mutations cause a reduction in autophagic flux can be improved. In particular, the precise method of how the western blots and the immunofluorescence data were quantified, with included controls, would enhance the quality of the data and offer more supportive evidence of the authors' conclusions.

Strengths:

The authors cloned novel dRTA causing kAE1 mutants into expression vectors to study the subcellular localization and transport properties of the variants. The immunofluorescence images are generally of high quality, and the authors do well to include multiple samples for all of their western blots.

Weaknesses:

Inconsistent results are reported for some of the variants. For example, R295H causes intracellular alkalinization but also has no effect on intracellular pH when measured by BCECF. The authors also appear to have performed these in vitro studies on mIMCD cells that were not polarized, and therefore, the localization of kAE1 to the basolateral membrane seems unlikely, based upon images included in the manuscript. Additionally, there is no in vivo work to demonstrate that these kAE1 variants alter intracellular pH, including the R607H mouse, which is available to the authors. The western blots are of varying quality, and it is often unclear which of the bands are being quantified. For example, LAMP1 is reported at 100kDa, the authors show three bands, and it is unclear which one(s) are used to quantify protein abundance. Strikingly, the authors report a nonsensical value for their quantification of LCRB II in Figure 2, where the ratio of LCRB II to total LCRB (I + II) is greater than one. The control experiments with starvation and bafilomyocin are not supportive and significantly reduce enthusiasm for the authors' findings regarding autophagy. There are labeling errors between the manuscript and the figures, which suggest a lack of vigilance in the drafting process.

Reviewer #3 (Public review):

Summary:

The response to lysosomal damage is a fast-moving and timely field. Besides repair and degradation pathways, increasing interest has been focusing on damaged-induced signaling. The authors conducted both transcriptomics and proteomics to characterize the cellular response to lysosomal damage. They identify a signaling pathway leading to activation of NFkappaB. Based on this and supported by Western blot and microscopy data, the authors nicely show that TAB2/3 and TAK1 are activated at damaged lysosomes and kick off the pathway to alter gene expression, which induces cytokines and protect from cell death. TAB2/3 activation is proposed to occur through K63 ubiquitin chain formation. Generally, this is a careful and well conducted study that nicely delineates the pathway under lysosomal stress. The "omics" data serves a valuable resource for the field. More work should be invested into how TAB2/3 are activated at the damaged lysosomes, also to increase novelty in light of previous reports.

Strengths:

Generally, this is a careful and well-conducted study that nicely delineates how the NFkB pathway is activated under lysosomal stress and modulates cell behavior. The "omics" data serves as a valuable resource for the field.

Weaknesses:

While activation of TAB2/3 by K63-linked Ub chains is convincing, more work needs to be done on how they are recruited by distinct damage types to probe relevance for different pathophysiological conditions."

Comments on revisions:

The authors have addressed much of my criticism. Specifically, they have put (with new experiments) the data on the TAB2/3-TAK1 pathway in perspective to the previously reported LUBAC-mediated activation of NFkB. They also addressed the question about the significance of K63-linked chains for TAB2/3 activation with new complementation experiments (a K63-specific NZF mutant failed to rescue).

The third point (types of damage as triggers) raises more questions, though. The authors find that, in contrast to LLOMe, GPN or DC661-induced damage does not activate TAK1 (consistent with lower damage levels). However, the authors still observe K63 ubiquitylation. This goes along with their finding that TAB2 is recruited in the absence of any ubiquitylation (blocked by TAK-243). It argues that TAB2 is recruited by an unknown cue (that may be damage-specific) and then activated by K63. The authors need to clarify whether TAB2 is or is not recruited in the GPN/DC661 conditions (in which K63 occurs, but TAK1 is not activated). The point about the effects of other damage types was also raised by reviewer #1 and should be solved. The fact that TAB2 is recruited independently of K63 should also be visualized in the model. The manuscript will then be an important contribution to the field.

Reviewer #3 (Public review):

Summary:

This study by Bushey et al. adapts and evaluates two newly developed red-shifted optogenetic inhibitors, A1ACR1 and HfACR1, collectively referred to as RubyACRs, for neuronal silencing in Drosophila melanogaster. Traditional optogenetic inhibitors such as GtACR1 and GtACR2 are activated by green (~515 nm) and blue (~470 nm) light, respectively, which poses several limitations in Drosophila. Specifically, shorter-wavelength light suffers from reduced tissue penetration and increased absorption, and is visible to flies, potentially confounding behavioral assays, particularly those involving visual processing. In contrast, RubyACRs are activated by red light (~610-660 nm), which penetrates the cuticle more effectively and thus can be more potent in manipulating fly behavior. In the current manuscript, the authors first demonstrate that both A1ACR1 and HfACR1 can be robustly expressed in fly neurons and are properly trafficked to the plasma membrane. Upon red-light stimulation, both opsins produce strong and sustained hyperpolarization in larval motor neurons, outperforming GtACR1 in both magnitude and temporal dynamics. Next, using two-photon calcium imaging in the visual system, the authors further demonstrate that activation of RubyACRs significantly reduces GCaMP6s signal, indicating that they can reliably inhibit neuronal activity. Importantly, unlike reported in some mammalian studies, RubyACRs do not appear to trigger paradoxical depolarization at axon terminals in the fly visual system, as no evidence of aberrant depolarization is observed in motion-detecting Mi1 neurons.

In the second part of the manuscript, the authors characterize the effects of RubyACRs on fly behavior (walking, learning, and courtship song). Using the inhibition of genetically labelled neurons that regulate these behaviors, the authors demonstrate that stimulation of RubyACRs leads to potent suppression of locomotion, courtship song, or dopamine-dependent associative learning.

Strengths:

Altogether, the experiments conducted in this manuscript demonstrate that RubyACRs are powerful tools for optogenetic inhibition in Drosophila, with advantages in spectral compatibility, behavioral specificity, and potential applications in vivo two-photon calcium imaging.

Weaknesses:

The manuscript is strong, but it can be further improved with a few additional analyses and minor revisions. Especially, a more detailed evaluation of RubyACRs with two-photon excitation will help clarify to what extent these opsins can be simultaneously used together with green GECIs, such as GCaMPs.

Reviewer #3 (Public review):

Summary:

In this manuscript titled "Mycobacterial Metallophosphatase MmpE Acts as a Nucleomodulin to Regulate Host Gene Expression and Promote Intracellular Survival", Chen et al describe biochemical characterisation, localisation and potential functions of the gene using a genetic approach in M. bovis BCG and perform macrophage and mice infections to understand the roles of this potentially secreted protein in the host cell nucleus. The findings demonstrate the role of a secreted phosphatase of M. bovis BCG in shaping the transcriptional profile of infected macrophages, potentially through nuclear localisation and direct binding to transcriptional start sites, thereby regulating the inflammatory response to infection.

Strengths:

The authors demonstrate using a transient transfection method that MmpE when expressed as a GFP-tagged protein in HEK293T cells, exhibits nuclear localisation. The authors identify two NLS motifs that together are required for nuclear localisation of the protein. A deletion of the gene in M. bovis BCG results in poorer survival compared to the wild-type parent strain, which is also killed by macrophages. Relative to the WT strain-infected macrophages, macrophages infected with the ∆mmpE strain exhibited differential gene expression. Overexpression of the gene in HEK293T led to occupancy of the transcription start site of several genes, including the Vitamin D Receptor. Expression of VDR in THP1 macrophages was lower in the case of ∆mmpE infection compared to WT infection. This data supports the utility of the overexpression system in identifying potential target loci of MmpE using the HEK293T transfection model. The authors also demonstrate that the protein is a phosphatase, and the phosphatase activity of the protein is partially required for bacterial survival but not for the regulation of the VDR gene expression.

Weaknesses:

(1) While the motifs can most certainly behave as NLSs, the overexpression of a mycobacterial protein in HEK293T cells can also result in artefacts of nuclear localisation. This is not unprecedented. Therefore, to prove that the protein is indeed secreted from BCG, and is able to elicit transcriptional changes during infection, I recommend that the authors (i) establish that the protein is indeed secreted into the host cell nucleus, and (ii) the NLS mutation prevents its localisation to the nucleus without disrupting its secretion.

Demonstration that the protein is secreted: Supplementary Figure 3 - Immunoblotting should be performed for a cytosolic protein, also to rule out detection of proteins from lysis of dead cells. Also, for detecting proteins in the secreted fraction, it would be better to use Sauton's media without detergent, and grow the cultures without agitation or with gentle agitation. The method used by the authors is not a recommended protocol for obtaining the secreted fraction of mycobacteria.

Demonstration that the protein localises to the host cell nucleus upon infection: Perform an infection followed by immunofluorescence to demonstrate that the endogenous protein of BCG can translocate to the host cell nucleus. This should be done for an NLS1-2 mutant expressing cell also.

(2) In the RNA-seq analysis, the directionality of change of each of the reported pathways is not apparent in the way the data have been presented. For example, are genes in the cytokine-cytokine receptor interaction or TNF signalling pathway expressed more, or less in the ∆mmpE strain?

(3) Several of these pathways are affected as a result of infection, while others are not induced by BCG infection. For example, BCG infection does not, on its own, produce changes in IL1β levels. As the authors did not compare the uninfected macrophages as a control, it is difficult to interpret whether ∆mmpE induced higher expression than the WT strain, or simply did not induce a gene while the WT strain suppressed expression of a gene. This is particularly important because the strain is attenuated. Does the attenuation have anything to do with the ability of the protein to induce lysosomal pathway genes? Does induction of this pathway lead to attenuation of the strain? Similarly, for pathways that seem to be downregulated in the ∆mmpE strain compared to the WT strain, these might have been induced upon infection with the WT strain but not sufficiently by the ∆mmpE strain due to its attenuation/ lower bacterial burden.

(4) CHIP-seq should be performed in THP1 macrophages, and not in HEK293T. Overexpression of a nuclear-localised protein in a non-relevant line is likely to lead to several transcriptional changes that do not inform us of the role of the gene as a transcriptional regulator during infection.

(5) I would not expect to see such large inflammatory reactions persisting 56 days post-infection with M. bovis BCG. Is this something peculiar for an intratracheal infection with 1x107 bacilli? For images of animal tissue, the authors should provide images of the entire lung lobe with the zoomed-in image indicated as an inset.

(6) For the qRT-PCR based validation, infections should be performed with the MmpE-complemented strain in the same experiments as those for the WT and ∆mmpE strain so that they can be on the same graph, in the main manuscript file. Supplementary Figure 4 has three complementary strains. Again, the absence of the uninfected, WT, and ∆mmpE infected condition makes interpretation of these data very difficult.

(7) The abstract mentions that MmpE represses the PI3K-Akt-mTOR pathway, which arrests phagosome maturation. There is not enough data in this manuscript in support of this claim. Supplementary Figure 5 does provide qRT-PCR validation of genes of this pathway, but the data do not indicate that higher expression of these pathways, whether by VDR repression or otherwise, is driving the growth restriction of the ∆mmpE strain.

(8) The relevance of the NLS and the phosphatase activity is not completely clear in the CFU assays and in the gene expression data. Firstly, there needs to be immunoblot data provided for the expression and secretion of the NLS-deficient and phosphatase mutants. Secondly, CFU data in Figure 3A, C, and E must consistently include both the WT and ∆mmpE strain.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors examine the processing stages involved in perceptual decision-making using a new approach to analysing EEG data, combined with a critical stimulus manipulation. This new EEG analysis method enables single-trial estimates of the timing and amplitude of transient changes in EEG time-series, recurrent across trials in a behavioural task. The authors find evidence for three events between stimulus onset and the response in a two-spatial-interval visual discrimination task. By analysing the timing and amplitude of these events in relation to behaviour and the stimulus manipulation, the authors interpret these events as related to separable processing stages for stimulus encoding, attention orientation, and decision (deliberation). This is largely consistent with previous findings from both event-related potentials (across trials) and single-trial estimates using decoding techniques and neural network approaches.

Strengths:

This work is not only important for the conceptual advance, but also in promoting this new analysis technique, which will likely prove useful in future research. For the broader picture, this work is an excellent example of the utility of neural measures for mental chronometry.

Weaknesses:

The manuscript would benefit from some conceptual clarifications, which are important for readers to understand this manuscript as a stand-alone work. This includes clearer definitions of Piéron's and Fechner's laws, and a fuller description of the EEG analysis technique. The manuscript, broadly, but the introduction especially, may be improved by clearly delineating the multiple aims of this project: examining the processes for decision-making, obtaining single-trial estimates of meaningful EEG-events, and whether central parietal positivity reflects ramping activity or steps averaged across trials. A fuller discussion of the limitations of the work, in particular, the absence of motor contributions to reaction time, would also be appreciated.

At times, the novelty of the work is perhaps overstated. Rather, readers may appreciate a more comprehensive discussion of the distinctions between the current work and previous techniques to gauge single-trial estimates of decision-related activity, as well as previous findings concerning distinct processing stages in decision-making. Moreover, a discussion of how the events described in this study might generalise to different decision-making tasks in different contexts (for example, in auditory perception, or even value-based decision-making) would also be appreciated.

Reviewer #3 (Public review):

Summary:

This manuscript presents a well-designed and insightful behavioural study examining human adaptation to room acoustics, building on prior work by Brandewie & Zahorik. The psychophysical results are convincing and add incremental but meaningful knowledge to our understanding of reverberation learning. However, I find the transcranial magnetic stimulation (TMS) component to be over-interpreted. The TMS protocol, while interesting, lacks sufficient anatomical specificity and mechanistic explanation to support the strong claims made regarding a unique role of the dorsolateral prefrontal cortex (dlPFC) in this learning process. More cautious interpretation is warranted, especially given the modest statistical effects, the fact that the main TMS result of interest is a null result, the imprecise targeting of dlPFC (which is not validated), and the lack of knowledge about the timescale of TMS effects in relation to the behavioural task. I recommend revising the manuscript to shift emphasis toward the stronger behavioural findings and to present a more measured and transparent discussion of the TMS results and their limitations.

Strengths:

(1) Well-designed acoustical stimuli and psychophysical task.

(2) Comparisons across room combinations are well conducted.

(3) The virtual acoustic environment is impressive and applied well here.

(4) A timely study with interesting behavioural results.

Weaknesses:

(1) Lack of hypotheses, particularly for TMS.

(2) Lack of evidence for targeting TMS in [brain] space and time.

(3) The most interesting effect of TMS is a null result compared to a weak statistical effect for "meta adaptation"

Reviewer #3 (Public review):

Summary:

The authors review published literature and propose that a visual cortical region in the mouse that is widely considered to contain multiple visual areas should be considered a single visual area.

Strengths:

The authors point out that relatively new data showing reversals of visual-field sign within known, single visual areas of some species require that a visual field sign change by itself should not be considered evidence for a border between visual areas.

Weaknesses:

The existing data are not consistent with the authors' proposal to consolidate multiple mouse areas into a single "V2". This is because the existing definition of a single area is that it cannot have redundant representations of the visual field. The authors ignore this requirement, as well as the data and definitions found in published manuscripts, and make an inaccurate claim that "higher order visual areas in the mouse do not have overlapping representations of the visual field". For quantification of the extent of overlap of representations between 11 mouse visual areas, see Figure 6G of Garrett et al. 2014. [Garrett, M.E., Nauhaus, I., Marshel, J.H., and Callaway, E.M. (2014). Topography and areal organization of mouse visual cortex. The Journal of neuroscience 34, 12587-12600. 10.1523/JNEUROSCI.1124-14.2014.

Reviewer #3 (Public review):

Summary:

The authors use calcium recordings from STN to measure STN activity during spontaneous movement and in a multi-stage avoidance paradigm. They also use optogenetic excitation, optogenetic inhibition, and lesion approaches to increase or decrease the activity of STN during the avoidance paradigm. The paper reports a large amount of data and makes many claims, some seem well supported to this Reviewer, others not so much.

Strengths:

Well-supported claims include data showing that during spontaneous movements, especially contraversive ones, STN calcium activity is increased using bulk photometry measurements. Single-cell measures back this claim but also show that it is only a modest minority of STN cells that respond strongly, with most showing no response during movement, and a similar number showing smaller inhibitions during movement.

Similar data during cued active avoidance procedures show that STN calcium activity sharply increases in response to auditory cues, and during cued movements to avoid a footshock. Optogenetic and lesion experiments are consistent with an important role for STN in generating cue-evoked avoidance. And a strength of these results is that multiple bi-directional approaches were used.

Weaknesses:

I found the experimental design and presentation convoluted and the results over-interpreted.

(1) I really don't understand or accept this idea that delayed movement is necessarily indicative of cautious movements. Is the distribution of responses multi-modal in a way that might support this idea, or do the authors simply take a normal distribution and assert that the slower responses represent 'caution'? Even if responses are multi-modal and clearly distinguished by 'type', why should readers think this that delayed responses imply cautious responding instead of say: habituation or sensitization to cue/shock, variability in attention, motivation, or stress; or merely uncertainty which seems plausible given what I understand of the task design where the same mice are repeatedly tested in changing conditions. This relates to a major claim (i.e., in the work's title).

(2) Related to the last, I'm struggling to understand the rationale for dividing cells into 'types' based the their physiological responses in some experiments (e.g., Figure 7).

(3) The description and discussion of orienting head movements were not well supported, but were much discussed in the avoidance datasets. The initial speed peaks to cue seem to be the supporting data upon which these claims rest, but nothing here suggests head movement or orientation responses.

(4) Similar to the last, the authors note in several places, including abstract, the importance of STN in response timing, i.e., particularly when there must be careful or precise timing, but I don't think their data or task design provides a strong basis for this claim.

(5) I think that other reports show that STN calcium activity is recruited by inescapable foot shock as well. What do these authors see? Is shock, independent of movement, contributing to sharp signals during escapes?

(6) In particular, and related to the last point, the following work is very relevant and should be cited: https://elifesciences.org/reviewed-preprints/104643#tab-content. Note that the focus of this other paper is on a subset of VGLUT2+ Tac1 neurons in paraSTN, but using VGLUT2-Cre to target STN will target both STN and paraSTN.

(7) In multiple other instances, claims that were more tangential to the main claims were made without clearly supporting data or statistics. E.g., claim that STN activation is related to translational more than rotational movement; claim that GCaMP and movement responses to auditory cues were small; claims that 'some animals' responded differently without showing individual data.

(8) In several figures, the number of subjects used was not described. This is necessary. Also necessary is some assessment of the variability across subjects. The only measure of error shown in many figures relates to trial-to-trial or event variability, which is minimal because, in many cases, it appears that hundreds of trials may have been averaged per animal, but this doesn't provide a strong view of biological variability. When bar/line plots are used to display data, I recommend showing individual animals where feasible.

(9) Can the authors consider the extent to which calcium imaging may be better suited to identify increases compared to decreases and how this may affect the results, particularly related to the GRIN data when similar numbers of cells show responses in both directions (e.g., Figure 3)?

(10) Raw example traces are not provided.

(11) The timeline of the spontaneous movement and avoidance sessions was not clear, nor was the number of events or sessions per animal nor how this was set. It is not clear if there was pre-training or habituation, if many or variable sessions were combined per animal, or what the time gaps between sessions were, or if or how any of these parameters might influence interpretation of the results.

(12) It is not clear if or how the spread of expression outside of the target STN was evaluated, and if or how many mice were excluded due to spread or fiber placements.

Reviewer #3 (Public review):

Summary:

The authors recorded brain responses while participants viewed images and captions. The images and captions were taken from the COCO dataset, so each image has a corresponding caption, and each caption has a corresponding image. This enabled the authors to extract features from either the presented stimulus or the corresponding stimulus in the other modality. The authors trained linear decoders to take brain responses and predict stimulus features. "Modality-specific" decoders were trained on brain responses to either images or captions, while "modality-agnostic" decoders were trained on brain responses to both stimulus modalities. The decoders were evaluated on brain responses while the participants viewed and imagined new stimuli, and prediction performance was quantified using pairwise accuracy. The authors reported the following results:

(1) Decoders trained on brain responses to both images and captions can predict new brain responses to either modality.

(2) Decoders trained on brain responses to both images and captions outperform decoders trained on brain responses to a single modality.

(3) Many cortical regions represent the same concepts in vision and language.

(4) Decoders trained on brain responses to both images and captions can decode brain responses to imagined scenes.

Strengths:

This is an interesting study that addresses important questions about modality-agnostic representations. Previous work has shown that decoders trained on brain responses to one modality can be used to decode brain responses to another modality. The authors build on these findings by collecting a new multimodal dataset and training decoders on brain responses to both modalities.

To my knowledge, SemReps-8K is the first dataset of brain responses to vision and language where each stimulus item has a corresponding stimulus item in the other modality. This means that brain responses to a stimulus item can be modeled using visual features of the image, linguistic features of the caption, or multimodal features derived from both the image and the caption. The authors also employed a multimodal one-back matching task, which forces the participants to activate modality-agnostic representations. Overall, SemReps-8K is a valuable resource that will help researchers answer more questions about modality-agnostic representations.

The analyses are also very comprehensive. The authors trained decoders on brain responses to images, captions, and both modalities, and they tested the decoders on brain responses to images, captions, and imagined scenes. They extracted stimulus features using a range of visual, linguistic, and multimodal models. The modeling framework appears rigorous, and the results offer new insights into the relationship between vision, language, and imagery. In particular, the authors found that decoders trained on brain responses to both images and captions were more effective at decoding brain responses to imagined scenes than decoders trained on brain responses to either modality in isolation. The authors also found that imagined scenes can be decoded from a broad network of cortical regions.

Weaknesses:

The characterization of "modality-agnostic" and "modality-specific" decoders seems a bit contradictory. There are three major choices when fitting a decoder: the modality of the training stimuli, the modality of the testing stimuli, and the model used to extract stimulus features. However, the authors characterize their decoders based on only the first choice-"modality-specific" decoders were trained on brain responses to either images or captions, while "modality-agnostic" decoders were trained on brain responses to both stimulus modalities. I think that this leads to some instances where the conclusions are inconsistent with the methods and results.

First, the authors suggest that "modality-specific decoders are not explicitly encouraged to pick up on modality-agnostic features during training" (line 137) while "modality-agnostic decoders may be more likely to leverage representations that are modality-agnostic" (line 140). However, whether a decoder is required to learn modality-agnostic representations depends on both the training responses and the stimulus features. Consider the case where the stimuli are represented using linguistic features of the captions. When you train a "modality-specific" decoder on image responses, the decoder is forced to rely on modality-agnostic information that is shared between the image responses and the caption features. On the other hand, when you train a "modality-agnostic" decoder on both image responses and caption responses, the decoder has access to the modality-specific information that is shared by the caption responses and the caption features, so it is not explicitly required to learn modality-agnostic features. As a result, while the authors show that "modality-agnostic" decoders outperform "modality-specific" decoders in most conditions, I am not convinced that this is because they are forced to learn more modality-agnostic features.

Second, the authors claim that "modality-specific decoders can be applied only in the modality that they were trained on, while "modality-agnostic decoders can be applied to decode stimuli from multiple modalities, even without knowing a priori the modality the stimulus was presented in" (line 47). While "modality-agnostic" decoders do outperform "modality-specific" decoders in the cross-modality conditions, it is important to note that "modality-specific" decoders still perform better than expected by chance (figure 5). It is also important to note that knowing about the input modality still improves decoding performance even for "modality-agnostic" decoders, since it determines the optimal feature space-it is better to decode brain responses to images using decoders trained on image features, and it is better to decode brain responses to captions using decoders trained on caption features.

Reviewer #3 (Public review):

Summary

This paper investigates how disinformation affects reward learning processes in the context of a two-armed bandit task, where feedback is provided by agents with varying reliability (with lying probability explicitly instructed). They find that people learn more from credible sources, but also deviate systematically from optimal Bayesian learning: They learned from uninformative random feedback, learned more from positive feedback, and updated too quickly from fully credible feedback (especially following low-credibility feedback). Overall, this study highlights how misinformation could distort basic reward learning processes, without appeal to higher order social constructs like identity.

Strengths

• The experimental design is simple and well-controlled; in particular, it isolates basic learning processes by abstracting away from social context
• Modeling and statistics meet or exceed standards of rigor
• Limitations are acknowledged where appropriate, especially those regarding external validity
• The comparison model, Bayes with biased credibility estimates, is strong; deviations are much more compelling than e.g. a purely optimal model
• The conclusions are of substantial interest from both a theoretical and applied perspective

Weaknesses

The authors have addressed most of my concerns with the initial submission. However, in my view, evidence for the conclusion that less credible feedback yields a stronger positivity bias remains weak. This is due to two issues.

Absolute or relative positivity bias?

The conclusion of greater positivity bias for lower credible feedback (Fig 5) hinges on the specific way in which positivity bias is defined. Specifically, we only see the effect when normalizing the difference in sensitivity to positive vs. negative feedback by the sum. I appreciate that the authors present both and add the caveat whenever they mention the conclusion. However, without an argument that the relative definition is more appropriate, the fact of the matter is that the evidence is equivocal.

There is also a good reason to think that the absolute definition is more appropriate. As expected, participants learn more from credible feedback. Thus, normalizing by average learning (as in the relative definition) amounts to dividing the absolute difference by increasingly large numbers for more credible feedback. If there is a fixed absolute positivity bias (or something that looks like it), the relative bias will necessarily be lower for more credible feedback. In fact, the authors own results demonstrate this phenomenon (see below). A reduction in relative bias thus provides weak evidence for the claim.

It is interesting that the discovery study shows evidence of a drop in absolute bias. However, for me, this just raises questions. Why is there a difference? Was one a just a fluke? If so, which one?

Positivity bias or perseveration?

Positivity bias and perseveration will both predict a stronger relationship between positive (vs. negative) feedback and future choice. They can thus be confused for each other when inferred from choice data. This potentially calls into question all the results on positivity bias.

The authors clearly identify this concern in the text and go to considerable lengths to rule it out. However, the new results (in revision 1) show that a perseveration-only model can in fact account for the qualitative pattern in the human data (the CA parameters). This contradicts the current conclusion:

Critically, however, these analyses also confirmed that perseveration cannot account for our main finding of increased positivity bias, relative to the overall extent of CA, for low-credibility feedback.

Figure 24c shows that the credibility-CA model does in fact show stronger positivity bias for less credible feedback. The model distribution for credibility 1 is visibly lower than for credibilities 0.5 and 0.75.

The authors need to be clear that it is the magnitude of the effect that the perseveration-only model cannot account for. Furthermore, they should additionally clarify that this is true only for models fit to data; it is possible that the credibility-CA model could capture the full size of the effect with different parameters (which could fit best if the model was implemented slightly differently).

The authors could make the new analyses somewhat stronger by using parameters optimized to capture just the pattern in CA parameters (for example by MSE). This would show that the models are in principle incapable of capturing the effect. However, this would be a marginal improvement because the conclusion would still rest on a quantitative difference that depends on specific modeling assumptions.

New simulations clearly demonstrate the confound in relative bias

Figure 24 also speaks to the relative vs. absolute question. The model without positivity bias shows a slightly stronger absolute "positivity bias" for the most credible feedback, but a weaker relative bias. This is exactly in line with the logic laid out above. In standard bandit tasks, perseveration can be quite well-captured by a fixed absolute positivity bias, which is roughly what we see in the simulations (I'm not sure what to make of the slight increase; perhaps a useful lead for the authors). However, when we divide by average credit assignment, we now see a reduction. This clearly demonstrates that a reduction in relative bias can emerge without any true differences in positivity bias.

Given everything above, I think it is unlikely that the present data can provide even "solid" evidence for the claim that positivity bias is greater with less credible feedback. This confound could be quickly ruled out, however, by a study in which feedback is sometimes provided in the absence of a choice. This would empirically isolate positivity bias from choice-related effects, including perseveration.

Reviewer #3 (Public review):

This manuscript investigates computational mechanisms underlying increased risk-taking behavior in adolescent patients with suicidal thoughts and behaviors. Using a well-established gambling task that incorporates momentary mood ratings and previously established computational modeling approaches, the authors identify particular aspects of choice behavior (which they term approach bias) and mood responsivity (to certain rewards) that differ as a function of suicidality. The authors replicate their findings on both clinical and large-scale non-clinical samples.

The main problem, however, is that the results do not seem to support a specific conclusion with regard to suicidality. The S+ and S- groups differ substantially in the severity of symptoms, as can be seen by all symptom questionnaires and the baseline and mean mood, where S- is closer to HC than it is to S+. The main analyses control for illness duration and medication but not for symptom severity. The supplementary analysis in Figure S11 is insufficient as it mistakes the absence of evidence (i.e., p > 0.05) for evidence of absence. Therefore, the results do not adequately deconfound suicidality from general symptom severity.

The second main issue is that the relationship between an increased approach bias and decreased mood response to CR is conceptually unclear. In this respect, it would be natural to test whether mood responses influence subsequent gambling choices. This could be done either within the model by having mood moderate the approach bias or outside the model using model-agnostic analyses.

Additionally, there is a conceptual inconsistency between the choice and mood findings that partly results from the analytic strategy. The approach bias is implemented in choice as a categorical value-independent effect, whereas the mood responses always scale linearly with the magnitude of outcomes. One way to make the models more conceptually related would be to include a categorical value-independent mood response to choosing to gamble/not to gamble.

The manuscript requires editing to improve clarity and precision. The use of terms such as "mood" and "approach motivation" is often inaccurate or not sufficiently specific. There are also many grammatical errors throughout the text.

Claims of clinical relevance should be toned down, given that the findings are based on noisy parameter estimates whose clinical utility for the treatment of an individual patient is doubtful at best.

Reviewer #3 (Public review):

Chen et al. identify endophilin A1 as a novel component of the inhibitory postsynaptic scaffold. Their data show impaired evoked inhibitory synaptic transmission in CA1 neurons of mice lacking endophilin A1, and an increased susceptibility to seizures. Endophilin can interact with the postsynaptic scaffold protein gephyrin and promotes assembly of the inhibitory postsynaptic element. Endophilin A1 is known to play a role in presynaptic terminals and in dendritic spines, but a role for endophilin A1 at inhibitory postsynaptic densities has not yet been described, providing a valuable addition to the field.

To investigate the role of endophilin A1 at inhibitory postsynapses, the authors used a broad array of experimental approaches, including tests of seizure susceptibility, electrophysiology, biochemistry, neuronal culture and image analysis. The authors have addressed the remaining concerns in their revision. Taken together, their results expand the synaptic role of endophilin-A1 to include the inhibitory post synaptic element.

Reviewer #3 (Public review):

This study provides insights into the growth kinetics of a diverse collection of Streptococcus pneumoniae, identifying capsule and lineage differences. It was not able to identify any specific loci from the GWAS that were associated with the growth features. It does provide a useful study linking phenotypic data with large scale genomic population data.

In the revised version, the authors have addressed the points raised by the reviewers. The authors have provided additional detail in the Introduction and Methods that both improves the general accessibility for the broad readership of eLife, and the ability of other researchers to reproduce the approaches used in this study. They have expanded the Results and Discussion text in some sections to provide greater clarity and accuracy in reporting their data.

The inclusion of a Data Availability statement was a useful addition and will help ensure the manuscript adheres to eLife's publishing policies.

Reviewer #3 (Public review):

Summary:

The authors aimed to develop Channelrhodopsins (ChRs), light-gated ion channels, with high potency and blue action spectra for use in multicolor (multiplex) optogenetics applications. To achieve this, they performed a bioinformatics analysis to identify ChR homologues in several protist species, focusing on ChRs from ancyromonads, which exhibited the highest photocurrents and the most blue-shifted action spectra among the tested candidates. Within the ancyromonad clade, the authors identified two new anion-conducting ChRs and one cation-conducting ChR. These were characterized in detail using a combination of manual and automated patch-clamp electrophysiology, absorption spectroscopy, and flash photolysis. The authors also explored sequence features that may explain the blue-shifted action spectra and differences in ion selectivity among closely related ChRs.

Strengths:

A key strength of this study is the high-quality experimental data, which were obtained using well-established techniques such as manual patch-clamp and absorption spectroscopy, complemented by modern automated patch-clamp approaches. These data convincingly support most of the claims. The newly characterized ChRs expand the optogenetics toolkit and will be of significant interest to researchers working with microbial rhodopsins, those developing new optogenetic tools, as well as neuro- and cardioscientists employing optogenetic methods.

Weaknesses:

This study does not exhibit major methodological weaknesses.

Reviewer #3 (Public review):

Summary:

This manuscript from Mudgett et al. explores the relative roles of PID and NPY1 in auxin-dependent floral initiation in Arabidopsis. Micro vectorial auxin flows directed by PIN1 are essential to flower initiation, and loss of PIN1 or two of its regulators, PID and NPY1 (in a yucca-deficient background) phenocopies the pinformed phenotype. This group has previously shown that PID-PIN1 interactions and function are dosage-dependent. The authors pick up this thread by demonstrating that a heterozygote containing a CRISPR deletion of one copy of PIN1 can restore quasi-wild type floral initiation to pid.

The authors then show that overexpression of NPY1 is sufficient to more or less restore wild-type floral initiation to the pid mutant. The authors claim that this result demonstrates that NPY1 functions downstream of PID, as this ectopic abundance of NPY1 resulted in phosphorylation of PIN1 at sites that differ from sites of action of PID. The authors pursue evidence that PID action via NPY1 is analogous to the mode of action by which phot1/2 act on NPH3 in seedling phototropism. Such a model is supported by the evidence presented herein that the C terminus of NPY1, which has abundant Ser/Thr content, is phosphorylated, and that the deletion of this domain prevents overexpression compensation of the pinformed phenotype.<br /> While the results presented support evidence in the literature that PID acts on NPY1 to regulate PIN1 function, it is also possible that NPY1 overexpression results in limited expansion of phosphorylation targets observed with other AGC kinases. And if the phot model is any indication, there may be other PID targets that modulate PIN1-dependent floral initiation.

However, overexpression of the NPY1 C-terminal deletion construct resulted in phosphorylation of both PIN1 and PIN2 and agravitropic root growth similar to what is observed in pin2 mutants. This suggests that direct PID phosphorylation of PINs and action via NPY1 can be distinguished by phosphorylation sites and by growth phenotypes.

Strengths:

A very important effort that places NPY1 downstream of PID in floral initiation.

Weaknesses:

As PID has been shown to act on sites that regulate PIN protein polarity as well as PIN protein function, it would be useful if the authors consider how their results would fit/not fit with a model where combinatorial function of NPY1 and PID regulate PIN1 in a manner similar to the way that PID appears to function combinatorially with D6PK on PIN3.

Reviewer #3 (Public review):

Summary:

The study explores a molecular mechanism by which C. elegans detects low-quality food through neuron-digestive crosstalk, offering new insights into food quality control systems. Liu and colleagues demonstrated that NSY-1, expressed in AWC neurons, is a key regulator for sensing Staphylococcus saprophyticus (SS), inducing avoidance behavior and shutting down the digestive system via intestinal BCF-1. They further revealed that INS-23, an insulin peptide, interacts with the DAF-2 receptor in the gut to modulate SS digestion. The study uncovers a food quality control system connecting neural and intestinal responses, enabling C. elegans to adapt to environmental challenges.

Strengths:

The study employs a genetic screening approach to identify nsy-1 as a critical regulator in detecting food quality and initiating adaptive responses in C. elegans. The use of RNA-seq analysis is particularly noteworthy, as it reveals distinct regulatory pathways involved in food sensing (Figure 4) and digestion of Staphylococcus saprophyticus (Figure 5). The strategic application of both positive and negative data mining enhances the depth of analysis. Importantly, the discovery that C. elegans halts digestion in response to harmful food and employs avoidance behavior highlights a physiological adaptation mechanism.

Weaknesses:

Major weaknesses have been addressed.

Reviewer #3 (Public review):

Hawes et al. combined behavioral, optical imaging, and activity manipulation techniques to investigate the role of striatal patch SPNs in locomotion regulation. Using Sepw1-Cre transgenic mice, they found that patch SPNs encode locomotion deceleration in a light-dark box procedure through optical imaging techniques. Moreover, genetic ablation of patch SPNs increased locomotion speed, while chemogenetic activation of these neurons decreased it. The authors concluded that a subtype of patch striatonigral neurons modulates locomotion speed based on external environmental cues.

In the revision, the authors have largely addressed my concerns with additional explanation and discussion, although some of the key experiments to strengthen the authors' claim by identifying the function of specific cell populations remain to be conducted due to technical challenges. Nevertheless, the current results remain valuable and interesting to a wide audience in the field.

Reviewer #3 (Public review):

Summary:

The authors present a novel method to measure passive joint torques - torques due to internal forces other than active muscle contraction - in the fruit fly: genetically inactivating all motor neurons in intact limb acted upon by a gravitational load results in a change in limb configuration; evaluating the moment equilibrium condition about the limb joints then yields a direct estimate of the passive joint torques. Deactivating all motor neurons in an intact standing fly provided two further conclusions: First, because deactivation causes the fly to drop to the floor, the passive joint torques are deemed insufficient to maintain rotational equilibrium against the body weight; using a multi-body-dynamics simulation, the authors estimate that the passive torques would need to be about 40-80 times higher to maintain a typical posture without active muscle action. Second, a delay between the motor neuron inactivation and the onset of the "free fall" motivates the authors to invoke a simple exponential decay model, which is then used to derive a time constant for muscle deactivation, in robust agreement with direct electro-physiological recordings.

Strengths:

The experimental design that permits determination of passive joint torques is elegant, effective, novel, and altogether excellent; it permits measurements previously impossible. A careful error analysis is presented, and a spectrum of technically challenging methods, including multi-body dynamics and e-phys, is deployed to further interpret and contextualise the results.

Weaknesses:

(1) Passive torques are measured, but only some short speculative statements, largely based on previous work, are offered on their functional significance; some of these claims are not well supported by experimental evidence or theoretical arguments. Passive forces are judged as "large" compared to the weight force of the limb, but the arguably more relevant force is the force limb muscles can generate, which, even in equilibrium conditions, is already about two orders of magnitude larger. The conclusion that passive forces are dynamically irrelevant seems natural, but contrasts with the assertion that "passive forces [...] will have a strong influence on limb kinematics". As a result, the functional significance of passive joint torques in the fruit fly, if any, remains unclear, and this ambiguity represents a missed opportunity. We now know the magnitude of passive joint torques - do they matter and for what? Are they helpful, for example, to maintain robust neuronal control, or a mechanical constraint that negatively impacts performance, e.g., because they present a sink for muscle work?

(2) The work is framed with a scaling argument, but the assumptions that underpin the associated claims are not explicit and can thus not be evaluated. This is problematic because at least some arguments appear to contradict textbook scaling theory or everyday experience. For example, active forces are assumed to scale with limb volume, when every textbook would have them scale with area instead; and the asserted scaling of passive forces involves some hidden assumptions that demand more explicit discussion to alert the reader to associated limitations. Passive forces are said to be important only in small animals, but a quick self-experiment confirms that they are sufficient to stabilize human fingers or ankles against gravity, systems orders of magnitude larger than an insect limb, in seeming contradiction with the alleged dominance of scale. Throughout the manuscript, there are such and similar inaccuracies or ambiguities in the mechanical framing and interpretation, making it hard to fairly evaluate some claims, and rendering others likely incorrect.

Reviewer #3 (Public review):

Summary:

In this manuscript Pinon et al. describe the development of a 3D model of human vasculature within a microchip to study Neisseria meningitidis (Nm)- host interactions and validate it through its comparison to the current gold-standard model consisting of human skin engrafted onto a mouse. There is a pressing need for robust biomimetic models with which to study Nm-host interactions because Nm is a human-specific pathogen for which research has been primarily limited to simple 2D human cell culture assays. Their investigation relies primarily on data derived from microscopy and its quantitative analysis, which support the authors' goal of validating their Vessel-on-Chip (VOC) as a useful tool for studying vascular infections by Nm, and by extension, other pathogens associated with blood vessels.

Strengths:<br /> • Introduces a novel human in vitro system that promotes control of experimental variables and permits greater quantitative analysis than previous models<br /> • The VOC model is validated by direct comparison to the state-of-the-art human skin graft on mouse model<br /> • The authors make significant efforts to quantify, model, and statistically analyze their data<br /> • The laser ablation approach permits defining custom vascular architecture<br /> • The VOC model permits the addition and/or alteration of cell types and microbes added to the model<br /> • The VOC model permits the establishment of an endothelium developed by shear stress and active infusion of reagents into the system

Weaknesses:<br /> • The work presented here is mostly descriptive, with little new information that is learned about the biology of Nm or endothelial cells. However, the goal of this study was to establish the VOC model, and the validation presented here is necessary for follow-on studies on Nm pathogenesis and host response.<br /> • The VOC model contains one cell type, human umbilical cord vascular endothelial cells (HUVECs), while true vasculature contains a number of other cell types that associate with and affect the endothelium, such as smooth muscle cells, pericytes, and components of the immune system. These and other shortcomings of the VOC model as it currently stands warrant additional discussion.

Impact:<br /> The VOC model presented by Pinon et al. is an exciting advancement in the set of tools available to study human pathogens interacting with the vasculature. This manuscript focuses on validating the model, and as such sets the foundation for impactful research in the future. Of particular value is the photoablation technique that permits the custom design of vascular architecture without the use of artificial scaffolding structures described in previously published works.

Reviewer #3 (Public review):

Summary:

This paper investigates changes in brain oscillations in V1 in response to experimentally manipulating visual stimulus features (size, contrast at optimal size) and examines whether these effects are of perceptual relevance. The results reveal prominent stimulus-related theta oscillations in V1 that match in frequency the rhythms of behavioural performance (response speed in detecting targets in the visual display). Phase analyses relate these fluctuations of detection performance more formally to opposite theta phase angles in V1.

Strengths:

The non-human primate model provides unique findings on how brain oscillations relate to rhythms in perception (in two rhesus monkeys) that align well with findings from human studies (as occurring in the theta band). However, theta rhythms in humans are typically associated with fronto-parietal activity in the domain of spatial orienting, attentional sampling, while here the focus is on V1. Importantly, microsaccade-controls seem to speak against a spatial orienting/ attentional sampling mechanism to explain the observed effects (at least regarding overt attention).

Weaknesses:

This study provides interesting clues on perceptually relevant brain oscillations. Despite the microsaccade-control, I believe it remains an open question whether the V1 rhythmicity is of pure V1 origin, or driven by top-down input, as it is conceivable that specific stimuli capture attention differently (and hence induce specific covert attentional (re)orienting patterns). For perceptually relevant (yet beta) rhythmicity over occipital areas that are top-down generated, see e.g., Veniero et al., 2019.

Reviewer #3 (Public review):

Summary:

This paper demonstrates that membrane depolarization induces a small increase in cell entry into mitosis. Based on previous work from another lab, the authors propose that ERK activation might be involved. They show convincingly using a combination of assays that ERK is activated by membrane depolarization. They show this is Ca2+ independent and is a result of activation of the whole K-Ras/ERK cascade which results from changed dynamics of phosphatidylserine in the plasma membrane that activates K-Ras. Although the activation of the Ras/ERK pathway by membrane depolarization is not new, linking it to an increase in cell proliferation is novel.

Strengths

A major strength of the study is the use of different techniques - live imaging with ERK reporters, as well as Western blotting to demonstrate ERK activation as well as different methods for inducing membrane depolarization. They also use a number of different cell lines. Via Western blotting the authors are also able to show that the whole MAPK cascade is activated.

Weaknesses

A weakness of the study is the data in Figure 1 showing that membrane depolarization results in an increase of cells entering mitosis. There are very few cells entering mitosis in their sample in any condition. This should be done with many more cells to increase the confidence in the results. The study also lacks a mechanistic link between ERK activation by membrane depolarization and increased cell proliferation.

The authors did achieve their aims with the caveat that the cell proliferation results could be strengthened. The results, for the most par,t support the conclusions.

This work suggests that alterations in membrane potential may have more physiological functions than action potential in the neural system as it has an effect on intracellular signalling and potentially cell proliferation.

In the revised manuscript, the authors have now addressed the issues with Figure 1, and the data presented are much clearer. They did also attempt to pinpoint when in the cell cycle ERK is having its activity, but unfortunately, this was not conclusive.

Reviewer #3 (Public review):

Summary:

The tissue regeneration enhancer elements (TREEs) identified in zebrafish have been shown to drive injury-activated temporal-spatial gene expression in mice and large animals. These findings increase the translational potential of findings in zebrafish to mammals. In this manuscript, the authors tested TREEs in combination with different adeno-associated viral (AAV) vectors using in vivo luciferase bioluminescent imaging that allows for longitudinal tracking. The TREE-driven luciferase delivered by a liver de-targeted AAV.cc84 decreased off-target transduction in liver. They further screened an AAV library to identify capsid variants that display enhanced transduction for infarcted myocardium post ischemia reperfusion and myocardial infarction. A new capsid variant, AAV.IR41, was found to show increased transduction post I/R and MI.

Strengths:

The authors injected AAV-cargo several days after ischemia/reperfusion (I/R) injury as a clinically relevant approach. Overall, this study is significant in that it identifies new AAV vectors that can be used to deliver promising genes as potential new gene therapies in the future. The manuscript is well-written and the data are also of high quality.

Weaknesses:

The authors have addressed my previous concerns.

Reviewer #3 (Public review):

Summary:

The authors seek to determine the underlying traits that support the exceptional capacity of Aspergillus oryzae to secrete enzymes and heterologous proteins. To do so, they leverage the availability of multiple domesticated isolates of A. oryzae along with other Aspergillus species to perform comparative imaging and genomic analysis.

Strengths:

The strength of this study lies in the use of multifaceted approaches to identify significant differences in hyphal morphology that correlate with enzyme secretion, which is then followed by the use of genomics to identify candidate functions that underlie these differences.

Weaknesses:

Although the image analysis and data interpretation is convincing, the genetic data supporting the author's model is somewhat more speculative and will likely require additional investigation.

Overall, the authors have achieved their aims in that they are able to clearly document the presence of two distinct hyphal forms in A. oryzae and other Aspergillus species, and to correlate the presence of the thicker rapidly growing form with enhanced enzyme secretion. The image analysis is convincing. The discovery that addition of yeast extract and specific amino acids can stimulate formation of the novel hyphal form is also notable. Although the conclusions are generally supported by the results, this is perhaps less so for the genetic analysis as it remains unclear how direct the role of RseA and the calcium transporters might be in supporting the formation of the thicker hyphae.

The results presented here will impact the field. The complexity of hyphal morphology and how it affects secretion are not well understood despite the importance of these processes for the fungal lifestyle. In addition, the description of approaches that can be used to facilitate the study of these different hyphal forms (i.e., stimulation using yeast extract or specific animo acids) will benefit future efforts to understand the molecular basis of their formation.

Reviewer #3 (Public review):

The manuscript is focused on local bulbar mechanisms to solve the flexibility-stability dilemma in contrast to long range interactions documented in other systems (hippocampus-cortex). The network performance is assessed in a perceptual learning task: the network is presented with alternating, similar artificial stimuli (defined as enrichment) and the authors assess its ability to discriminate between these stimuli by comparing the mitral cell representations quantified by Fisher discriminant analysis. The authors use enhancement in discriminability between stimuli as function of the degree of specificity of connectivity in the network to quantify the formation of an odor-specific network structure which as such has memory - they quantify memory as the specificity of that connectivity.

The focus on neurogenesis, excitability and synaptic connectivity of abGCs is topical, and the authors systematically built their model, clearly stating their assumptions and setting up the questions and answers. In my opinion, the combination of latent dendritic representations, excitability and apoptosis in an age-dependent manner is interesting and as the authors point out leads to experimentally testable hypotheses.

In the revised manuscript, the authors have systematically addressed my previous concerns. In particular, they now refer to previous work on granule cells-mitral cell interactions more generally, they explain the pros and cons for usage of specificity in connectivity as a proxy for memory capacity, and the biological plausibility of the model.

Reviewer #3 (Public review):

Summary:

This manuscript by Fontana et al. sets out to fill a critical gap in our understanding of how individuality in fear responses corresponds to changes in brain activity. Previous work has shown in myriad species that fear behaviors are highly variable, and these variabilities correlate with sex and strain, with epigenetic modifications, and neural activity in specific regions of the brain, such as the amygdala. However, a whole-brain functional assessment of whether activity in different regions of the brain is associated with fear behavior has been difficult to assess, in part due to the large size and opacity of the brain. The Kenney group overcomes these limitations using the zebrafish, together with powerful behavioral and brain imaging approaches pioneered by their lab. To overcome the technical obstacles of delivering a reproducible unconditioned stimulus in water and quantifying nuanced behavioral responses, the authors developed a three-day conditioning paradigm in which fish were repeatedly exposed to CAS in one tank context and to control water in another. Leveraging automated cluster analysis across over 300 individuals from four inbred strains, they identified four distinct memory-recall phenotypes - non-reactive, evaders, evading freezers, and freezers - demonstrating both the robustness of their assay and the influence of genetic background and sex on fear learning. Finally, whole-brain imaging using the AZBA atlas (Kenney et al. eLife) and cfos mapping coupled with multivariate analysis revealed that although all fish reengaged telencephalic regions during recall, high-freezing phenotypes uniquely recruited cerebellar, preglomerular, and pretectal nuclei, whereas mixed evasion-freezing fish showed preferential activation of preoptic and hypothalamic areas - a finding that lays the groundwork for dissecting the distributed neural substrates of associative fear in zebrafish.

Strengths:

The strengths of the study lie in the use of zeberarish and the innovative behavioral, modeling, and brain imaging tools applied to address this question. The question of how brain-wide activity correlates with variations in fear behavior is fundamental, and arguably, this system is the only system that could be used to address this. The statistics are appropriate, and the study is well reasoned. Overall, I like this manuscript very much and think it adds invaluable information to the field of fear/anxiety.

Weaknesses:

I have a few questions and suggestions.

(1) The three-day contextual fear paradigm, as implemented - one CAS pairing on day 2 followed by a single recall test on day 3 - inevitably conflates acquisition and long-term memory, making it impossible to know whether strains like TU truly recall the association poorly or simply learn it more slowly. For example, given that TU fish extinguish fear faster than AB or TL strains in extended protocols, they may simply require additional or repeated CAS pairings to achieve the same asymptotic performance. To disentangle learning kinetics from recall strength, the assay could be revised to include multiple acquisition trials (e.g., conditioning on two or more consecutive days) with an immediate post-conditioning probe to assess acquisition independent of consolidation, and continuous measurement of freezing and evasive behaviors across each trial to fit learning curves for each strain. Such refinements - even if on a subset of the strains - would reveal whether "non-reactive" phenotypes reflect genuine recall deficits or merely delayed acquisition.

(2) My second major question is with respect to Figure 3 panel B. This is a complex figure, and I can understand the gist of what the authors are attempting to show, but it is difficult to understand as it is. Can this be represented in a way that is clearer and explained a bit more easily?

(3) The brain mapping is by far one of the most interesting aspects of this study, and the methods that the group used are interesting. The brain mapping, however, relies on generating "contrasting" groups (Figure 6A), and I was not clear as to how these two groups were formed. Could the authors elaborate a bit?

Reviewer #3 (Public review):

Summary:

The heat shock response (HSR) is an inducible transcriptional program that has provided paradigmatic insight into how stress cues feed information into the control of gene expression. The recent elucidation that the chaperone Hsp70 controls the DNA binding activity of the central HSR transcription factor Hsf1 by direct binding has spurred the question how such a general chaperone obtains specificity. This study has addressed the next logical question, how J-domain proteins execute this task in budding yeast, the leading cell model for studying the HSR. While an involvement and in part overlapping function of general class A and B J-domain proteins, Ydj1 and Sis1 are indicated by the genetic analysis a highly specific role for the class A Apj1 in displacing Hsf1 from the promoters is found unveiling specificity in the system.

Strengths

The central strong point of the paper is the identification of class A J-domain protein Apj1 as a specific regulator of the attenuation of the HSR by removing Hsf1 from HSEs at the promoters. The genetic evidence and the ChIP data strongly support this claim. This identification of a specific role for a lowly expressed nuclear J-domain protein changes how the wiring of the HSR should be viewed. It also raises important questions regarding the model of chaperone titration, the concept that a chaperone with limiting availability is involved in a thug of war involving competing interactions with misfolded protein substrates and regulatory interactions with Hsf1. Perhaps Apj1 with its low levels and interactions with misfolded and aggregated proteins in the nucleus is the titrated Hsp70 (co)chaperone that determines the extent of the HSR? This would mean that Apj1 is at the nexus of the chaperone titration mechanism. Although Apj1 is not a highly conserved J domain protein among eukaryotes the strength of the study is that is provides a conceptual framework for what may be required for chaperone titration in other eukaryotes: One or more nuclear J-domain proteins with low nuclear levels that has an affinity for Hsf1 and that can become limiting due to interactions with misfolded Hsp70 proteins. The provides a pathway for how these may be identified using for example ChIP-seq.

Weakness

A built-in challenge when studying the mechanism of the HSR is the general role of Hsp70 chaperone system and its J domain proteins. Indeed, a weakness of the study is that it is unclear what of the phenotypic effects have to do with directly recruiting Hsp70 to Hsf1 dependent on a J domain protein and what instead is an indirect effect of protein misfolding caused by the mutation. This interpretation problem is clearly and appropriately dealt with in the manuscript text and in experiments but is of such fundamental nature that it cannot easily be fully ruled out.

Reviewer #3 (Public review):

Summary:

In this paper, it is established that high fever-like 39{degree sign}C temperatures cause parasite-infected red blood cells to become stickier. It is thought that high temperatures might help the spleen to destroy parasite-infected cells, and they become stickier in order to remain trapped in blood vessels, so they stop passing through the spleen.

Strengths:

The strength of this research is that it shows that fever-like temperatures can cause parasite-infected red blood cells to stick to surfaces designed to mimic the walls of small blood vessels. In a natural infection, this would cause parasite-infected red blood cells to stop circulating through the spleen, where the parasites would be destroyed by the immune system. It is thought that fevers could lead to infected red blood cells becoming stiffer and therefore more easily destroyed in the spleen. Parasites respond to fevers by making their red blood cells stickier, so they stop flowing around the body and into the spleen. The experiments here prove that fever temperatures increase the export of Velcro-like sticky proteins onto the surface of the infected red blood cells and are very thorough and convincing.

Weaknesses:

A minor weakness of the paper is that the effects of fever on the stiffness of infected red blood cells were not measured. This can be easily done in the laboratory by measuring how the passage of infected red blood cells through a bed of tiny metal balls is delayed under fever-like temperatures.

Reviewer #3 (Public review):

Summary:

The manuscript by Chen et al examines the structure of the inactive LRRK2 bound to microtubules using cryo-EM tomography. Mutations in this protein have been shown to be linked to Parkinson's Disease. It is already shown that the active-like conformation of LRRK2 binds to the MT lattice, but this investigation shows that full-length LRRk2 can oligomerize on MTs in its autoinhibited state with different helical parameters than were observed with active-like state. The structural studies suggest that the autoinhibited state is less stable on MTs.

Strengths:

The protein of interest is very important biomedically and a novel conformational binding to microtubules in proposed

The authors have addressed my original critique.

Reviewer #3 (Public review):

Summary:

The authors build and analyze recurrent neural network (RNN) models of brain-computer interface (BCI) multi-task learning, developing a valuable theoretical understanding of learning-related neural population phenomena ("memory traces" and "uniform shifts") that have been reported in recent experimental studies of BCI and motor learning. The authors find that both phenomena emerge in their RNN models, and both correlate in some manner to learning-related behavioral phenomena ("savings" and "forgetting"). The authors also reveal that RNN training details, in particular, incorporating a task-indicating contextual input, can impact these population-level signatures of learning in RNN activity and their relation to those behavioral phenomena.

Strengths:

The text is well written, and the figures are clearly composed to convey the core concepts and findings. The RNN studies are elegant in their ability to recapitulate the memory trace and uniform shift phenomena, and further allow evaluations of novel scenarios that were not tested in the original corpus of the modeled animal experiments. The authors assess the sensitivity of their results to multiple approaches to RNN training, including training connectivity within a model of motor cortex, training only an upstream model that provides inputs to the motor cortex model, and providing task-indicating contextual inputs.

Weaknesses:

(1) It is unclear to what extent these RNN models operate in regimes relevant to biological neural networks (e.g., motor cortex), even at the neural-population level of abstraction studied here. Can the authors speak to how sensitive their results are to details that might speak to these operating regimes (e.g., signal-to-noise ratios or dimensionality of the RNN activities)?

(2) The work could be further strengthened by analyses demonstrating a more direct link between the neural population phenomena (memory trace and uniform shift) and the behavioral phenomena (savings, forgetting, etc). While in animal experiments, it can be exceedingly difficult to demonstrate links beyond correlative effects, the promise of a model is the relative tractability of implementing manipulations that might establish something closer to a causal link between phenomena. Is it the case that the memory trace is a task-dependent, mean-preserving rotation of the across-target task-relevant activity space? And that the uniform shift is a translation (non-mean-preserving) of that space? If so, could the authors design regularization schemes that specifically target each of these effects, enabling a more direct test of the functional role the effects play in driving behavioral phenomena?

Minor Comments:

The current study is based on BCI learning of center-out tasks, analogous to the Losey et al. task that initially reported the memory trace phenomena. However, a rather different behavioral task - involving arm movements through curl force fields - was employed by the Sun, O'Shea, et al. study that originally reported the uniform shift phenomena. How should readers interpret the current study's findings related to the uniform shift? To what extent might the behavioral implications of the uniform shift depend on the demands of the task, e.g., the biomechanics, day-to-day experiencing of different curl-field perturbations, etc.?

Reviewer #3 (Public review):

Summary:

In this well-written manuscript, Unitt and colleagues propose a new, hierarchical nomenclature system for the pathogen Neisseria gonorrhoeae. The proposed nomenclature addresses a longstanding problem in N. gonorrhoeae genomics, namely that the highly recombinant population complicates typing schemes based on only a few loci and that previous typing systems, even those based on the core genome, group strains at only one level of genomic divergence without a system for clustering sequence types together. In this work, the authors have revised the core genome MLST scheme for N. gonorrhoeae and devised life identification numbers (LIN) codes to describe the N. gonorrhoeae population structure.

Strengths:

The LIN codes proposed in this manuscript are congruent with previous typing methods for Neisseria gonorrhoeae, like cgMLST groups, Ng-STAR, and NG-MAST. Importantly, they improve upon many of these methods as the LIN codes are also congruent with the phylogeny and represent monophyletic lineages/sublineages.

The LIN code assignment has been implemented in PubMLST, allowing other researchers to assign LIN codes to new assemblies and put genomes of interest in context with global datasets.

Weaknesses:

The authors correctly highlight that cgMLST-based clusters can be fused due to "intermediate isolates" generated through processes like horizontal gene transfer. However, the LIN codes proposed here are also based on single linkage clustering of cgMLST at multiple levels. It is unclear if future recombination or sequencing of previously unsampled diversity within N. gonorrhoeae merges together higher-level clusters, and if so, how this will impact the stability of the nomenclature.

The authors have defined higher resolution thresholds for the LIN code scheme. However, they do not investigate how these levels correspond to previously identified transmission clusters from genomic epidemiology studies. It would be useful for future users of the scheme to know the relevant LIN code thresholds for these investigations.

Reviewer #3 (Public review):

Summary:

The manuscript by Atchou et al. investigates the role of the microtubule cytoskeleton in sporozoites of Plasmodium berghei, including possible functions of microtubule post-translational modifications (tyrosination and polyglutamylation) in the development of sporozoites in the liver. They also assessed the development of sporozoites in the mosquito. Using cell culture models and in vivo infections with parasites that contain tubulin mutants deficient in certain PTMs, they show that may aspects of the life cycle progression are impaired. The main conclusion is that microtubule PTMs play a major role in the differentiation processes of the parasites.

However, there are a number of major and minor points of criticism that relate to the interpretation of some of the data.

Comments:

(1) The first paragraph of "Results" almost suggests that the presence of a subpellicular MT-array in sporozoites is a new discovery. This is not the case, see e.g. the recent publication by Ferreira et al. (Nature Communications, 2023).

(2) Why were HeLa cells and not hepatocytes (as in Figure 3) used for measuring infection rates of the mutants in Figure 5H and 5L? As I understand, HeLa cells are not natural host cells for invading sporozoites. HeLa cells are epithelial cells derived from a cervical tumour. I am not an expert in Plasmodium biology, but is a HeLa infection an accepted surrogate model for liver stage development?

(3) The tubulin staining in Figures 1A and 1B is confusing and doesn't seem to make sense. Whereas in 1A the antibody nicely stains host and parasite tubulin, in 1B, only parasite tubulin is visible. If the same antibody and the same host cells have been used, HeLa cytoplasmic microtubules should be visible in 1B. In fact, they should be the predominant antigen. The same applies to Figure 2, where host microtubules are also not visible.

(4) In Figures 2A and B, the host nuclei appear to have very different sizes in the DMSO controls and in the drug-treated cells. For example, in the 20 µM (-) image (bottom right), the nuclei are much larger than in the DMSO (-) control (top left). If this is the case, expansion microscopy hasn't worked reproducibly, and therefore, quantification of fluorescence is problematic. The scalebar is the same for all panels.

(5) I don't quite follow the argument that spindles and the LSPMB are dynamic structures (e.g., lines 145, 174). That is a trivial statement for the spindle, as it is always dynamic, but beyond that, it has only been shown that the structure is sensitive to oryzalin. That says little about any "natural" dynamic behaviour. Any microtubule structure can be destroyed by a particular physical or chemical treatment, but that doesn't mean all structures are dynamic. It also depends on the definition of "dynamic" in a particular context, for example, the time scale of dynamic behaviour (changes within seconds, minutes, or hours).

(6) I am not sure what part in the story EB1 plays. The data are only shown in the Supplements and don't seem to be of particular relevance. EB1 is a ubiquitous protein associated with microtubule plus ends. The statement (line 192) that it "may play a broader role..." is unsubstantiated and cannot be based merely on the observation that it is expressed in a particular life cycle stage.

(7) Line 196 onwards: The antibody IN105 is better known in the field as polyE. Maybe that should be added in Materials and Methods. Also, the antibody T9028 against tyrosinated tubulin is poorly validated in the literature and rarely used. Usually, researchers in this field use the monoclonal antibody YL1/2. I am not sure why this unusual antibody was chosen in this study. In fact, has its specificity against tyrosinated α-tubulin from Plasmodium berghei ever been shown? The original antigen was human and had the sequence EGEEY. The Plasmodium sequence is YEADY and hence very different. It is stated that the LSPMB is both polyglutamylated and tyrosinated. This is unusual because polyglutamylated microtubules are usually indicative of stable microtubules, whereas tyrosinated microtubules are found on freshly polymerised and dynamic microtubules. However, a co-localisation within the same cell has not been attempted. This is, however, possible since polyE is a rabbit antibody and T9028 is a mouse antibody. I suspect that differences or gradients along the LSPMB would have been noticed. Also, in lines 207/208, it is said that tyrosination disappears after hepatocyte invasion, which is shown in Figure 3. However, in Figure 3A, quite a lot of positive signals for tyrosination are visible in the 54 and 56 hpi panels.

(8) In line 229, it is stated that tyrosination "has previously been associated with stable microtubule in motility". This statement is not correct. In fact, none of the cited references that apparently support this statement show that this is the case. On the contrary, stable microtubules, such as flagellar axonemes, are almost completely detyrosinated. Therefore, tyrosination is a marker for dynamic microtubules, whereas detyrosinated microtubules are indicative of stable microtubules. This is an established fact, and it is odd that the authors claim the opposite.

(9) Line 236 onwards: Concerning the generation of tubulin mutants, I think it is necessary to demonstrate successful replacement of the wild-type allele by the mutant allele. I am sure the authors have done this by amplification and subsequent sequencing of the genomic locus using PCR primers outside the plasmid sequences. I suggest including this information, e.g., by displaying the chromatograph trace in a supplementary figure. Or are the sequences displayed in Figure S3B already derived from sequenced genomic DNA? This is not described in the Legend or in Materials and Methods. The left PCR products obtained for Figure S3 B would be a suitable template for sequencing.

(10) It is also important to be aware of the fact that glutamylation also occurs on β-tubulin. This signal will also be detected by polyE (IN105). Therefore, it is surprising that IN105 immunofluorescence is negative on the C-term Δ cells (Figure S3 D). Is there anything known about confirmed polyglutamylation sites on both α- and β-tubulins in Plasmodium, e.g., by MS? In Toxoplasma, both α- and β-tubulin have been shown to be polyglutamylated.

(11) Figure S3 is very confusing. In the legend, certain intron deletions are mentioned. How does this relate to posttranslational tubulin modifications? The corresponding section in Results (lines 288-292) is also not very helpful in understanding this.

(12) Figure 4E doesn't look like brightfield microscopy but like some sort of fluorescent imaging. In Figure 4C, were the control (NoΔ) cells with an integrated cassette, but no mutations, or non-transgenic cells?

(13) It is difficult to understand why the TyΔ and the CtΔ mutants still show quite a strong signal using the anti-tyrosination antibody. If the mutants have replaced all wild-type alleles, the signal should be completely absent, unless the antibody (see my comment above concerning T9028) cross-reacts with detyrosinated microtubules. Therefore, the quantitation in Figures 5F and 5G is actually indicative of something that shouldn't be like that. The quantitation of 5F is at odds with the microscopy image in 5D. If this image is representative, the anti-Ty staining in TyΔ is as strong as in the control NoΔ.

(14) The statement that the failure of CtΔ mutants to generate viable sporozoites is due to the lack of microtubule PTMs (lines 295-296) is speculative. The lack of the entire C-terminal tail could have a number of consequences, such as impaired microtubule assembly or failure to recruit and bind associated proteins. This is not necessarily linked to PTMs. Also, it has been shown in yeast that for microtubules to form properly and exquisite regulation (proteostasis) of the ratio between α- and β-tubulin is essential (Wethekam and Moore, 2023). I am not sure, but according to Materials and Methods (line 423), the gene cassettes for replacing the wild-type tubulin gene with the mutant versions contain a selectable marker gene for pyrimethamine selection. Are there qPCR data that show that expression levels of mutant α-tubulin are more or less the same as the wild-type levels?

(15) In the Discussion, my impression is that two recent studies, the superb Expansion Microscopy study by Bertiaux et al. (2021) and the cryo-EM study by Ferreira et al. (2023), are not sufficiently recognised (although they are cited elsewhere in the manuscript). The latter study includes a detailed description of the microtubule cytoskeleton in sporozoites. However, the present study clearly expands the knowledge about the structure of the cytoskeleton in liver stage parasites and is one of the few studies addressing the distribution and function of microtubule post-translational modifications in Plasmodium.

(16) I somewhat disagree with the statement of a co-occurrence of polyglutamylated and tyrosinated microtubules. I think the resolution is too low to reach that conclusion. As this is a bold claim, and would be contrary to what is known from other organisms, it would require a more rigorous validation. Given the apparent problems with the anti-Ty antibody (signal in the TyΔ mutant), one should be very cautious with this claim.

(17) In the Discussion (lines 311 and 377), it is again claimed that tyrosinated microtubules are "a well-known marker of stable microtubules". This statement is completely incorrect, and I am surprised by this serious mistake. A few lines later, the authors say that polyglutamylated is "commonly associated with dynamic microtubule behaviour". Again, this is completely incorrect and is the opposite of what is firmly established in the literature. Polyglutamylation and detyrosination are markers of stable microtubules.

(18) In line 339, the authors interpret the residual antibody staining after the introduction of the mutant tubulin as a compensatory mechanism. There is no evidence for this. More likely explanations are firstly the quality of the anti-Ty-antibody used (see comment above), and the fact that also β-tubulin carries C-terminal polyglutamylation sites, which haven't been investigated in this study. PTMs on β-tubulin are not compensatory, but normal PTMs, at least in all other organisms where microtubule PTMs have been investigated.

Reviewer #3 (Public review):

Asthma is a complex disease that includes endogenous epithelial, immune and neural components that respond to environmental stimuli. Small airborne particles with diameters in the range of 2.5 micrometers or less, so-called PM2.5, are thought to contribute to some forms of asthma. These forms of asthma may have neutrophils, eosinophils and macrophages in bronchoalveolar lavage. Here, Wang and colleagues build on a recent model that incorporated PM2.5 which was found to have a neutrophilic component. Wang altered the model to provide an extra kick via the incorporation of ovalbumin. The major strength of this work is that silencing TRPV1-expressing neurons either pharmacologically or genetically, modulated inflammation and the motility of neutrophils. By examining bronchoalveolar lavage fluid, they found not only that levels of a number of cytokines were increased, but also that artemin, a protein that supports neuronal development and function, was elevated, which did not occur in nociceptor- ablated mice. Their data strengthens links between pollutants, immune and neural interactions.

Comments on revisions:

The manuscript has been revised extensively, including the addition of new experiments, such as intravital microscopy. Did the comments from the reviewers, manifest by additional experiments and modifying how some of the data was presented, result in any changes in the hypotheses or the interpretation of such?

Reviewer #3 (Public review):

Genetically encoded calcium indicators (GECIs) are essential tools in neurobiology and physiology. Technological constraints in targeting and kinetics of previous versions of GECIs have limited their application at the subcellular level. Chen et al. present a set of novel tools that overcome many of these limitations. Through systematic testing in the Drosophila NMJ, they demonstrate improved targeting of GCaMP variants to synaptic compartments and report enhanced brightness and temporal fidelity using members of the GCaMP8 series. These advancements are likely to facilitate more precise investigation of synaptic physiology.

This is a comprehensive and detailed manuscript that introduces and validates new GECI tools optimized for the study of neurotransmission and neuronal excitability. These tools are likely to be highly impactful across neuroscience subfields. The authors are commended for publicly sharing their imaging software.

This manuscript could be improved by further testing the GECIs across physiologically relevant ranges of activity, including at high frequency and over long imaging sessions. The authors provide a custom software package (CaFire) for Ca2+ imaging analysis; however, to improve clarity and utility for future users, we recommend providing references to existing Ca2+ imaging tools for context and elaborating on some conceptual and methodological aspects, with more guidance for broader usability. These enhancements would strengthen this already strong manuscript.

Reviewer #3 (Public review):

Summary:

The authors use a combination of a head-fixed grooming paradigm, single-photon mesoscale, and wide-field-of-view two-photon calcium imaging to characterize cortical activity patterns during evoked grooming. Previous work has shown that grooming behavior does not require cortex, but that there are neuronal representations of grooming in motor cortex. The authors extend these findings by showing cortex-wide activation patterns at the meso-scale that relate to distinct grooming elements. This activation is strongest at grooming onset, but declines over the course of extended grooming periods. They also find similar activity patterns during licking/drinking behavior. Two-photon imaging further revealed that individual neurons across the cortex are preferentially activated by grooming. While their activity also declines after grooming onset, they remain active throughout grooming periods. This work extends previous findings by revealing that grooming and other subcortically-generated behaviors may be represented not only in motor cortex, but across dorsal cortex, both on the mesoscale and single neuron levels. These findings may lead to further investigation into the role of cortical activity during subcortically generated behaviors.

Strengths:

(1) Detailed characterization of grooming behavior in a head-fixed paradigm.

(2) Combination of single photon mesoscale and two-photon wide field-of-view imaging to characterize grooming (and licking)-related activity across dorsal cortex on multiple levels

Weaknesses:

(1) The behavior observed in the head-fixed grooming paradigm only partially resembles spontaneous grooming, lacking typical elements of the syntactic chain, while additionally evoking non-typical behaviors, resembling unilateral reaches, making the interpretation of the observations and their relevance to natural behaviors difficult. Furthermore, the nature of the non-typical movements (which may be cortex-dependent while typical grooming is not) is not explored.

(2) Two important findings in relation to the neural representations of individual grooming behaviors remain unclear:

a) The authors state that individual grooming behaviors did not have distinct neuronal representations (except unilateral grooming; Figure 4G) - it remains unclear how this fits with the observation of distinct activation maps during the different grooming behaviors. Should this differential activation not also correspond to distinct activation patterns of 'grooming' neurons across the cortex? Or do they mean that the activity in the 'grooming' neurons is not consistent across grooming instances and therefore no distinct representation can be detected?

b) The authors state that the 'typical' grooming behaviors do not have consistent activation patterns across animals (Figure 3 and supplements). It remains, therefore, unclear what the averaged activation maps really represent. Furthermore, this observation leaves several open questions: Are the activation patterns consistent in individual animals? Do differences across animals emerge due to differences in their behavior? And most importantly, can the actual behavior be decoded from the activation patterns?

(3) Multiple statements/conclusions are not supported by quantification of the data, but only by qualitative assessments, e.g.: lines 433-435: "In general, the maximally activated networks involved in licking and unilateral grooming behaviors 'appeared' to be the most consistent across animals compared to the bilateral grooming movements (Figure 3G)."; 436-437: "Averaged cortical activation maps associated with licking and elliptical behaviors were 'qualitatively similar' between evoked and spontaneous sessions, where the water drop was not applied".; 480-482: "The unique explained variance maps for the licking behavior 'differed' in the drinking context compared to the grooming context (Figure 3-figure supplement 3F)." The lack of quantification leaves the significance of these observations unclear.

(4) It remains unclear what the ongoing activity in 'grooming' neurons represents, since there is no detailed analysis of the relationship between activity and the detailed kinematics of the grooming movements.

The authors show that neuronal representations of grooming and other subcortical behaviors can be found across dorsal cortex and that these representations are at least to some degree specific to distinct behavioral elements. While this study does not reveal functional insights into the role of cortical representations of subcortically-generated behaviors, it is a step towards more in-depth studies. In the future, it will be important to determine whether these representations are efference copies or sensory-driven, or whether they affect the behavior, and if so, under which circumstances.

Reviewer #3 (Public review):

Summary:

This study used transcranial direct current stimulation administered using small 'high definition' electrodes to modulate neural activity within the non-human primate prefrontal cortex during both wakefulness and anaesthesia. Functional magnetic resonance imaging (fMRI) was used to assess neuromodulatory effects of stimulation. The authors report on modification of brain dynamics during and following anodal and cathodal stimulation during wakefulness and following anodal stimulation at two intensities (1 mA, 2 mA) during anaesthesia. This study provides some support that prefrontal direct current stimulation can alter neural activity patterns across wakefulness and sedation in monkeys.

Strengths and Weaknesses:

A key strength of this work is the use of fMRI-based methods to track changes in brain activity with good spatial precision. Another strength is the exploration of stimulation effects across wakefulness and sedation, which has the potential to provide novel information on the impact of electrical stimulation across states of consciousness. The authors should be commended for undertaking this challenging and important work.

The lack of a sham stimulation condition is a limitation of the study, as it somewhat restricts the certainty with which the results can be attributed to the active stimulation as opposed to other external factors such as drowsiness or fatigue as a result of the experimental procedure? Nevertheless, I acknowledge the demanding nature of performing this work in non-human primates and that only runs with high fixation rates were included, which should have helped reduce any fatigue-related effects.

In the anaesthesia condition, the authors investigated the effects of two intensities of stimulation (1 mA and 2 mA). However, it is possible that the initial 1 mA stimulation block might have caused some level of plasticity-related changes in neural activity that could have potentially interfered with the following 2 mA block due to the lack of a sufficient wash-out period. This potentially limits the findings from the 2 mA block as they cannot be interpreted as completely separate to the initial 1 mA stimulation period, given that they were administered consecutively. However, I do acknowledge the author's point that differences between the 1 mA post-stimulation and baseline conditions were not significantly different, which provides some evidence against this possibility.

The different electrode placement for the two anaesthetised monkeys (i.e., Monkey R: F3/O2 montage, Monkey N: F4/O1 montage) is potentially problematic, as it might have resulted in stimulation over different brain regions. Electric field models of brain current flow for the monkeys would really be needed to understand with reasonable certainty, however, I recognise that these models are generally designed for human studies making their integration into non-human primate studies challenging.

Finally, the sample size is obviously small. However, I realise this is often a limitation in non-human primate research, which can be both expensive and labour intensive.

Assessment:

This manuscript presents some novel insights into the effects of transcranial direct current stimulation on functional brain dynamics in awake and anaesthetised monkeys using MRI-based connectivity indices. Overall, the study presents several interesting and potentially impactful findings regarding stimulation-induced changes in brain activity. There are some limitations, such as the small sample size, lack of a sham stimulation control, and lack of electric field models, which, if included, would have, in my view, further helped improve the rigour of the study. Nevertheless, the manuscript presents several important findings, which warrant further analysis in future work.

Reviewer #3 (Public review):

Summary:

The authors sought to determine, at the level of individual presubiculum pyramidal cells, how allocentric spatial information from retrosplenial cortex was integrated with egocentric information from the anterior thalamic nuclei. Employing a dual opsin optogenetic approach with patch clamp electrophysiology, Richevaux and colleagues found that around three quarters of layer 3 pyramidal cells in presubiculum receive monosynaptic input from both brain regions. While some interesting questions remain (e.g. the role of inhibitory interneurons in gating the information flow and through different layers of presubiculum, this paper provides valuable insights into the microcircuitry of this brain region and the role that it may play in spatial navigation.

Strengths:

One of the main strengths of this manuscript was that the dual opsin approach allowed the direct comparison of different inputs within an individual neuron, helping to control for what might otherwise have been an important source of variation. The experiments were well-executed and the data rigorously analysed. The conclusions were appropriate to the experimental questions and were well-supported by the results. These data will help to inform in vivo experiments aimed at understanding the contribution of different brain regions in spatial navigation and could be valuable for computational modelling.

Weaknesses:

Some attempts were made to gain mechanistic insights into how inhibitory neurotransmission may affect processing in presubiuclum (e.g. figure 5) but these experiments were a little underpowered and the analysis carried out could have been more comprehensively undertaken, as was done for other experiments in the manuscript.

Comments on revised version:

The authors have addressed all of my comments and I have nothing further to add. Well done for an interesting and valuable contribution!

Reviewer #3 (Public review):

Summary:

The authors present a new computational method (OPT) for predicting off-target probe binding in the commercial 10X Xenium spatial transcriptomics platform. They identified 28 genes in the 10x xenium human breast cancer gene panel (280 genes) that are not accurately detected at the single-molecule level. They validated the predicted off-target binding using reference data from single-cell RNA-seq and 3'-sequencing-based Visium RNA-seq. This work provides a practical resource and will serve as a valuable reference for future data interpretation.

Strengths:

(1) Provides a toolbox for the community to identify off-target probes.

(2) Validates the predictions using single-cell RNA-seq and sequencing-based Visium RNA-seq datasets.

Weaknesses:

(1) Does not apply the OPT method to the most widely used Xenium gene panels (e.g., pan-Human, pan-Mouse panels with ~5,000 genes each).

(2) Lacks clarity on how the confidence level of off-target predictions is calculated.

Reviewer #3 (Public review):

Kim, Lognon et al. present an important finding on pro-locomotor effects of optogenetic activation of the A13 region, which they identify as a dopamine-containing area of the medial zona incerta that undergoes profound remodeling in terms of afferent and efferent connectivity after administration of 6-OHDA to the MFB. The authors claim to address a model of PD-related gait dysfunction, a contentious problem that can be difficult to treat by dopaminergic medication or DBS in conventional targets. They make use of an impressive array of technologies to gain insight into the role of A13 remodeling in the 6-OHDA model of PD. The evidence provided is solid and the paper is well written, but there are several general issues that reduce the value of the paper in its current form, and a number of specific, more minor ones. Also some suggestions, that may improve the paper compared to its recent form, come to mind.

The most fundamental issue that needs to be addressed is the relation of the structural to the behavioral findings. It would be very interesting to see whether the structural heterogeneity in afferent/effects projections induced by 6-OHDA is related to the degree of symptom severity and motor improvement during A13 stimulation.

The authors provide extensive interrogation of large-scale changes in the organization of the A13 region afferent and efferent distributions. It remains unclear how many animals were included to produce Fig 4 and 5. Fig S5 suggests that only 3 animals were used, is that correct? Please provide details about the heterogeneity between animals. Please provide a table detailing how many animals were used for which experiment. Were the same animals used for several experiments?

While the authors provide evidence that photoactivation of the A13 is sufficient in driving locomotion in the OFT, this pro-locomotor effect seems to be independent of 6-OHDA induced pathophysiology. Only in the pole test do they find that there seems to be a difference between Sham vs 6-OHDA concerning effects of photoactivation of the A13. Because of these behavioral findings, optogenic activation of A13 may represent a gain of function rather than disease-specific rescue. This needs to be highlighted more explicitly in the title, abstract and conclusion.

The authors claim that A13 may be a possible target for DBS to treat gait dysfunction. However, the experimental evidence provided (imparticular lack of disease-specific changes in the OFT) seem insufficient to draw such conclusions. It needs to be highlighted that optogenetic activation does not necessarily have the same effects as DBS (see the recent review from Neumann et al. in Brain: https://pubmed.ncbi.nlm.nih.gov/37450573/). This is important because ZI-DBS so far had very mixed clinical effects. The authors should provide plausible reasons for these discrepancies. Is cell-specificity, that only optogenetic interventions can achieve, necessary? Can new forms of cyclic burst DBS achieve similar specificity (Spix et al, Science 2021)? Please comment.

In a recent study, Jeon et al (Topographic connectivity and cellular profiling reveal detailed input pathways and functionally distinct cell types in the subthalamic nucleus, 2022, Cell Reports) provided evidence on the topographically graded organization of STN afferents and McElvain et al. (Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon, 2021, Neuron) have shown similar topographical resolution for SNr efferents. Can a similar topographical organization of efferents and afferents be derived for the A13/ ZI in total?

In conclusion, this is an interesting study that can be improved taking into consideration the points mentioned above.

Reviewer #3 (Public review):

Summary:

The authors aim to identify the peripheral end-organ origin in the fly's wing of all sensory neurons in the anterior dorsomedial nerve. They reconstruct the neurons and their downstream partners in an electron microscopy volume of a female ventral nerve cord, analyse the resulting connectome, and identify their origin with a review of the literature and imaging of genetic driver lines. While some of the neurons were already known through previous work, the authors expand on the identification and create a near-complete map of the wing mechanosensory neurons at synapse resolution.

Strengths:

The authors elegantly combine electron microscopy, neuron morphology, connectomics, and light microscopy methods to bridge the gap between fly wing sensory neuron anatomy and ventral nerve cord morphology. Further, they use EM ultrastructural observations to make predictions on the signaling modality of some of the sensory neurons and thus their function in flight.

The work is as comprehensive as state-of-the-art methods allow to create a near-complete map of the wing mechanosensory neurons. This work will be of importance to the field of fly connectomics and modelling of fly behavior, as well as a useful resource to the Drosophila research community.

Through this comprehensive mapping of neurons to the connectome, the authors create a lot of hypotheses on neuronal function, partially already confirmed with the literature and partially to be tested in the future. The authors achieved their aim of mapping the periphery of the fly's wing to axonal projections in the ventral nerve cord, beautifully laying out their results to support their mapping.

The authors identify the neurons in a previously published connectome of a male fly ventral nerve cord to enable cross-individual analysis of connections. Further, together with their companion paper, Dhawan et al. 2025, describing the haltere sensory neurons in the same EM dataset, they cover the entire mechanosensory space involved in Drosophila flight.

Weaknesses:

The connectomic data are only available upon request; the inclusion of a connectivity table of the reconstructed neurons would aid analysis reproducibility and cross-dataset comparisons.

Reviewer #3 (Public review):

Summary:

This study found that ADF serotonergic neurons have a significant role in extending lifespan mediated by HIF-1, as well as serotonin receptor SER-7 in the GABAergic RIS interneurons. The author focuses on the sufficiency and necessity of components from the central nervous system and how they contribute to aging upon hypoxia.

Previous work from the lab has identified that the stabilization of HIF-1 in neurons is sufficient to extend lifespan through the serotonin receptor, SER-7, which subsequently activates fmo-2 in the intestine and leads to lifespan extension. Building on this, the author sought to determine which serotonergic neurons are involved and found that serotonin signaling in ADF neurons is required for lifespan extension mediated by HIF-1.

The author next tested which subset of neurons requires Ser-7 expression to rescue hypoxic response. They found that ser-7 expression in multiple neurons is sufficient to induce fmo-2, with the top candidate being the RIS neuron. Ablation of the RIS neuron did not extend lifespan, suggesting that ser-7 expression in the RIS neuron is required for lifespan extension, positioning it as a key component in the longevity signaling pathway.

The author also investigated neurotransmitters and found that GABA and tyramine are important components in this circuit. They showed that the tyramine receptor called tyra-3 is required for vhl-1-mediated longevity. Given that tyra-3 is expressed in oxygen- and carbon dioxide-sensing neurons, the author demonstrated that these sensing neurons work downstream of serotonin signaling. Lastly, the author screened neuropeptide/receptor binding pairs and identified NLP-17 as playing a role in hypoxia-mediated longevity.

Originality and Significance:

This research is significant in that it uncovers components that are sufficient and necessary for lifespan extension via the hypoxic response. It provides comprehensive data supporting longevity induced by HIF-1-mediated hypoxic response, in conjunction with fmo-2, a longevity gene, as demonstrated in previous work from the lab. Moreover, it provides a number of new transgenic worm tools for C. elegans and aging communities.

Data and Methodology:

(1) The experiments were thoroughly conducted, especially the generations of strains using different neuron-type promoters and crossing into mutant strains to demonstrate sufficiency and necessity.

(2) Some figure legends from the text do not match what the data show. (Figure 6E, F, G).

(3) The lifespan graph legends are confusing and could use some revamping for better clarification.

Conclusions:

This study provides insights into how hypoxic response regulates aging in a cell non-autonomous manner, outlining a potential circuit involving neurons, neurotransmitters, and neuropeptides.

Reviewer #3 (Public review):

Summary:

In this work, the authors aim to improve neural encoding models for naturalistic video stimuli by integrating temporally aligned multimodal features derived from a deep learning model (VALOR) to predict fMRI responses during movie viewing.

Strengths:

The major strength of the study lies in its systematic comparison across unimodal and multimodal models using large-scale, high-resolution fMRI datasets. The VALOR model demonstrates improved predictive accuracy and cross-dataset generalization. The model also reveals inherent semantic dimensions of cortical organization and can be used to evaluate the integration timescale of predictive coding.

This study demonstrates the utility of modern multimodal pretrained models for improving brain encoding in naturalistic contexts. While not conceptually novel, the application is technically sound, and the data and modeling pipeline may serve as a valuable benchmark for future studies.

Weaknesses:

The overall framework of using data-driven features derived from pretrained AI models to predict neural response has been well studied and accepted by the field of neuroAI for over a decade. The demonstrated improvements in prediction accuracy, generalization, and semantic mapping are largely attributable to the richer temporal and multimodal representations provided by the VALOR model, not a novel neural modeling framework per se. As such, the work may be viewed as an incremental application of recent advances in multimodal AI to a well-established neural encoding pipeline, rather than a conceptual advance in modeling neural mechanisms.

Several key claims are overstated or lack sufficient justification:

(1) Lines 95-96: The authors claim that "cortical areas share a common space," citing references [22-24]. However, these references primarily support the notion that different modalities or representations can be aligned in a common embedding space from a modeling perspective, rather than providing direct evidence that cortical areas themselves are aligned in a shared neural representational space.

(2) The authors discuss semantic annotation as if it is still a critical component of encoding models. However, recent advances in AI-based encoding methods rely on features derived from large-scale pretrained models (e.g., CLIP, GPT), which automatically capture semantic structure without requiring explicit annotation. While the manuscript does not systematically address this transition, it is important to clarify that the use of such pretrained models is now standard in the field and should not be positioned as an innovation of the present work. Additionally, the citation of Huth et al. (2012, Neuron) to justify the use of WordNet-based annotation omits the important methodological shift in Huth et al. (2016, Nature), which moved away from manual semantic labeling altogether.

Since the 2012 dataset is used primarily to enable comparison in study 3, the emphasis should not be placed on reiterating the disadvantages of semantic annotation, which have already been addressed in prior work. Instead, the manuscript's strength lies in its direct comparison between data-driven feature representations and semantic annotation based on WordNet categories. The authors should place greater emphasis on analyzing and discussing the differences revealed by these two approaches, rather than focusing mainly on the general advantage of automated semantic mapping.

(3) The authors use subject-specific encoding models trained on the HCP dataset to predict group-level mean responses in an independent in-house dataset. While this analysis is framed as testing model generalization, it is important to clarify that it is not assessing traditional out-of-distribution (OOD) generalization, where the same subject is tested on novel stimuli, but rather evaluating which encoding model's feature space contains more stimulus-specific and cross-subject-consistent information that can transfer across datasets.

Within this setup, the finding that VALOR outperforms CLIP, AlexNet, and WordNet is somewhat expected. VALOR encodes rich spatiotemporal information from videos, making it more aligned with movie-based neural responses. CLIP and AlexNet are static image-based models and thus lack temporal context, while WordNet only provides coarse categorical labels with no stimulus-specific detail. Therefore, the results primarily reflect the advantage of temporally-aware features in capturing shared neural dynamics, rather than revealing surprising model generalization. A direct comparison to pure video-based models, such as Video Swin Transformers or other more recent video models, would help strengthen the argument.

Moreover, while WordNet-based encoding models perform reasonably well within-subject in the HCP dataset, their generalization to group-level responses in the Short Fun Movies (SFM) dataset is markedly poorer. This could indicate that these models capture a considerable amount of subject-specific variance, which fails to translate to consistent group-level activity. This observation highlights the importance of distinguishing between encoding models that capture stimulus-driven representations and those that overfit to individual heterogeneities.

Reviewer #3 (Public review):

Summary:

This manuscript presents a series of experiments that further investigate the roles of the BLA and PRH in sensory preconditioning, with a particular focus on understanding their differential involvement in the association of S1 and S2 with shock.

Strengths:

The motivation for the study is clearly articulated, and the experimental designs are thoughtfully constructed. I especially appreciate the inclusion of Table 1, which makes the designs easy to follow. The results are clearly presented, and the statistical analyses are rigorous. My comments below mainly concern areas where the writing could be improved to help readers more easily grasp the logic behind the experiments.

Weaknesses:

(1) Lines 56-58: The two previous findings should be more clearly summarized. Specifically, it's unclear whether the "mediated S2-shock" association occurred during Stage 2 or Stage 3. I assume the authors mean Stage 2, but Stage 2 alone would not yet involve "fear of S2," making this expression a bit confusing.

(2) Line 61: The phrase "Pavlovian fear conditioning" is ambiguous in this context. I assume it refers to S1-shock or S2-shock conditioning. If so, it would be clearer to state this explicitly.

(3) Regarding the distinction between having or not having Stage 1 S2-S1 pairings, is "novel vs. familiar" the most accurate way to frame this? This terminology could be misleading, especially since one might wonder why S2 couldn't just be presented alone on Stage 1 if novelty is the critical factor. Would "outcome relevance" or "predictability" be more appropriate descriptors? If the authors choose to retain the "novel vs. familiar" framing, I suggest providing a clear explanation of this rationale before introducing the predictions around Line 118.

(4) Line 121: This statement should refer to S1, not S2.

(5) Line 124: This one should refer to S2, not S1.

(6) Additionally, the rationale for Experiment 4 is not introduced before the Results section. While it is understandable that Experiment 4 functions as a follow-up to Experiment 3, it would be helpful to briefly explain the reasoning behind its inclusion.

Reviewer #3 (Public review):

Summary:

Krwawicz et al., present evidence that expression of DNMTs in E. coli results in (1) introduction of alkylation damage that is repaired by AlkB; (2) confers hypersensitivity to alkylating agents such as MMS (and exacerbated by loss of AlkB); (3) confers hypersensitivity to oxidative stress (H2O2 exposure); (4) results in a modest increase in ROS in the absence of exogenous H2O2 exposure; and (5) results in the production of oxidation products of 5mC, namely 5hmC and 5fC, leading to cellular toxicity. The findings reported here have interesting implications for the concept that such genotoxic and potentially mutagenic consequences of DNMT expression (resulting in 5mC) could be selectively disadvantageous for certain organisms. The other aspect of this work which is important for understanding the biological endpoints of genotoxic stress is the notion that DNA damage per se somehow induces elevated levels of ROS.

Strengths:

The manuscript is well-written, and the experiments have been carefully executed providing data that support the authors' proposed model presented in Fig. 7 (Discussion, sources of DNA damage due to DNMT expression).

Weaknesses:

(1) The authors have established an informative system relying on expression of DNMTs to gauge the effects of such expression and subsequent induction of 3mC and 5mC on cell survival and sensitivity to an alkylating agent (MMS) and exogenous oxidative stress (H2O2 exposure). The authors state (p4) that Fig. 2 shows that "Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to WT C2523, supporting the conclusion that the expression of DNMTs increased the levels of alkylation damage." This is a confusing statement and requires revision as Fig. 2 does ALL cells shown in Fig. 2 are expressing DNMTs and have been treated with MMS. It is the absence of AlkB and the expression of DNMTs that that causes the MMS sensitivity.

(2) It would be important to know whether the increased sensitivity (toxicity) to DNMT expression and MMS is also accompanied by substantial increases in mutagenicity. The authors should explain in the text why mutation frequencies were not also measured in these experiments.

(3) Materials and Methods. ROS production monitoring. The "Total Reactive Oxygen Species (ROS) Assay Kit" has not been adequately described. Who is the Vendor? What is the nature of the ROS probes employed in this assay? Which specific ROS correspond to "total ROS"?

(4) The demonstration (Fig. 4) that DNMT expression results in elevated ROS and its further synergistic increase when cells are also exposed to H2O2 is the basis for the authors' discussion of DNA damage-induced increases in cellular ROS. S. cerevisiae does not possess DNMTs/5mC, yet exposure to MMS also results in substantial increases in intracellular ROS (Rowe et al, (2008) Free Rad. Biol. Med. 45:1167-1177. PMC2643028). The authors should be aware of previous studies that have linked DNA damage to intracellular increases in ROS in other organisms and should comment on this in the text.

Comments for the revised manuscript:

In this revised manuscript, the authors have satisfactorily addressed the issues raised in the review of the original submission and have significantly improved these studies.

Reviewer #3 (Public review):

This manuscript presents a study combining molecular dynamics simulations and Hedgehog (Hh) pathway assays to investigate cholesterol translocation pathways to Smoothened (SMO), a G protein-coupled receptor central to Hedgehog signal transduction. The authors identify and characterize two putative cholesterol access routes to the transmembrane domain (TMD) of SMO and propose a model whereby cholesterol traverses through the TMD to the cysteine-rich domain (CRD), which is presented as the primary site of SMO activation.

The MD simulations and biochemical experiments are carefully executed and provide useful data. However, the manuscript is significantly weakened by a narrow and selective interpretation of the literature, overstatement of certain conclusions, and a lack of appropriate engagement with alternative models that are well-supported by published data-including data from prior work by several of the coauthors of this manuscript. In its current form, the manuscript gives a biased impression of the field and overemphasizes the role of the CRD in cholesterol-mediated SMO activation. Below, I provide specific points where revisions are needed to ensure a more accurate and comprehensive treatment of the biology.

Major Comments:

(1) Overstatement of the CRD as the Orthosteric Site of SMO Activation

The manuscript repeatedly implies or states that the CRD is the orthosteric site of SMO activation, without adequate acknowledgment of alternative models. To give just a few examples (of many in this manuscript):

a) "PTCH is proposed to modulate the Hh signal by decreasing the ability of membrane cholesterol to access SMO's extracellular cysteine-rich domain (CRD)" (p. 3).

b) "In recent years there has been a vigorous debate on the orthosteric site of SMO" (p. 3).

c) "cholesterol must travel through the SMO TMD to reach the orthosteric site in the CRD" (p. 4).

d) "we observe cholesterol moving along TM6 to the TMD-CRD interface (common pathway, Fig. 1d) to access the orthosteric binding site in the CRD" (p. 6).

While the second quote in this list at least acknowledges a debate, the surrounding text suggests that this debate has been entirely resolved in favor of the CRD model. This is misleading and not reflective of the views of other investigators in the field (see, for example, a recent comprehensive review from Zhang and Beachy, Nature Reviews Molecular and Cell Biology 2023, which makes the point that both the CRD and 7TM sites are critical for cholesterol activation of SMO as well as PTCH-mediated regulation of SMO-cholesterol interactions).

In contrast, a large body of literature supports a dual-site model in which both the CRD and the TMD are bona fide cholesterol-binding sites essential for SMO activation. Examples include:

a) Byrne et al., Nature 2016: point mutation of the CRD cholesterol binding site impairs-but does not abolish-SMO activation by cholesterol (SMO D99A, Y134F, and combination mutants - Fig 3 of the 2016 study).

b) Myers et al., Dev Cell 2013 and PNAS 2017: CRD deletion mutants retain responsiveness to PTCH regulation and cholesterol mimetics (similar Hh responsiveness of a CRD deletion mutant is also observed in Fig 4 Byrne et al, Nature 2016).

c) Deshpande et al., Nature 2019: mutation of residues in the TMD cholesterol binding site blocks SMO activation entirely, strongly implicating the TMD as a required site, in contrast to the partial effects of mutating or deleting the CRD site.

Qi et al., Nature 2019, and Deshpande et al., Nature 2019, both reported cholesterol binding at the TMD site based on high-resolution structural data. Oddly, Deshpande et al., Nature 2019, is not cited in the discussion of TMD binding on p. 3, despite being one of the first papers to describe cholesterol in the TMD site and its necessity for activation (the authors only cite it regarding activation of SMO by synthetic small molecules).

Kinnebrew et al., Sci Adv 2022 report that CRD deletion abolished PTCH regulation, which is seemingly at odds with several studies above (e.g., Byrne et al, Nature 2016; Myers et al, Dev Cell 2013); but this difference may reflect the use of an N-terminal GFP fusion to SMO in the Kinnebrew et al 2022, which could alter SMO activation properties by sterically hindering activation at the TMD site by cholesterol (but not synthetic SMO agonists like SAG); in contrast, the earlier work by Byrne et al is not subject to this caveat because it used an untagged, unmodified form of SMO.

Although overexpression of PTCH1 and SMO (wild-type or mutant) has been noted as a caveat in studies of CRD-independent SMO activation by cholesterol, this reviewer points out that several of the studies listed above include experiments with endogenous PTCH1 and low-level SMO expression, demonstrating that SMO can clearly undergo activation by cholesterol (as well as regulation by PTCH1) in a manner that does not require the CRD.

Recommendation:

The authors should revise the manuscript to provide a more balanced overview of the field and explicitly acknowledge that the CRD is not the sole activation site. Instead, a dual-site model is more consistent with available structural, mutational, and functional data. In addition, the authors should reframe their interpretation of their MD studies to reflect this broader and more accurate view of how cholesterol binds and activates SMO.

(2) Bias in Presentation of Translocation Pathways

The manuscript presents the model of cholesterol translocation through SMO to the CRD as the predominant (if not sole) mechanism of activation. Statements such as: "Cholesterol traverses SMO to ultimately reach the CRD binding site" (p. 6) suggest an exclusivity that is not supported by prior literature in the field. Indeed, the authors' own MD data presented here demonstrate more stable cholesterol binding at the TMD than at the CRD (p 17), and binding of cholesterol to the TMD site is essential for SMO activation. As such, it is appropriate to acknowledge that cholesterol may activate SMO by translocating through the TM5/6 tunnel, then binding to the TMD site, as this is a likely route of SMO activation in addition to the CRD translocation route they highlight in their discussion.

The authors describe two possible translocation pathways (Pathway 1: TM2/3 entry to TMD; Pathway 2: TM5/6 entry and direct CRD transfer), but do not sufficiently acknowledge that their own empirical data support Pathway 2 as more relevant. Indeed, because their experimental data suggest Pathway 2 is more strongly linked to SMO activation, this pathway should be weighted more heavily in the authors' discussion. In addition, Pathway 2 is linked to cholesterol binding to both the TMD and CRD sites (the former because the TMD binding site is at the terminus of the hydrophobic tunnel, the latter via the translocation pathway described in the present manuscript), so it is appropriate that Pathway 2 figure more prominently than Pathway 1 into the authors' discussion.

The authors also claim that "there is no experimental structure with cholesterol in the inner leaflet region of SMO TMD" (p 16). However, a structural study of apo-SMO from the Manglik and Cheng labs (Zhang et al., Nat Comm, 2022) identified a cholesterol molecule docked at the TM5/6 interface and also proposed a "squeezing" mechanism by which cholesterol could enter the TM5/6 pocket from the membrane. The authors do not take this SMO conformation into account in their models, nor do they discuss the possibility that conformational dynamics at the TM5/6 interface could facilitate cholesterol flipping and translocation into the hydrophobic conduit, even though both possibilities have precedent in the 2022 empirical cryoEM structural analysis.

Recommendation:

The authors should avoid oversimplification of the SMO cholesterol activation process, either by tempering these claims or broadening their discussion to better reflect the complexity and multiplicity of cholesterol access and activation routes for SMO, and consider the 2022 apo-SMO cryoEM structure in their analysis of the TM5/6 translocation pathway.

(3) Alternative Possibility: Direct Membrane Access to CRD

The possibility that the CRD extracts cholesterol directly from the membrane outer leaflet is not considered. While the crystal structures place the CRD in a stable pose above the membrane, multiple cryo-EM studies suggest that the CRD is dynamic and adopts a variety of conformations, raising the possibility that the stability of the CRD in the crystal structures is a result of crystal packing and that the CRD may be far more dynamic under more physiological conditions.

Recommendation:

The authors should explicitly acknowledge and evaluate this potential mechanism and, if feasible, assess its plausibility through MD simulations.

(4) Inconsistent Framing of Study Scope and Limitations

The discussion contains some contradictory and misleading language. For example, the authors state that "In this study we only focused on the cholesterol movement from the membrane to CRD binding site." and then several sentences later state that "We outline the entire translocation mechanism from a kinetic and thermodynamic perspective.". These statements are at odds. The former appropriately (albeit briefly) notes the limited scope of the modeling, while the latter overstates the generality of the findings.

In addition, the authors' narrow focus on the CRD site constitutes a major caveat to the entire work. It should be acknowledged much earlier in the manuscript, preferably in the introduction, rather than mentioned as an aside in the penultimate paragraph of the conclusion.

Recommendation:<br /> The authors should clarify the scope of the study and expand the discussion of its limitations. They should explicitly acknowledge that the study models one of several cholesterol access routes and that the findings do not rule out alternative pathways.

Summary:

This study has the potential to make a useful contribution to our understanding of cholesterol translocation and SMO activation. However, in its current form, the manuscript presents an overly narrow and, at times, misleading view of the literature and biological models; as such, it is not nearly as impactful as it could be. I strongly encourage the authors to revise the manuscript to include:

(1) A more balanced discussion of the CRD vs. TMD binding sites.

(2) Acknowledgment of alternative cholesterol access pathways.

(3) More comprehensive citation of prior structural and functional studies.

(4) Clarification of assumptions and scope.

Of note, the above suggestions require little to no additional MD simulations or experimental studies, but would significantly enhance the rigor and impact of the work.

Reviewer #3 (Public review):

Summary:

This study addresses the role of MIRO1 in vascular smooth muscle cell proliferation, proposing a link between MIRO1 loss and altered growth due to disrupted mitochondrial dynamics and function. While the findings are potentially useful for understanding the importance of mitochondrial positioning and function in this specific cell type within health and disease contexts, the evidence presented appears incomplete, with key bioenergetic and mechanistic claims lacking adequate support.

Strengths:

(1) The study focuses on an important regulatory protein, MIRO1, and its role in vascular smooth muscle cell (VSMC) proliferation, a relatively underexplored context.

(2) It explores the link between smooth muscle cell growth, mitochondrial dynamics, and bioenergetics, which is a potentially significant area for both basic and translational biology.

(3) The use of both in vivo and in vitro systems provides a potentially useful experimental framework to interrogate MIRO1 function in this context.

Weaknesses:

(1) The central claim that MIRO1 loss impairs mitochondrial bioenergetics is not convincingly demonstrated, with only modest changes in respiratory parameters and no direct evidence of functional respiratory chain deficiency.

(2) The proposed link between MIRO1 and respiratory supercomplex assembly or function is speculative, lacking mechanistic detail and supported by incomplete or inconsistent biochemical data.

(3) Key mitochondrial assays are either insufficiently controlled or poorly interpreted, undermining the strength of the conclusions regarding oxidative phosphorylation.

(4) The study does not adequately assess mitochondrial content or biogenesis, which could confound interpretations of changes in respiratory activity.

(5) Overall, the evidence for a direct impact of MIRO1 on mitochondrial respiratory function in the experimental setting is weak, and the conclusions overreach the data.

Reviewer #3 (Public review):

Summary:

Metabolons are multisubunit complexes that promote the physical association of sequential enzymes within a metabolic pathway. Such complexes are proposed to increase metabolic flux and efficiency by channeling reaction intermediates between enzymes. The TCA cycle enzymes malate dehydrogenase (MDH1) and citrate synthase (CIT1) have been linked to metabolon formation, yet the conditions under which these enzymes interact, and whether such interactions are dynamic in response to metabolic cues, remain unclear, particularly in the native cellular context. This study uses a nanoBIT protein-protein interaction assay to map the dynamic behavior of the MDH1-CIT1 interaction in response to multiple metabolic stimuli and challenges in yeast. Beyond mapping these interactions in real time, the authors also performed GC-MS metabolomics to map whole-cell metabolite alterations across experimental conditions. Finally, the authors use microscale thermophoresis to determine components that alter the MDH1-CIT1 interaction in vitro. Collectively, the authors synthesize their collected data into a model in which the MDH1-CIT1 metabolon dissociates in conditions of low respiratory flux, and is stimulated during conditions of high respiratory flux. While their data largely support these models, some key exceptions are found that suggest this model is likely oversimplified and will require further work to understand the complexities associated with MDH1-CIT1 interaction dynamics. Nonetheless, the authors put forth an interesting and timely toolkit to begin to understand the interaction kinetics and dynamics of key metabolic enzymes that should serve as a platform to begin disentangling these important yet understudied aspects of metabolic regulation.

Strengths:

(1) The authors address an important question: how do metabolon-associated protein-protein interactions change across altered metabolic conditions?

(2) The development and validation of the MDH1-CIT1 nanoBIT assay provides an important tool to allow the quantification of this protein-protein interaction in vivo. Importantly, the authors demonstrate that the assay allows kinetic and real time assessment of these protein interactions, which reveal interesting and dynamic behavior across conditions.

(3) The use of classic biochemical techniques to confirm that pH and various metabolites can alter the MDH1-CIT1 interaction in vitro is rigorous and supports the model put forth by the authors.

Weaknesses:

(1) Some of the data collected seem to be merely reported rather than synthesized and interpreted for the reader. This is particularly true for data that seem to reflect more complex trends, such as the GC-MS experiments that map metabolites across multiple experiments, or treatments that show somewhat counterintuitive results, such as the antimycin A treatment, which promotes rather than disrupts the MDH1-CIT1 interaction.

(2) Some of the assertions put forth in the manuscript are not substantiated by the data presented, and the authors are at times overly reliant on previous findings from the literature to support their claims. This is particularly notable for claims about "TCA cycle flux"; the authors do not perform flux analysis anywhere in their study and should be cautious when insinuating correlations between their observations and "flux".

(3) The manuscript presentation could be improved. For figures, at times, the axes do not have intuitive labels (example, Figure 1A), data points and details about the number of samples analyzed are missing (bar graphs and box plots), and molecular weight markers are not reported on western blots. The authors refer to the figures out of order in the text, which makes the manuscript challenging to navigate as a reader.

Reviewer #3 (Public review):

Summary:

The authors present a new method for detecting and identifying proline hydroxylation sites within the proteome. This tool utilizes traditional LC-MS technology with optimized parameters, combined with HILIC-based separation techniques. The authors show that they pick up known hydroxy-proline sites and also validate a new site discovered through their pipeline.

Strengths:

The manuscript utilizes state-of-the-art mass spectrometric techniques with optimized collision parameters to ensure proper detection of the immonium ions, which is an advance compared to other similar approaches before. The use of synthetic control peptides on the HILIC separation step clearly demonstrates the ability of the method to reliably distinguish hydroxy-proline from oxidized methionine - containing peptides. Using this method, they identify a site on CDCA2, which they go on to validate in vitro and also study its role in regulation of mitotic progression in an associated manuscript.

Weaknesses:

Despite the authors' claim about the specificity of this method in picking up the intended peptides, there is a good amount of potential false positives that also happen to get picked (owing to the limitations of MS-based readout), and the authors' criteria for downstream filtering of such peptides require further clarification. In the same vein, greater and more diverse cell-based validation approach will be helpful to substantiate the claims regarding enrichment of peptides in the described pathway analyses.

Reviewer #3 (Public review):

Summary:

Type II IRES, such as those from encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus (FMDV), mediate cap-independent translation initiation by using the full complement of eukaryotic initiation factors (eIFs), except the cap-binding protein eIF4E. The molecular details of how IRES type II interacts with the ribosome and initiation factors to promote recruitment have remained unclear. Das and Hussain used cryo-electron microscopy to determine the structure of a translation initiation complex assembled on the EMCV IRES. The structure reveals a direct interaction between the IRES and the 40S ribosomal subunit, offering mechanistic insight into how type II IRES elements recruit the ribosome.

Strengths:

The structure reveals a direct interaction between the IRES and the 40S ribosomal subunit, offering mechanistic insight into how type II IRES elements recruit the ribosome.

Weaknesses:

While this reviewer acknowledges the technical challenges inherent in determining the structure of such a highly flexible complex, the overall resolution remains insufficient to fully support the authors' conclusions, particularly given that cryo-EM is the sole experimental approach presented in the manuscript.

The study is biologically significant; however, the authors should improve the resolution or include complementary biochemical validation.

Reviewer #3 (Public review):

Bru et al. investigated how inorganic phosphate (Pi) is buffered in cells using S. cerevisiae as a model. Pi is stored in cells in the form of polyphosphates in acidocalcisomes. In S. cerevisiae, the vacuole, which is the yeast lysosome, also fulfills the function of Pi storage organelle. Therefore, yeast is an ideal system to study Pi storage and mobilization.

They can recapitulate in their previously established system, using isolated yeast vacuoles, findings from their own and other groups. They integrate the available data and propose a working model of feedback loops to control the level of Pi on the cellular level.

This is a solid study, in which the biological significance of their findings is not entirely clear. The data analysis and statistical significance need to be improved and included, respectively. The manuscript would have benefited from rigorously testing the model, which would also have increased the impact of the study.

Reviewer #3 (Public review):

Summary:

In this paper, Bhandari, Keglovits, et al. explore the representational structure of task encoding in the lateral prefrontal cortex. Through an impressive fMRI data-collection effort, they compare and contrast neural representations across tasks with different high-level stimulus-response structures. They find that the lateral prefrontal cortex shows enhanced encoding of task-relevant information, but that most of these representations do not generalize across conditions (i.e., have low abstraction). This appears to be driven in part by the representation of task conditions being clustered by the higher-order task properties ('global' representations), with poor generalization across these clusters ('local' representations). Overall, this paper provides an interesting account of how task representations are encoded in the PFC.

Strengths:

(1) Impressive dataset, which may provide further opportunities for investigating prefrontal representations.

(2) Clever task design, allowing the authors to confound several features within a complex paradigm.

(3) Best-practice analysis for decoding, similarity analyses, and assessments of representational geometry.

(4) Extensive analyses to quantify the structure of PFC task representations.

Weaknesses:

(1) The paper would benefit from improved presentational clarity: more scaffolding of design and analysis decisions, clearer grounding to understand the high-level interpretations of the analyses (e.g., context, cluster, abstraction), and better visualizations of the key findings.

(2) The paper would benefit from stronger theoretical motivation for the experimental design, as well as a refined discussion on the implications of these findings for theories of cognitive control.

Reviewer #3 (Public review):

Summary:

In this study, the authors have developed a new Ca indicator conjugated to the peptide, which likely recognizes synaptic ribbons and have measured microdomain Ca near synaptic ribbons at retinal bipolar cells. This interesting approach allows one to measure Ca close to transmitter release sites, which may be relevant for synaptic vesicle fusion and replenishment. Though microdomain Ca at the active zone of ribbon synapses has been measured by Hudspeth and Moser, the new study uses the peptide recognizing synaptic ribbons, potentially measuring the Ca concentration relatively proximal to the release sites.

Strengths:

The study is, in principle, technically well done, and the peptide approach is technically interesting, which allows one to image Ca near the particular protein complexes. The approach is potentially applicable to other types of imaging.

Weaknesses:

Peptides may not be entirely specific, and genetic approach tagging particular active zone proteins with fluorescent Ca indicator proteins may well be more specific. The readers should be aware of this, when interpreting the results.

Reviewer #3 (Public review):

Summary:

This paper presents a framework for a multilevel agent-based model of the drosophila larva, using a simplified larval body and locomotor equations coupled to oscillators and sensory input. The model itself is built upon significant existing literature, particularly Wystrach, Lagogiannis, and Webb 2016 and Jürgensen et al. 2024. The aim is to generate an easily configurable, well-documented platform for organism-scale behavioral simulation in specific experiments. The authors demonstrate qualitative similarity between in vivo behavioral experiments to calibrated models.

Strengths:

The goal is excellent - a system to rapidly run computational experiments that align naturally with behavioral experiments would be well-suited to develop intuitions and cut through hypotheses. The authors provide quantitative descriptions that show that the best-fit parameters in their models produce results that agree with several properties of larval locomotion.

The description of model calibration in the appendix is clear and explains several aspects of the model better than the main text.

In addition, the code is well-organized using contemporary Python tooling and the documentation is nicely in progress (although it remains incomplete). However, see notes for difficulties with installation.

Weaknesses:

(1) As presented here the modeling itself is described in an unclear fashion and without a particular scientific question. The majority of the effort appears to be calibrating modest extensions of existing models and applying them to very simple experiments. This could be an effective first part of a paper on the software tool, but the paper needs to point to a scientific question or, if it is a tool paper, a gap in the current state of modeling tools needed to address scientific goals. While the manuscript has a good overview of larval behavioral papers, the discussion of modeling is more of an afterthought. However, the paper is a modeling paper and the contribution is to modeling and particularly with this work's minor adaptions of existing models, it is unclear what the principle contribution is intended to be.

(2) While the models presented do qualitatively agree with experimental data in specific situations, there is no effort to challenge the model assumptions or compare them to alternative models. Simply because the data is consistent in a small number of simple experiments does not mean that the models are correct. Moreover, given the highly empirical nature of the modeling, I wonder what results are largely the model putting out what was put in, particularly with regards to kinematic results like frequency and body length or the effect of learning simply changing the sensory gain constant. It is difficult to imagine how at this level of empirical modeling, it would appear quite difficult to integrate the type of cell-type-specific perturbation or functional observation that is common in larval experiments.

(3) The central framing of a "layered control architecture" does not have a significant impact on the work presented here and the paper would do better with less emphasis on it. Given the limited empirical models, there are only so many parameters where different components can influence one another, and as best as I can tell from the paper there is only chemotaxis and modulation of a chemotactic gain constant that are incorporated so far. However, since these are empirical functions it says little about how the layers are actually controlled by the nervous system - indeed, the larval nervous system appears to have many levels of local and long-range module of circuits at both the sensory and motor layers. It is not clear how this aspect would contribute beyond the well-appreciated concept of a relatively finite set of behavioral primitives in an insect brain, particularly for the fly larva. What would be a contradictory model and how would the authors differentiate between that and the one they currently propose? If focusing only on olfactory learning and chemotaxis, how does the current framing add to the existing understanding?

(4) The paper uses experimental data to calibrate the models, however, the experiments are not described at all in the text.

Reviewer #3 (Public review):

Summary:

This work provides graphical tools for reconstructing the detailed anatomy of a nervous system from a series of sections imaged by electron microscopy. Contact between neuronal processes can direct outgrowth and is necessary for connectivity, thus function. A bioinformatic approach is used to group neurons according to shared features (e.g., contact, synapses) in a hierarchy of "relatedness" that can be interrogated at each step. In this work, Koonze et al analyze vEM data sets for the C. elegans nerve ring (NR), a dense fascicle of processes from181 neurons. In a bioinformatic approach, the clustering algorithm Diffusion Condensation (DC) groups neurons according to similar cell biological features in iterations that remove chunks of differences in feature data with each step ultimately merging all NR neurons in one cluster. DC results are displayed with C-Phate a 3D visualization tool to produce a trajectory that can be interrogated for cell identities and other features at each iterative step. In previous work by these authors, this approach was utilized to identify subgroups of neuronal processes or "strata" in the NR that can be grouped by physical contact and connectivity. Here they expand their analysis to include a series of available vEM data sets across C. elegans larval development. This approach suggests that strata initially established during embryonic development are largely preserved in the adult. Importantly, exceptions involving stage specific-specific reorganization of neuronal placement in specific strata were also detected. A case study featured in the paper demonstrates the utility of this approach for visualizing the integration of newly generated neurons into the existing NR anatomy. Visualization tools used in this work are publicly available at NeuroSCAN.

Strengths:

A web-based app, NeuroSCAN, that individual researchers can use to interrogate the structure and organization of the C. elegans nerve ring across development.

Weaknesses:

minor revisions

Comments on Revisions:

The authors have satisfactorily addressed my critiques.

Reviewer #3 (Public review):

In the present study, the authors describe the development of new tools and imaging strategies to assess the concomitant development of excitatory and inhibitory synapses in dissociated neuron cultures. To this end, they generate fluorescently tagged constructs of excitatory and inhibitory synapse marker proteins using either conventional overexpression or CRISPR-based strategies. They then image these marker proteins over a timespan of 15 hours to assess synaptic dynamics at different developmental timepoints. Based on their data, they conclude that excitatory and inhibitory synapse development occur in concert to maintain a functional balance despite individual synapse turnover.

Overall, this study addresses an interesting question, i.e., the interplay between the development of excitatory and inhibitory synapses, which has important implications, particularly for neurodevelopmental disorders in which the balance of excitation and inhibition is disrupted. The experiments are technically solid and well-executed, and the individual images are highly compelling.

However, a number of aspects remain to be addressed in order for the study to support the claims made by the authors. First, the novelty aspect of the development of the fluorescently tagged synaptic proteins is unclear, since reporters of this nature are in routine use in many labs. Second, the analysis of the acquired images often seems incomplete, with only example images but no quantification shown, or the distinction between spatial and temporal dynamics appearing unclear. Third, given this incomplete analysis, the interpretations of the authors are not always convincingly supported by the data presented. In conclusion, substantial improvements are required to render the main messages of the study clear and compelling.

Reviewer #3 (Public review):

This paper investigates invariance to natural background noise in the auditory cortex of ferrets and humans. The authors first replicate, in ferrets, a finding from human neuroimaging showing that invariance to background noise increases along the cortical hierarchy (i.e. from primary to non-primary auditory cortex). Next, the authors ask whether this pattern of invariance could be explained by differences in tuning to low-level acoustic features across primary and non-primary regions. The authors conclude that this tuning can explain the spatial organization of background invariance in ferrets, but not in humans. The conclusions of the paper are well supported by the data.

The paper is very straightforwardly written, with a generally clear presentation including well-designed and visually appealing figures. Not only does this paper provide an important replication in a non-human animal model commonly used in auditory neuroscience, but also it extends the original findings in three ways. First, the authors reveal a more fine-grained gradient of background invariance by showing that background invariance increases across primary, secondary and tertiary cortical regions. Second, the authors address a potential mechanism that might underlie this pattern of invariance by considering whether differences in tuning to frequency and spectrotemporal modulations across regions could account for the observed pattern of invariance. The spectrotemporal modulation encoding model used here is a well-established approach in auditory neuroscience and seems appropriate for exploring potential mechanisms underlying invariance in auditory cortex, particularly in ferrets. Third, the authors provide a more complete picture of invariance by additionally analyzing foreground invariance, a complementary measure not explored in the original study.

Comments on author revisions:

The authors have thoroughly addressed the concerns raised in my initial review.

Reviewer #3 (Public review):

In this revised manuscript, the authors explore how Mtb can infect hepatocytes and create a favorable niche associated with upregulation of the transcription factor PPARγ which presumably allows the bacteria to scavenge lipids from lipid droplets in host cells and upregulate drug-metabolizing enzymes to protect against its elimination. In response to the review, the authors have performed some additional immunostaining of hepatocytes, added more detail to figure legends, added experiments somewhat showing improved colocalization and staining, clarified several points and paragraphs, and updated the referenced literature and discussion.

The current manuscript provides evidence that human miliary TB patients have infection of hepatocytes with Mtb, with evidence that the bacteria survive at least partially through upregulation of PPARγ, which significantly changes the lipid milieu of the cells. There is also an examination of transcriptomics and lipid metabolism in response to Mtb infection, as well as drug tolerance of Mtb inside hepatocytes. The current manuscript is an improvement over the previous one.

However, although the manuscript is improved, tissue immunophenotyping of the various cells in the liver remains weak and unconvincing. This is truly a missed opportunity and lessens the rigor of the central findings and conclusions. As pointed out by another reviewer, literature has described different fates of Mtb in the liver. Given the tissue available to the authors, carefully dissecting the various cells that the bacteria are in (esp. hepatocytes versus Kupffer cells) is critical. The authors use only 2 generic markers and do not distinguish among cell types within the tissue slices. A review of the literature shows a variety of both human and mouse antibody markers. In fact, a liver atlas based on immunophenotyping has been published. Likewise, the authors comment on liver granulomas, but this is not justified without immunophenotyping.

Reviewer #3 (Public review):

Summary:

This is an exciting, comprehensive paper that demonstrates the role of GATA4 on OA-like changes in chondrocytes. The authors present elegant reverse translational experiments that justify this mechanism and demonstrate the sufficiency of GATA4 in a mouse model of osteoarthritis (DMM), where GATA4 drove cartilage degeneration and pain in a manner that was significantly worse than DMM alone. This could pave the way for new therapies for OA that account for both structural changes and pain.

Strengths:

(1) GATA4 was identified from human chondrocytes.

(2) IHC and sequencing confirmed GATA4 presence.

(3) Activation of SMADs is clearly shown in vitro with GATA4 overexpression.

(4) The role of GATA4 was functionally assessed in vivo using the mouse DMM model, where the authors uncovered that GATA4 worsens OA structure and hyperalgesia in male mice.

(5) It is interesting that GATA4 is largely known to be found in cardiac cells and to have a role in cardiac repair, metabolism, and inflammation, among other things listed by the authors in the discussion (in liver, lung, pancreas). What could this new knowledge of GATA4 mean for OA as a potentially systemically mediated disease, where cardiac disease and metabolic syndrome are often co-morbid?

Weaknesses:

(1) It would be useful to explain why GATA4 was chosen over HIF1a, which was the most differentially expressed.

(2) In Figure 5, it would be useful to demonstrate the non-surgical or naive limbs to help contextualize OARSI scores and knee hyperalgesia changes.

(3) While there appear to be GATA4 small molecule inhibitors in various stages of development that could be used to assess the effects in age-related OA, those experiments are out of scope for the current study.

Comments on revised version:

I do not have further comments. Thank you for addressing the previously mentioned concerns.

Reviewer #3 (Public review):

Summary:

In their article "Range geography and temperature variability explain cross-continental convergence in range and phenology shifts in a model insect taxon" the authors rigorously investigate the spatial and temporal trends in the occurrence of odonate species and their potential drivers. Specifically, they examine whether species shift their geographic ranges poleward or alter their phenology to cope with changing conditions. Leveraging opportunistic observations of European and North American odonates, they find that species showing significant range shifts also exhibited shifts to earlier emergence. Considering a broad range of potential predictors, their results reveal that geographical factors, but not functional traits, are associated with these shifts.

Strengths:

The article addresses an important topic in ecology and conservation that is particularly timely in the face of reports of substantial insects declines in North America and Europe over the past decades. Through data integration the authors leverage the rich natural history record for odonates, broadening the taxonomic scope of analyses of temporal trends in phenology and distribution. The combination of phenological and range shifts in one framework presents an elegant way to reconcile previous findings and informs about the drivers of biodiversity loss.

Weaknesses:

To better understand whether species shifting both their ranges and phenology are more successful, or as stated here are 'clear winners', and hence whether those that do neither are more vulnerable would require integrating population trends alongside the discussed response. The ~10% species that have not shifted their distribution or phenology might have not declined in abundance, if they have rapidly adapted to local changes in climatic conditions (i.e. they might show a plastic response). These species might be the real 'winners', while species that have recently shifted their ranges or phenology may eventually reach hard limits. The authors are discussing this limitation but might want to adapt their wording, given the potential for misinterpretation. The finding that species with more northern ranges showed lesser northward shifts would speak to the fact that some species have already reached such a geographical range limit.

Achievements and impact:

The results support broad differences in the response of odonate species to climate change, and the prediction that range geography and temperature seasonality are more important predictors of these changes than functional traits. Simultaneously addressing range and phenological shifts highlights that most species exhibit coupled responses but also identifies a significant portion of species that do not respond in these ways that are of critical conservation concern. These results are important for improving forecasts of species' responses to climate change and identifying species of particularly conservation concern. Although not exhaustive regarding abundance trends, the study presents an important step towards a general framework for investigating the drivers of multifaceted species responses.

Reviewer #3 (Public review):

Summary:

Canelo et al. used a combination of mathematical modeling and behavioral experiments to ask how flies orient to visual features and stabilize their gaze. In particular, the authors propose three models of visuomotor control, which lead to specific experimental predictions. With the goal of teasing out the suggested models, the authors design three flight experiments: 1) a bar-background experiment, 2) a looming-background experiment, and 3) a bar-background statistics experiment. The authors claim that: experiment 1 data favor the addition-only and graded EC model; experiment 2 data favor the all-or-none EC model; experiment 3 appears to suggest a graded EC model.

While the study is interesting, there are major issues with the conceptual framework. In general, there is a major disconnect between model and animal data. The manuscript lacks a statistical framework to support or refute the proposed models. In the end, it is unclear what are the main conclusions of the manuscript and contributions to the field.

Strengths:

They ask a significant question related to efference copies during volitional movement.

The figures are overall clear and salient.

Weaknesses:

Comparison of model to fly data:<br /> In general, the manuscript suffers from a lack of quantitative comparisons between proposed models and fly data, which compromises the main findings of the work. While Figure 1-Fig. supplement 1 shows a direct comparison between experiment and model predictions, puzzlingly there is no such quantitative comparison in the main manuscript for the faster moving stimuli. Please overlay model predictions and experimental data and provide statistical comparisons throughout. The 3 proposed models are hypotheses, but there is no statistical framework to reject or support the models/hypotheses. Further, there is a disconnect between the new flight experiments and models. In fact, we do not see the model predictions for the set of experimental conditions tested in Figs. 5-7.

Concerns about mechanical model: I have several concerns regarding the biomechanics block in Figure 2:

(1) The inertia coefficient, derived from free flight studies. does not take into account the fact that the center of rotation and center of mass do not align in the magnetic tether (see Bender & Dickinson, 2006 for estimates). This must be corrected using the parallel axis theorem. As the authors compare the model prediction to experimental data in a magnetic tether, it is critical that they revise their analysis.

(2) According to their chosen inertia and damping constants, they would estimate that the I/C time constant is ~1E-3 ms, which is much much smaller than what has been estimated for yaw turns in the magnetic tether (200 ms; Bender & Dickinson, 2006) or free flight saccades (~17 ms; see Cheng et al., 2010; 10.1242/jeb.038778). The bottom line is that the current model underestimates the influence of inertia in turn manoeuvres, i.e. the aerodynamic damping is cranked up too high relative to yaw inertia. This may explain the mismatch between data and model that the authors posit, "What causes the fly to undershoot the movement of the target object in the magnetically tethered assay? One hypothesis is that strong upward magnetic force or a blunt top end of the steel pin significantly dampens the flies' flight turns."

Loom response experiment:<br /> As nicely shown by 10.1242/jeb.02369, visual stimulation of looming stimuli in the magnetic tether evokes saccades. Is it the case as well in Fig. 6? Without showing individual trials, it is not possible to know whether this is the case. If indeed saccades are present, then the authors will have to reframe their results given the physiological evidence for saccade-related cancellation signals and the three proposed models.

Minor comments:

Missing Equation 13 for saccade model in Methods.

For the discussion and results related to flight responses to the mismatch between expected and actual visual feedback, which is germane to the proposed models, the authors should integrate a discussion of a recent paper which directly tested this idea through an augmented reality system: 10.1016/j.cub.2023.11.045. In particular, the authors argue that the optomotor response is not particularly flexible because it may not rely on an internal model, as suggested by recent physiological evidence (Fenk et al.). How do these findings relate to the 3 proposed models within your work?

Reviewer #3 (Public review):

Summary:

In this paper, the authors used fMRI to determine whether peripherally viewed objects could be decoded from the foveal cortex, even when the objects themselves were never viewed foveally. Specifically, they investigated whether pre-saccadic target attributes (shape, semantic category) could be decoded from the foveal cortex. They found that object shape, but not semantic category, could be decoded, providing evidence that foveal feedback relies on low-mid-level information. The authors claim that this provides evidence for a mechanism underlying visual stability and object recognition across saccades.

Strengths:

I think this is another nice demonstration that peripheral information can be decoded from / is processed in the foveal cortex - the methods seem appropriate, and the experiments and analyses are carefully conducted, and the main results seem convincing. The paper itself was very clear and well-written.

Weaknesses:

There are a couple of reasons why I think the main theoretical conclusions drawn from the study might not be supported, and why a more thorough investigation might be needed to draw these conclusions.

(1) The authors used a blocked design, with each object being shown repeatedly in the same block. This meant that the stimulus was entirely predictable on each block, which weakens the authors' claims about this being a predictive mechanism that facilitates object recognition - if the stimulus is 100% predictable, there is no aspect of recognition or discrimination actually being tested. I think to strengthen these claims, an experiment would need to have unpredictable stimuli, and potentially combine behavioural reports with decoding to see whether this mechanism can be linked to facilitating object recognition across saccades.

(2) Given that foveal feedback has been found in previous studies that don't incorporate saccades, how is this a mechanism that might specifically contribute to stability across saccades, rather than just being a general mechanism that aids the processing/discrimination of peripherally-viewed stimuli? I don't think this paper addresses this point, which would seem to be crucial to differentiate the results from those of previous studies.

Reviewer #3 (Public review):

This paper used computational modeling of infants' performance in a reversal learning paradigm to identify two subgroups of infants, one that initially learned a bit faster but then perseverated more and failed to switch after the reversal (yellow cluster), and those who sampled more before the switch but then perseverated less/switched better (magenta cluster - though see below for comments about infants' overall weak performance). The authors describe magenta babies as showing a profile of greater cognitive flexibility, which they note in adults is linked to better outcomes and a lower incidence of psychiatric disorder. Indeed, the yellow cluster scored less well on several scales of the Vineland and showed lower surgency on the IBQ than the magenta cluster. The authors argue that this paper paves the way for the field of "infant computational neuropsychiatry."

In general, I think this is a fun and intriguing paper. That said, I have a number of concerns with how it is currently written.

First, the role of pupil dilation in the models was really unclear -- I've read it through a few times and came away with different impressions each time. I am now pretty sure the models were only based on infants' behavioural responses (e.g., choice for the correct versus incorrect location) rather than differences in pupil size, but pupil size kept popping up throughout, and so I initially thought the clusters were based on that. The authors should clarify this so other readers are not confused. (One thing that might help is avoiding the word "behaviour" on its own, unless it is further specified as looking behaviour or not, as I assume that some would characterize pupil dilation as a behaviour as well.)

If clusters were NOT based on pupil size (e.g., reaction to prediction error), why not? Was this attempted, and did no clusters emerge? Did the yellow and magenta group also differ in reaction to prediction error, or not? It seems like the argument that this work will be the basis of infant computational psychiatry would require that there not simply be a link between behaviour in an infant study and other measurements of their functioning - because many other papers to date have demonstrated such relationships, many longitudinally - but instead with the link to something where the neurobiology of the behaviour being studied is better understood. I assume this is why pupil dilation kept coming up, but again, it didn't actually seem to be part of the modelling unless I missed something. That is, although I think that this is a nice finding, currently I think the novelty of the finding, as well as the suggestion that it will start a whole new field, may be overblown. I certainly think the pupillometry data has promise, as does the LUMO data, which the authors alluded to being in the works. But perhaps the implications should be toned down a bit in this paper, until those data are further along.

My final substantial comment (a few more minimal ones below) is that overall, babies did quite poorly at this task. Even after 9 post-switch trials, the magenta group was still responding at chance, and the yellow group seemed not to switch at all. Infants then all seemed to perform very well again during block 2, which makes it seem like they still had the original contingency in mind. That said, from what I could see, no data was provided about how many babies looked to the original correct first during Block 2. But based on the data, I assume they basically all went back to predicting on the first side, as otherwise their return to high levels of successful trials would not make sense, unless they somehow forgot the entire thing. It would be good to know for sure, and to have that data (specifically, how many babies looked to the original side again at the start of block 2) in the main paper. Given this overall lack of sensitive performance in the paradigm, even despite the cues signaling where the rewarding video would be changing completely (that is, the contingency between cue and outcome did not itself switch, the cues themselves did), it seems odd to discuss things like statistical or even skillful learning alongside these data.

Reviewer #3 (Public review):

Summary:

Marantos et al. showed that for some coliphages, the energetic state of the bacterial host cell has a strong impact on whether phage infection is initiated. The authors drew this conclusion from the observation that there are more free phages remaining in the medium after infection of arsenate-azide-treated cells as compared to after infection of untreated cells. These data were analyzed and reported both as ratios of the treated vs. untreated conditions and using a mass-action kinetic model of phage-cell collision in the infection mixture. The data supported the findings that for four phages infecting Escherichia coli bacteria, namely, phages λ, 𝜙80, m13, and T6, the phages are less likely to initiate infection if the host bacteria are energy-depleted. However, for phage T5, the authors found that their infection propensity is not impacted.

Strengths:

The data presented by the authors clearly supported the principal conclusion of the study ("Viral commitment to infection depends on host metabolism"). The five phages chosen by the authors represent different viral lifestyles and infection mechanisms, highlighting the potential applicability to other Escherichia coli phages. Finally, the authors successfully used a classic mass-action model of phage-cell collision to interpret their data. The simplicity of their experimental assay, combined with the use of this mathematical model, offers other investigators who study phage-bacterial interactions in other contexts a potentially useful toolkit to examine infection in general, and specifically, the dependence of phage infection on the host's metabolic state.

Weaknesses:

(1) The authors isolated and measured the numbers of free phages in the medium after infection of bacteria under different treatments. These measurements were analyzed in two different ways: (1) simply as ratios (corrected/normalized using different controls), and (2) fitted using a simple mathematical model. I have concerns regarding both analyses.

1.1) For the first method, having different time points at which the sample of each phage is collected critically complicates data interpretation. As one incubates the phage-bacteria mixture for a longer time, more infection occurs, and the number of phages collected from the mixture decreases. Therefore, the different incubation time forfeits the goal of "a systematic and quantitative comparison across different phages [...]" (line 81), just as the authors self-criticized. Conceivably, the authors could have used the shortest measurement time for all phages (i.e., 10 minutes, as for phage λ). Alternatively, the authors could have applied a systematic criterion such as half (or any other fraction) of the latent period of each phage, which would still "maximize the incubation period while ensuring that manipulations were completed before the first infection cycle concluded" (lines 126-127). In my view, the seemingly arbitrary measurement time for each phage renders the entire first analysis very challenging to interpret. It also goes against the author's proposition that the protocol was "standardized" (line 92) or "consistent" (line 200). It is not clear what the readers are supposed to take away from this first analysis, or rather, which evidence, finding, or conclusion the manuscript would lose if the authors only presented the modeling-based analysis.

1.2) The second method of analysis sought to remove the dependence of the measurements on time. I completely agree with this goal, and the findings extracted from this analysis significantly contributed to the merits of this manuscript. However, the authors achieved this goal using a single time point for each phage to calculate the infection rate (η). As shown in Figure S3, each of the phage depletion curves is anchored by only one data point (note that the P(t)/P(0) = 1 at t = 0 is assumed, not measured). This goes against the typical way this collision model is used in the literature, where a time series is measured and used to fit the model (e.g., DOI 10.1007/978-1-60327-164-6 18, or more recently, PMID 39700139). This practice in the current manuscript reduced the robustness of the inferred η values. This problem is exacerbated by assumptions used by the authors in formulating this model. For instance, the authors used a constant value for the bacterial concentration, B, because "bacterial growth and lysis were negligible" (lines 135-136). However, considering that the bacteria were cultured at 37oC in a very rich medium (first in YT broth, then in 2% glucose), the measurement times of 20, 30, and 55 minutes are most likely one or a few generations of bacterial growth and division.

Related note: I suggest that one of the panels in Figure S3 should be moved to the main text, since it is critical to the second method of analysis.

(2) The data were able to distinguish phages that successfully infected bacteria and those that remained free in the medium, and the authors appropriately interpreted the data as such throughout the Results section. However, in the Discussion (starting from the very first sentence, line 172), the authors used terms that include "adsorption" and "entry" more interchangeably (for example, see the three sentences in lines 310-313, for "viral entry efficiency is shaped by [...]", then "adsorption kinetics modeling"). I do not see how the authors' data could distinguish between adsorption (the phage particles attaching to the outside of the cell) and entry (the phage DNA being injected into the cell). Conceivably, any phage particles that irreversibly attach to a cell but do not yet inject their genome into the cell would still be removed from the medium and therefore not quantified. Another example: in lines 189-191, the authors interpreted that "[...] when the bacterium is in a low metabolic state, the phage does not bind irreversibly to the host", but how do the authors eliminate the case of no phage binding (i.e., the reversible step) to begin with? Similarly, in lines 283-293, how do the authors delineate whether energy depletion would increase the k_off term or decrease the k_inj term, because either would result in more free phages in the medium as observed in the data? I believe that the writing of the Discussion, as it stands now, is doing a disservice to the conclusions presented in the Results section.

(3) The authors presented an argument that performing infection of all five phages in the same condition is an advantage, allowing for comparison across different phages. While this goal is a completely valid one, it is difficult to reconcile that with the fact that different phages require different optimal conditions for successful infection. For instance, phage T5 famously requires Ca2+ for successful infection into the host bacterium (and later successful replication); see PMID 13174489. However, all infections were performed in TMG, which lacks Ca2+. Perhaps the absence of T5 dependence on the host metabolism is because the infection condition used by the authors was not optimal for T5 to begin with? Similar arguments could be made for other phages.

(4) Whereas the manuscript examined five coliphages, only phage T5 and phage λ were discussed extensively. I believe some discussion points for these two phages need clarification.

4.1) Phage T5: The data obtained by the authors show that the infection rate of phage T5 is not impacted by the metabolic state of the host cell. Considering that the authors used the terms "infection", "adsorption", and "entry" interchangeably to refer to the irreversible commitment of a phage to a host cell (see point 2), this discussion regarding phage T5 lacks one critical literature context: DNA entry of phage T5 is known to occur in two phases (first-step transfer and second-step transfer). Critically, the second step can only occur if phage proteins encoded by the phage DNA transferred in the first step are expressed (see PMID 10577483 and the cited papers therein). In that context, metabolic poisoning of the host bacteria should have impeded T5 infection. The authors should comment on this point.

4.2) Phage λ: The experiment using phage λ in this current study shares many resemblances to that in Brown et al. 2022. That feature alone is not a problem, but at many places in the text, the writing is ambiguous as to whether it is discussing the results in Brown et al. 2022 or in the current manuscript. I am giving three examples below, but this is not exhaustive: (i) Lines 67-69, there is no Brown et al. 2022 reference immediately after "a mutant phage variant (λh) could bypass this dependency [...]" (not just in the previous sentence); (ii) Line 228 should clearly say "Our previous findings suggested that phage λ is capable of [...]", since it concerns Brown et al., 2022, not the current study; and (iii) Lines 245-246, there is no Brown et al., 2022 reference immediately after "we observed that a mutant variant [...] even energy-depleted host" (without a reference, it reads like the authors "observed" that finding in this current manuscript).

Also, regarding phage λ: The discussion between line 230 and line 249 is very interesting, but since it concerns the differences between λ PaPa and Ur-λ, the authors should consider mentioning and discussing a very relevant recent study, PMCID: PMC6312755.

(5) Control experiments, or references to prior studies, are needed to support that the As/Az treatment at this concentration and duration (at least 10 minutes) is sufficient to deplete the metabolic state of the cell. For instance, this can be shown by impeded or null cell growth, arrested motility (using a standard swimming assay), or a fluorescent reporter for the energetic state of the cell.

Reviewer #3 (Public review):

Summary:

This is a strong and important report that presents a framework for understanding cortical contributions to neonatal respiration. Overall, the authors successfully achieved their goal of linking cortical activity to respiratory drive. Despite the correlational nature of this study, it is a crucial step in establishing a foundation for future work to elucidate the interaction between cortical activity and breathing.

Strengths:

(1) The introduction and use of workflows that establish correlational relationships between breathing and brain activity.

(2) The execution of these workflows in human neonates.

Weaknesses:

Interpretations related to causal inference, confounds of sleep and caffeine, and the spatial interpretation of EEG data need to be addressed to ensure that the data appropriately support the conclusions.

Reviewer #3 (Public review):

Summary:

In the present work, Yinyin Lv et al offer evidence for the therapeutic potential of trained immunity in the context of inflammatory bowel disease (IBD). Prior research has demonstrated that innate cells pre-treated (trained) with β-glucan show an enhanced pro-inflammatory response upon a second challenge.

While an increased immune response can be beneficial and protect against bacterial infections, there is also the risk that it will worsen symptoms in various inflammatory disorders. In the present study, the authors show that mice preconditioned with β-glucan have enhanced resistance to Staphylococcus aureus infection, indicating heightened immune responses.

The authors demonstrate that β-glucan training of bone marrow hematopoietic progenitors and peripheral monocytes mitigates the pro-inflammatory effects of colitis, with protection extending to naïve recipients of the trained cells.

Using a dextran sulfate sodium (DSS)-induced model of colitis, β-glucan pre-treatment significantly dampens disease severity. Importantly, the use of Rag1^-/- mice, which lack adaptive immune cells, confirms that the protective effects of β-glucan are mediated by innate immune mechanisms. Further, experiments using Ccr2^-/- mice underline the necessity of monocyte recruitment in mediating this protection, highlighting CCR2 as a key factor in the mobilization of β-glucan-trained monocytes to inflamed tissues. Transcriptomic profiling reveals that β-glucan training upregulates genes associated with pattern recognition, antimicrobial defense, immunomodulation, and interferon signaling pathways, suggesting broad functional reprogramming of the innate immune compartment. In addition, β-glucan training induces a distinct monocyte subpopulation with enhanced activation and phagocytic capacity. These monocytes exhibit an increased ability to infiltrate inflamed colonic tissue and differentiate into macrophages, marked by increased expression of Cx3cr1. Moreover, among these trained monocyte and macrophage subsets, other gene expression signatures are associated with tissue and mucosal repair, suggesting a role in promoting resolution and regeneration following inflammatory insult.

Strengths:

(1) Overall, the authors present a mechanistically insightful investigation that advances our understanding of trained immunity in IBD.

(2) By employing a range of well-characterized murine models, the authors investigate specific mechanisms involved in the effects of β-glucan training.

(3) Furthermore, the study provides functional evidence that the protection conferred by the trained cells persists within the hematopoietic progenitors and can be transferred to naïve recipients. The integration of transcriptomic profiling allows the identification of changes in key genes and molecular pathways underlying the trained immune phenotype.

(4) This is an important study that demonstrates that β-glucan-trained innate cells confer protection against colitis and promote mucosal repair, and these findings underscore the potential of harnessing innate immune memory as a therapeutic approach for chronic inflammatory diseases.

Weaknesses:

However, FPKM is not ideal for between-sample comparisons due to its within-sample normalization approach. Best practices recommend using raw counts (with DESeq2) for more robust statistical inference.

Reviewer #3 (Public review):

Summary:

The paper by Li et al. describes the crystal structure of a complex of Sld3-Cdc45-binding domain (CBD) with Cdc45 and a model of the dimer of an Sld3-binding protein, Sld7, with two Sld3-CBD-Cdc45 for the tethering. In addition, the authors showed the genetic analysis of the amino acid substitution of residues of Sld3 in the interface with Cdc45 and biochemical analysis of the protein interaction between Sld3 and Cdc45 as well as DNA binding activity of Sld3 to the single-strand DNAs of the ARS sequence.

for - research - pregnancy - 3 immune systems

Reviewer #3 (Public review):

Summary:

The manuscript is a comprehensive molecular and cell biological characterisation of the effects of P604 hydroxylation by PHD1 on RepoMan, a regulatory subunit of the PPIgamma complex. The identification and molecular characterisation of the hydroxylation site have been written up and deposited in BioRxiv in a separate manuscript. I reviewed the data and came to the conclusion that the hydroxylation site has been identified and characterised to a very high standard by LC-MS, in cells and in vitro reactions. I conclude that we should have no question about the validity of the PHD1-mediated hydroxylation.

In the context of the presented manuscript, the authors postulate that hydroxylation on P604 by PHD1 leads to the inactivation of the complex, resulting in the retention of pThr3 in H3.

Strengths:

Compelling data, characterisation of how P604 hydroxylation is likely to induce the interaction between RepoMan and a phosphatase complex, resulting in loading of RepoMan on Chromatin. Loss of the regulation of the hydroxylation site by PHD1 results in mitotic defects.

Weaknesses:

Reliance on a Proline-Alanine mutation in RepoMan to mimic an unhydroxylatable protein. The mutation will introduce structural alterations, and inhibition or knockdown of PHD1 would be necessary to strengthen the data on how hydroxylates regulate chromatin loading and interactions with B56/PP2A.

Reviewer #3 (Public review):

The manuscript contains a carefully designed fMRI study, using MVPA patter analysis to investigate which high-level associate cortices contain target-related information to guide visual search. A special focus is hereby on so-called 'target-associated' information, that has previously been shown to help in guiding attention during visual search. For this purpose the author trained their participants and made them learn specific target-associations, in order to then test which brain regions may contain neural representations of those learnt associations. They found that at least some of the associations tested were encoded in prefrontal cortex during the cue and delay period.

The manuscript is very carefully prepared. As far as I can see, the statistical analyses are all sound and the results integrate well with previous findings.

I have no strong objections against the presented results and their interpretation.

The authors have addressed all my previous comments and questions in their revision of the text.

Reviewer #3 (Public review):

Pattern formation is responsible for generating the spatial organization of cells, tissues, and organs during embryogenesis. It operates within a multifactorial system including initial conditions, gene regulatory networks, extracellular signals, mechanical forces, stochastic noise, and environmental inputs. Finally, it ensures the functional anatomy of an organism.

This study focuses on the one central aspect in pattern formation: how spatial heterogeneity arises from an initial condition and evolves into a more complex or distinct spatial pattern (non-trivial pattern formation, as they termed). The authors made efforts to explore and characterize all possible ways to achieve the pattern formation. They do this by discussing how extracellular signals spread, how individual cells respond to those signals, and how those responses, in turn, modulate signal propagation.

Finally, their comprehensive analysis summarizes that there are three classes of interactions between extracellular signals and intracellular responses, corresponding to previously known mechanisms that can generate spatial patterns: difference in morphogen concentrations in space, noise-amplification, and Turing pattern.

Reviewer #3 (Public review):

Summary:

The study adapts CRISPR-based detection toolkit (SHERLOCK assay) using conserved and species-specific targets for the detection of some members of the Trypanosomatidae family of veterinary importance and species-specific assays to differentiate between the six most common animal trypanosomes species responsible for AAT (SHERLOCK4AAT). The assays were able to discriminate between Trypanozoon (T. b. brucei, T. evansi and T. equiperdum), T. congolense (Savanah, Forest Kilifi and Dzanga sangha), T. vivax, T. theileri, T. simiae and T. suis. The design of both broad and species-specific assays was based primarily on sequences of the 18S rRNA, GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and invariant flagellum antigen (IFX) genes for species identification. Most importantly the authors showed varying limit of detection for the different SHERLOCK assays which is somewhat comparable to PCR-derived molecular techniques currently used for detecting animal trypanosomes even though some of these methodologies have used other primers that target genes such as ITS1 and 7SL sRNA.

The data presented in the study are particularly useful and of significant interest for diagnosis of AAT in affected areas.

Strengths:

The assays convincingly allow for the analysis and detection of most trypanosomes in AAT

Weaknesses:

Inability for the assay to distinguish T. b. brucei, T. evansi and T. equiperdum using the 18S rRNA gene as well as the IFX gene not achieving the sensitivity requirements for detection of T. vivax. Both T. brucei brucei and T. vivax are the most predominant infective species in animals (in addition to T. congolense), therefore a reliable assay should be able to convincingly detect these to allow for proper use of diagnostic assay.

Reviewer #4 (Public review):

Summary:

In this paper, Derkaloustian et al. look at the important topic of what affects fine touch perception. The observations that there may be some level of correlation with instabilities are intriguing. They attempted to characterize different materials by counting the frequency (occurrence #, not of vibration) of instabilities at various speeds and forces of a PDMS slab pulled lengthwise over the material. They then had humans perform the same vertical motion to discriminate between these samples. They correlated the % correct in discrimination with differences in frequency of steady sliding over the design space as well as other traditional parameters such as friction coefficient and roughness.

The authors pose an interesting hypothesis and make an interesting observation about the occurrences of instability regimes in different materials while in contact with PDMS, which is interesting for the community to see in publication. It should be noted however that the finger is complex, and there are many factors that may be over simplified, and perhaps even incorrect, with the use of the PDMS finger. There are trends, such as the trend of surfaces that are more similar in PDMS friction coefficient being easier to discriminate than those with more different PDMS friction coefficient, that contradict multiple other papers in the literature (Fehlberg et al., 2024; Smith and Scott, 1996). This may be due to the PDMS finger not being representative of the real finger conditions. A measurement of friction and the instabilities with a human finger, or demonstration that the PDMS finger is producing the same results (friction coefficient, instabilities, etc.) as a human finger, is needed.

Strengths:

The strength of this paper is in its intriguing hypothesis and important observation that instabilities may contribute to what humans are detecting as differences in these apparently similar samples.

Weaknesses:

There is are significant weaknesses in the representativeness of the PDMS finger, the vertical motion, and the speed of sliding to real human exploration. The real finger has multiple layers with different moduli. In fact, the stratum corneum cells, which are the outer layer at the interface and determine the friction, have much higher modulus than PDMS. In addition, the flat contact area can cause shifting of contact points. Both can contribute to making the PDMS finger have much more stick slip than a real finger. In fact, if you look at the regime maps, there is very little space that has steady sliding. This does not represent well human exploration of surfaces. We do not tend to use force and velocity that will cause extensive stick slip (frequent regions of 100% stick slip) and, in fact, the speeds used in the study are on the slow side, which also contributes to more stick slip. At higher speeds and lower forces, all of the materials had steady sliding regions. Further, on these very smooth surfaces, the friction and stiction are more complex and cannot dismiss considerations such as finger material property change with sweat pore occlusion and sweat capillary forces. Also, the vertical motion of both the PDMS finger and the instructed human subjects is not the motion that humans typically use to discriminate between surfaces.

This all leads to the critical question, why is the friction, normal force, and velocity not measured during the measured human exploration using the real human finger? An alternative would be showing that the PDMS finger reproduces the results of the human finger. I have checked the author's previous papers with this setup and did not find one that showed that the PDMS finger produced the same results as a human finger (Carpenter et al., 2018; Dhong et al., 2018; Nolin et al., 2022, 2021). The reviewer is not asking to do a more detailed psychophysical study with a decision-making model. All that is being asked is to use a human finger for the friction coefficient and instability measurements at typical human forces and speeds, or at least doing these measurements with both for one or two samples to show that the PDMS finger produces the same results as a human finger. The authors posed an extremely interesting hypothesis that humans may alter their speed to feel the instability transition regions. This is something that could be measured with a real finger but is not likely to be correlated accurately enough to match regime boundaries determined with such a simplified artificial finger.

References

Carpenter CW, Dhong C, Root NB, Rodriquez D, Abdo EE, Skelil K, Alkhadra MA, Ramírez J, Ramachandran VS, Lipomi DJ. 2018. Human ability to discriminate surface chemistry by touch. Mater Horiz 5:70-77. doi:10.1039/C7MH00800G<br /> Dhong C, Kayser LV, Arroyo R, Shin A, Finn M, Kleinschmidt AT, Lipomi DJ. 2018. Role of fingerprint-inspired relief structures in elastomeric slabs for detecting frictional differences arising from surface monolayers. Soft Matter 14:7483-7491. doi:10.1039/C8SM01233D<br /> Fehlberg M, Monfort E, Saikumar S, Drewing K, Bennewitz R. 2024. Perceptual Constancy in the Speed Dependence of Friction During Active Tactile Exploration. IEEE Transactions on Haptics 17:957-963. doi:10.1109/TOH.2024.3493421<br /> Nolin A, Licht A, Pierson K, Lo C-Y, Kayser LV, Dhong C. 2021. Predicting human touch sensitivity to single atom substitutions in surface monolayers for molecular control in tactile interfaces. Soft Matter 17:5050-5060. doi:10.1039/D1SM00451D<br /> Nolin A, Pierson K, Hlibok R, Lo C-Y, Kayser LV, Dhong C. 2022. Controlling fine touch sensations with polymer tacticity and crystallinity. Soft Matter 18:3928-3940. doi:10.1039/D2SM00264G<br /> Smith AM, Scott SH. 1996. Subjective scaling of smooth surface friction. Journal of Neurophysiology 75:1957-1962. doi:10.1152/jn.1996.75.5.1957

Reviewer #3 (Public review):

Summary:

In this study, authors utilize biophysical modeling to investigate differences in free energies and nucleosomal configuration probability density of CpG islands and nonmethylated regions in the genome. Toward this goal, they develop and apply the cgNA+ coarse-grained model, an extension of their prior molecular modeling framework.

Strengths:

The study utilizes biophysical modeling to gain mechanistic insight into nucleosomal occupancy differences in CpG and nonmethylated regions in the genome.

Weaknesses:

Although the overall study is interesting, the manuscripts need more clarity in places. Moreover, the rationale and conclusion for some of the analyses are not well described.

Reviewer #3 (Public review):

Summary:

In their manuscript, the authors investigate how glutaminolysis (GLS) and mitochondrial pyruvate import (MPC2) jointly shape B cell fate and the humoral immune response. Using inducible knockout systems and metabolic inhibitors, they uncover a "synthetic auxotrophy": When GLS activity/glutaminolysis is lost together with either GLUT1-mediated glucose uptake or MPC2, B cells fail to upregulate mitochondrial respiration, IL 21/STAT3 and IFN/STAT1 signaling is impaired, and the plasma cell output and antigen-specific antibody titers drop significantly. This work thus demonstrates the promotion of plasma cell differentiation and cytokine signaling through parallel activation of two metabolic pathways. The dataset is technically comprehensive and conceptually novel, but some aspects leave the in vivo and translational significance uncertain.

Strengths:

(1) Conceptual novelty: the study goes beyond single-enzyme deletions to reveal conditional metabolic vulnerabilities and fate-deciding mechanisms in B cells.

(2) Mechanistic depth: the study uncovers a novel "metabolic bottleneck" that impairs mitochondrial respiration and elevates ROS, and directly ties these changes to cytokine-receptor signaling. This is both mechanistically compelling and potentially clinically relevant.

(3) Breadth of models and methods: inducible genetics, pharmacology, metabolomics, seahorse assay, ELISpot/ELISA, RNA-seq, two immunization models.

(4) Potential clinical angle: the synergy of CB839 with UK5099 and/or hydroxychloroquine hints at a druggable pathway targeting autoantibody-driven diseases.

Weaknesses:

(1) Physiological relevance of "synthetic auxotrophy"

The manuscript demonstrates that GLS loss is only crippling when glucose influx or mitochondrial pyruvate import is concurrently reduced, which the authors name "synthetic auxotrophy". I think it would help readers to clarify the terminology more and add a concise definition of "synthetic auxotrophy" versus "synthetic lethality" early in the manuscript and justify its relevance for B cells.

While the overall findings, especially the subset specificity and the clinical implications, are generally interesting, the "synthetic auxotrophy" condition feels a little engineered. Therefore, the findings strongly raise the question of the likelihood of such a "double hit" in vivo and whether there are conditions, disease states, or drug regimens that would realistically generate such a "bottleneck". Hence, the authors should document or at least discuss whether GC or inflamed niches naturally show simultaneous downregulation/lack of glutamine and/or pyruvate. The authors should also aim to provide evidence that infections (e.g., influenza), hypoxia, treatments (e.g., rapamycin), or inflammatory diseases like lupus co-limit these pathways.

It would hence also be beneficial to test the CB839 + UK5099/HCQ combinations in a short, proof-of-concept treatment in vivo, e.g., shortly before and after the booster immunization or in an autoimmune model. Likewise, it may also be insightful to discuss potential effects of existing treatments (especially CB839, HCQ) on human memory B cell or PC pools.

(2) Cell survival versus differentiation phenotype

Claims that the phenotypes (e.g., reduced PC numbers) are "independent of death" and are not merely the result of artificial cell stress would benefit from Annexin-V/active-caspase 3 analyses of GC B cells and plasmablasts. Please also show viability curves for inhibitor-treated cells.

(3) Subset specificity of the metabolic phenotype

Could the metabolic differences, mitochondrial ROS, and membrane-potential changes shown for activated pan-B cells (Figure 5) also be demonstrated ex vivo for KO mouse-derived GC B cells and plasma cells? This would also be insightful to investigate following NP-immunization (e.g., NP+ GC B cells 10 days after NP-OVA immunization).

(4) Memory B cell gating strategy

I am not fully convinced that the memory-B-cell gate in Supplementary Figure 2d is appropriate. The legend implies the population is defined simply as CD19+GL7-CD38+ (or CD19+CD38++?), with no further restriction to NP-binding cells. Such a gate could also capture naïve or recently activated B cells. From the descriptions in the figure and the figure legend, it is hard to verify that the events plotted truly represent memory B cells. Please clarify the full gating hierarchy and, ideally, restrict the MBC gate to NP+CD19+GL7-CD38+ B cells (or add additional markers such as CD80 and CD273). Generally, the manuscript would benefit from a more transparent presentation of gating strategies.

(5) Deletion efficiency

mRNA data show residual GLS/MPC2 transcripts (Supplementary Figure 8). Please quantify deletion efficiency in GC B cells and plasmablasts.

Reviewer #3 (Public review):

In this manuscript, Yang et al. characterize the endocytic accessory protein CCDC32, which has implications in cardio-facio-neuro-developmental syndrome (CFNDS). The authors clearly demonstrate that the protein CCDC32 has a role in the early stages of endocytosis, mainly through the interaction with the major endocytic adaptor protein AP2, and they identify regions taking part in this recognition. Through live cell fluorescence imaging and electron microscopy of endocytic pits, the authors characterize the lifetimes of endocytic sites, the formation rate of endocytic sites and pits and the invagination depth, in addition to transferrin receptor (TfnR) uptake experiments. Binding between CCDC32 and CCDC32 mutants to the AP2 alpha appendage domain is assessed by pull down experiments.

Together, these experiments allow deriving a phenotype of CCDC32 knock-down and CCDC32 mutants within endocytosis, which is a very robust system, in which defects are not so easily detected. A mutation of CCDC32, mimicking CFNDS mutations, is also addressed in this study and shown to have endocytic defects.

An experimental proof for the resistance of the different CCDC32 mutants to siRNA treatment would have helped to strengthen the conclusions.

In summary, the authors present a strong combination of techniques, assessing the impact of CCDC32 in clathrin mediated endocytosis and its binding to AP2.

Reviewer #3 (Public review):

The manuscript by Ono et al describes the application of prime editors to introduce precise genetic changes in the zebrafish model system. Probably the most important observation is that, compared to the "standard" PE2, the prime editor with full nuclease activity appears to be more efficient at introducing insertions into the genome. Although many laboratories around the world have successfully used oligonucleotide-mediated HDR to insert short exogenous sequences such as epitope tags or loxP sites into the zebrafish genome, the method suffers from a high frequency of indels at the edit site. Thus, additional tools are badly needed, making this manuscript very important. Length of the longer reported insertion (+30) is quite close to the range of V5 (14 amino acids) and ALFA (12 amino acids without "spacer" prolines) epitope tags, as well as loxP site (34 nucleotides). Conclusions drawn in the paper are supported by compelling evidence. I only have a few minor comments:

(1) The logic for introducing two nucleotide changes (at +3 and +10) to change a single amino acid (I378) should be explicitly explained in the main body of the manuscript. It is indeed self-explanatory when looking at Supplementary Figure 1. One way of doing it could be to include Supplementary Figure 1a in Figure 1.

(2) It is not clear why a 3-nucleotide insertion was used to generate W722X. The human W720X is a single-nucleotide polymorphism, and it should be possible to make a corresponding zebrafish mutant by introducing two nucleotide changes.

(3) Lines 137-138: T7 Endonuclease assay used in Figure 2d detects all polymorphisms, both precise changes and indels. Thus, if this assay were performed on embryos shown in Figure 1c-d, the overall percentage of modified alleles would be similarly higher for PEn over PE2 (add up precise prime edits and indels). The conclusion in the last sentence of the paragraph is, therefore, incorrect, I believe.

(4) Use of terminology. "Germline transmission" is typically used to refer to the fraction of F0s transmitting desired changes (or transgenes) to their progeny, while "germline mosaicism" refers to the fraction of F1s with the desired change in the progeny of a given F0. "Germline transmission" in line 217 should be replaced with "germline mosaicism".

(5) Lines 253-255: The fraction of injected embryos that had mosaic nuclear expression of GFP, indicative of NLS insertion, should be clarified. It should also be clarified whether embryos positive for nuclear GFP were preselected for amplicon sequencing and germline transmission analyses. This is extremely important for extrapolation to scenarios like epitope tagging, where preselection is not possible.

(6) Statistical analyses. It would be helpful to clarify why different statistical tests are sometimes used to assess seemingly very similar datasets (Figures 1c, 1d, 2b, 2c, 2f).

(7) Discussion. Since authors suggest that PEn might be especially beneficial for insertion of additional sequences, it is important to stress locus-to-locus variability of success. While the precise +3 insertion was indeed tremendously efficient at both tested loci (ror2 and adgrf3b), +12 addition into adgrf3b was over 10 times less efficient (lines 193-194). In contrast, +30 into smyhc:GFP using the shorter pegRNA was highly efficient again with an average of 8.5% of sequence reads indicating precise integration (line 257, Figure 5c). Longer pegRNA did not work nearly as well (Figure 5c), but was still much better than +12 into adgrf3b. As dangerous as it is to extrapolate from small datasets, perhaps these observations indicate that optimization of RT template and PBS may be needed for each new locus in order to significantly outperform oligonucleotide-mediated HDR? If so, would the cost of ordering several pegRNAs and the effort needed to compare them factor in when deciding which method to use? Reported germline transmission rates for both ror2 W722X (+3, Figure 4a) and smyhc:NLS-GFP (+30, Figure 5f) are tantalizingly high.

Reviewer #3 (Public review):

Summary:

In the face of emerging antibiotic resistance and slow pace of drug discovery, strategies that can enhance the efficacy of existing clinically used antibiotics are highly sought after. In this manuscript, through genetic manipulation of a model bacterium (Escherichia coli) and clinically isolated and antibiotic resistant strains of concern (Pseudomonas, Burkholderia, Stenotrophomonas), an additional drug target to combat resistance and potentiate existing drugs is put forward. These observations were validated in both pure cultures, mixed bacterial cultures and in worm models. The drug target investigated in this study appears to be broadly relevant to the challenge posed by lactamases enzyme that render lactam antibiotics ineffective in the clinic. The compounds that target this enzyme are being developed already, some of which were tested in this study displaying promising results and potential for further optimization by medicinal chemists.

Strengths:

The work is well designed and well executed and targets an urgent area of research with the unprecedented increase in antibiotic resistance.

Weaknesses:

The impact of the work can be strengthened by demonstrating increased efficacy of antibiotics in mice models or wound models for Pseudomonas infections. Worm models are relevant, but still distant from investigations in animal models.

Reviewer #3 (Public review):

Strengths:

This work focuses on a problem of deep significance: quantifying the structure-tension relationship and underlying mechanism for the mechanosensitive Piezo 1 and 2 channels. Such an objective is challenging for molecular dynamics simulations, due to the relatively large size of each membrane-protein system. Nonetheless, the approach chosen here is based on methodology that is, in principle, established and widely accessible. Therefore, another group of practitioners would likely be able to reproduce these findings with reasonable effort.

More specifically, while acknowledging the limitations of the MARTINI force field, this work makes a significant improvement compared to previous simulations of Piezo proteins by adopting a range of membrane tensions that includes physiologically relevant values (below 10 mN/m).

Weaknesses:

The two main results of this paper are (1) that both channels exhibit a flatter structure compared to cryo-EM measurements, and (2) their estimated force vs. displacement relationship. Although the former correlates at least quantitatively with prior experimental work, the latter relies exclusively on simulation results and model parameters.

My remaining technical concerns in the revised manuscript are as follows:

(1) At each membrane tension, all concurrent atomistic simulations were initialized from the same snapshot of a previous CG simulation: in my opinion, it is inaccurate to refer to those atomistic simulations as "independent" from each other (as is done twice in the caption of Figure 3, as well as in the text).

(2) Continuum mechanics calculations were employed to model the membrane's curvature energetics. The bending modulus, k, was not determined for the specific lipid composition used in this study, but was instead taken from previous MARTINI simulations involving the same primary lipid, POPC. Given that these calculations are intended to describe MARTINI simulations specifically, this approximation may be acceptable. However, it does not account for the increased stiffness observed in POPC/cholesterol mixtures-an effect measured experimentally but not reproduced by the MARTINI model-nor does it reflect the asymmetric conditions, as all referenced simulations involve symmetric bilayers. As a result, the bending energies and forces shown in Figure 5(c,d) are internally consistent within the model, but they probably correspond to real values up to an unknown multiplicative factor.

Reviewer #3 (Public review):

This is an excellent, very interesting paper. There is a groundbreaking analysis of the data, going from typical picture presentation paradigms to more realistic conditions. I would like to ask the authors to consider a few points in the comments below.

(1) From Figure 2, I understand that there are 7 neurons responding to the character Summer, but then in line 157, we learn that there are 46. Are the other 39 from other areas (not parahippocampal)? If this is the case, it would be important to see examples of these responses, as one of the main claims is that it is possible to decode as good or better with non-responsive compared to single responsive neurons, which is, in principle, surprising.

(2) Also in Figure 2, there seem to be relatively very few neurons responding to Summer (1.88%) and to outdoor scenes (1.07%). Is this significant? Isn't it also a bit surprising, particularly for outdoor scenes, considering a previous paper of Mormann showing many outdoor scene responses in this area? It would be nice if the authors could comment on this.

(3) I was also surprised to see that there are many fewer responses to scene cuts (6.7%) compared to camera cuts (51%) because every scene cut involves a camera cut. Could this have been a result of the much larger number of camera cuts? (A way to test this would be to subsample the camera cuts.)

(4) Line 201. The analysis of decoding on a per-patient basis is important, but it should be done on a per-session basis - i.e., considering only simultaneously recorded neurons, without any pooling. This is because pooling can overestimate decoding performances (see e.g. Quian Quiroga and Panzeri NRN 2009). If there was only one session per patient, then this should be called 'per-session' rather than 'per-patient' to make it clear that there was no pooling.

(5) In general, the decoding results are quite interesting, and I was wondering if the authors could give a bit more insight by showing confusion matrices, with the predictions of the appearance of each of the characters, etc. Some of the characters may appear together, so this could be another entry of the decoder (say, predicting person A, B, C, A&B, A&C, B&C, A&B&C). I guess this could also show the power of analyzing the population activity.

(6) Lines 406-407. The claim that stimulus-selective responses to characters did not account for the decoding of the same character is very surprising. If I understood it correctly, the response criterion the authors used gives 'responsiveness' but not 'selectivity'. So, were people's responses selective (e.g., firing only to Summer) or non-selective (firing to a few characters)? This could explain why they didn't get good decoding results with responsive neurons. Again, it would be nice to see confusion matrices with the decoding of the characters. Another reason for this is that what are labelled as responsive neurons have relatively weak and variable responses.

(7) Line 455. The claim that 500 neurons drive decoding performance is very subjective. 500 neurons gives a performance of 0.38, and 50 neurons gives 0.33.

(8) Lines 492-494. I disagree with the claim that "character decoding does not rely on individual cells, as removing neurons that responded strongly to character onset had little impact on performance". I have not seen strong responses to characters in the paper. In particular, the response to Summer in Figure 2 looks very variable and relatively weak. If there are stronger responses to characters, please show them to make a convincing argument. It is fine to argue that you can get information from the population, but in my view, there are no good single-cell responses (perhaps because the actors and the movie were unknown to the subjects) to make this claim. Also, an older paper (Quian Quiroga et al J. Neurophysiol. 2007) showed that the decoding of individual stimuli in a picture presentation paradigm was determined by the responsive neurons and that the non-responsive neurons did not add any information. The results here could be different due to the use of movies instead of picture presentations, but most likely due to the fact that, in the picture presentation paradigm, the pictures were of famous people for which there were strong single neuron responses, unlike with the relatively unknown persons in this paper.

DOI: 10.1038/s44319-025-00511-8

Resource: RRID:ZFIN_ZDB-ALT-090424-3

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-ALT-090424-3

What is this?

DOI: 10.1038/s44319-025-00511-8

Resource: RRID:ZFIN_ZDB-GENO-140806-3

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-140806-3

What is this?

Reviewer #3 (Public review):

Summary:

In this work, Ryan et al. have performed a state-of-the-art full genome CRISP-based screen of iNEurons expressing a teggd version of TDP-43 in order to determine expression modifiers of this protein. Unexpectedly, using this approach the authors have uncovered a previously undescribed role of the BORC complex in affecting the levels of TDP-43 protein, but not mRNA expression. Taken together, these findings represent a very solid piece of work that will certainly be important for the field.

Strengths:

BORC is a novel TDP-43 expression modifier that has never been described before and it seemingly acts on regulating protein half life rather than transcriptome level. It has been long known that different labs have reported different half-lives for TDP-43 depending on the experimental system but no work has ever explained these discrepancies. Now, the work of Ryan et al. has for the time identified one of these factors which could account for these differences and play an important role in disease (although this is left to be determined in future studies).

The genome wide CRISPR screening has demonstrated to yield novel results with high reproducibility and could eventually be used to search for expression modifiers of many other proteins involved in neurodegeneration or other diseases

Reviewer #3 (Public review):

Summary:

The authors propose a new version of idTracker.ai for animal tracking. Specifically, they apply contrastive learning to embed cropped images of animals into a feature space where clusters correspond to individual animal identities.

Strengths:

By doing this, the new software alleviates the requirement for so-called global fragments - segments of the video, in which all entities are visible/detected at the same time - which was necessary in the previous version of the method. In general, the new method reduces the tracking time compared to the previous versions, while also increasing the average accuracy of assigning the identity labels.

Weaknesses:

The general impression of the paper is that, in its current form, it is difficult to disentangle the old from the new method and understand the method in detail. The manuscript would benefit from a major reorganization and rewriting of its parts. There are also certain concerns about the accuracy metric and reducing the computational time.

Reviewer #3 (Public review):

Summary:

The authors investigated the assembly and polar localization of the chemosensory cluster in P. aeruginosa. They discovered that a certain protein (FlhF) is required for the polar localization of the chemosensory cluster while core motor structures are necessary for the assembly of the cluster. They found that flagella and chemosensory clusters always co-localize in the cell; either at the cell pole in wild type cells or randomly-located in the cell in FlhF mutant cells. They hypothesize that this co-localization is required to keep the level of another protein (CheY-P), which controls motor switching, at low levels as the presence of high-levels of this protein (if the flagella and chemosensory clusters were not co-localized) is associated with high-levels of c-di-GMP and cell aggregations.

Strengths:

The manuscript is clearly-written and straightforward. The authors applied multiple techniques to study the bacterial motility system including fluorescence light microscopy and gene editing. In general, the work enhances our understanding of the subtlety of interaction between the chemosensory cluster and the flagellar motor to regulate cell motility. This work will be of interest to bacteriologists and cell biologists in general.

Reviewer #3 (Public review):

Mondal et al. use computational modeling to investigate how activity-dependent shifts in voltage-dependent (in)activation curves can complement changes in ion channel conductance to support homeostatic plasticity. While it is well established that the voltage-dependent properties of ion channels influence neuronal excitability, their potential role in homeostatic regulation, alongside conductance changes, has remained largely unexplored. The results presented here demonstrate that activity-dependent regulation of voltage dependence can interact with conductance plasticity to enable neurons to attain and maintain target activity patterns, in this case, intrinsic bursting. Notably, the timescale of these voltage-dependent shifts influences the final steady-state configuration of the model, shaping both channel parameters and activity features such as burst period and duration. A major conclusion of the study is that altering this timescale can seamlessly modulate a neuron's intrinsic properties, which the authors suggest may be a mechanism for adaptation to perturbations.

While this conclusion is largely well-supported, additional analyses could help clarify its scope. For instance, the effects of timescale alterations are clearly demonstrated when the model transitions from an initial state that does not meet the target activity pattern to a new stable state. However, Fig. 6 and the accompanying discussion appear to suggest that changing the timescale alone is sufficient to shift neuronal activity more generally. It would be helpful to clarify that this effect primarily applies during periods of adaptation, such as neurodevelopment or in response to perturbations, and not necessarily once the system has reached a stable, steady state. As currently presented, the simulations do not test whether modifying the timescale can influence activity after the model has stabilized. In such conditions, changes in timescale are unlikely to affect network dynamics unless they somehow alter the stability of the solution, which is not shown here. That said, it seems plausible that real neurons experience ongoing small perturbations which, in conjunction with changes in timescale, could allow gradual shifts toward new solutions. This possibility is not discussed but could be a fruitful direction for future work.