Reviewer #2 (Public review):

This second revision has partially addressed criticisms previously raised; however, substantial inadequacies, particularly concerning rigorous validation and model justification, remain unresolved. While recognizing evident strength, novelty, and technical complexity of this work, the authors have yet to fully resolve key major concerns explicitly pointed out during revision in a satisfactory manner. As currently written, the manuscript does not yet provide sufficiently robust validation, methodological rigour, or clarity required for complete acceptance in a top-tier scientific journal.

Summary of Authors' Aim:

In this revised version, the authors aimed to address prior reviewer critiques harshly pinpointing the need for greater clarity in the manuscript's logical flow, rigorous external validation, clearer explanation of methodological normalization choices, and deeper elaboration of diffusion MRI method relevance and potential translation. The authors present a diffusion-weighted MRS approach paired with complex biophysical modelling to elucidate differential developmental trajectories of cellular structures in cerebellum and thalamus in rat neonates, providing a novel, non-invasive avenue for monitoring cellular microstructure.

Major Comments:

Rigorous Validation (Reviewer #1 - point R1.1, Reviewer #2 - point R2.2):

The major concern previously raised and reiterated here is the insufficient external cross-validation of the dMRS-derived interpretations about cellular changes, including the particularly speculative interpretation that taurine undergoes compartment switching between neuronal and glial compartments in the thalamus. The authors acknowledge this important shortcoming (R1.1, R2.2) but attempt to mitigate these concerns merely through additional contextual comparisons from existing literature (page 23, lines 877-878, Figure S11, Table S2). While better contextualization is welcome, the modified manuscript still falls notably short of the level of rigour necessary to validate such striking switches in compartmentalization. To justify claims of metabolites changing cellular compartments, explicit verification against independent molecular/histological data, ideally with additional immunohistochemical staining for cellular markers (e.g., glial fibrillary acidic protein, NeuN), is necessary. The mere presence of literature correlations (such as the reported visual comparisons to morphometric reconstructions, page 24, lines 883-884) does not constitute rigorous validation at the required standard for high-impact publication. The revised manuscript remains fundamentally weakened without such validation. To properly improve, the authors must consider incorporating independent ex vivo experiments or, if this is no longer feasible, extensively temper their compartment-switching claims, acknowledging explicitly and prominently the speculative nature of current interpretations.

Normalization of Metabolite Concentrations (Reviewer #1 - point R1.3):

The authors clearly responded to a reviewer wish for justification of metabolite normalisation to macromolecular concentrations (page 13, lines 493-503, Figure S2). However, the rationale provided remains only partially convincing. While the authors appropriately acknowledge the unusual nature of their methodological choice and possible confounding factors, they opt to supplement rather than substitute this approach with a more standard method (normalisation by water) in the main body of the manuscript. The additional supplementary Figure S2 is helpful, yet the conclusions derived with macromolecular normalization still remain potentially confounded by age-dependent macromolecular changes (Tkac et al., 2003). The justification given in the revised manuscript remains vague, unsatisfactory, and somewhat contradictory-authors accept macromolecules changes likely with age, yet largely overlook this effect. At least, the comparison between normalization by macromolecules and water should be explicitly discussed in the main text, and conclusions drawn from macromolecular normalization must be cautiously framed.

Choice and Justification of Biophysical Model (Reviewer #1 - point R1.4):

The reviewers questioned model assumptions, particularly ignoring macroscopic anisotropy effects due to white matter presence, myelination, and fibre orientation dispersion in the cerebellar voxel. Authors provided newly included DTI data and acknowledged this limitation explicitly (R1.4, Figure S8, page 25, lines 921-924). However, the addition of these poor-quality DTI data with limited interpretability paradoxically weakens rather than strengthens the manuscript as a whole, since the authors now present unclear supplementary results with little additional interpretative value. Recognizing poor data quality in this scenario, although intellectually honest, does not substantially increase the current robustness of their chosen model nor improve justification. To address this fully, either higher-quality data should be collected to robustly probe anisotropy or fibre dispersion effects, or the authors must much further restrict their interpretations in view of this clear limitation. Currently, the solution proposed is incomplete and insufficient to clarify the consequences of their chosen model.

Logical Flow and Clarity (Reviewer #2 - points R2.1 and R2.3):

The authors attempted to respond to reviewer comments on logical flow and accessibility (page 3, introduction restructuring). While the manuscript readability has improved, the introduction and discussion remain overly intricate, and at times, detail-oriented without clear links into central claims. In particular, the biological rationale for choosing the specific metabolite markers (especially tCho, Ins, Tau, etc.) and their known relevance must be further streamlined and simplified to increase accessibility and directness. Although some helpful restructuring was carried out, further careful paragraph-level revision for logical flow and readability remains necessary.

Translation to Human Studies (Reviewer #2 - point R2.4):

The authors have extended contextual discussion on translational potential regarding taurine as a developmental marker in humans (pages 24-25, lines 906-917). However, mention remains vague and cursory, without presenting sufficiently solid arguments nor drawing from human developmental studies adequately. Translational potential must be assessed within the realistic limitations inherent in clinical translation of MRS studies, particularly given the technical complexities clearly identified even in preclinical studies of this paper. Discussion remains relatively superficial, and if retained, must be expanded to fully discuss realistic human translational hurdles and requirements.

Reviewer #1 (Public review):

Summary:

This study by Torok et al. takes a creative approach to studying circuit perturbations in a sensorimotor region for vocalization control, in a songbird species, the zebra finch. By expressing the light chain of tetanus toxin in neurons in a sensorimotor region HVC, the authors constrain neural firing and study the resulting degradation and then recovery of song, after a protracted (> 70-day) period. Recording data suggest a form of synaptic homeostasis emergent in both HVC and RA as a result of the profound loss of (inhibitory?) tone in HVC. The methods to analyze changes in song are particularly strong here, using dimension reduction and visualization techniques. Single-cell sequencing data showed accompanying changes in microglia abundance, as well as several other markers that were not observed in control viral injections. LFP analyses in birds during the tetanus onset phase showed clear dysregulation of typical voltage deflections and spectral power, each of which showed recovery in parallel with song recovery. Lastly, the authors present data indicating that the anterior forebrain region LMAN is not critical for the song degradation process, pointing instead to the direct relationship between HVC and RA in song plasticity in adults. The methods are generally well established, but my main concerns regard the validation of the viral construct, the lack of direct confirmation of tetanus toxin on inhibitory neurons or E/I balance in HVC, and a missed opportunity to look at song syllable sequence degradation and recovery.

Strengths:

The species under investigation is the premier model for the neural basis of vocal learning, and the telencephalic brain regions investigated are well mapped out for their control of vocal learning behavior. The methods for electrophysiology recording and analysis, song analysis, scRNAseq, and in situ hybridization pose no concern as they are well established for this group of co-authors.

Weaknesses:

The introduction lays out a case for pursuing long-term E/I imbalances, vis-à-vis transient perturbations that have shown effects on the behavior. However, the rationale is not clearly stated. Why should the reader care that "prolonged E/I imbalances" may occur? Do they occur naturally or in some disease states (as alluded to in the first paragraph)? Without this rationale, the reader is left with an impression that the experiments were done because of a technical capability rather than a conceptual thrust.

The cited works for the statement the "AAV viral vector expressing TeNT undre the human dlx promoter, which is selective for HVC inhibitory interneurons" (reference 5 Kosche et al., 2016; and reference 10 Vallentin et al 2016) do not substantiate the targeting of this dlx5 promoter for interneurons in zebra finch HVC. Neither of these cited studies used viral vectors, and so this is a misattribution of the dlx5 promoter as targeting HVC inhibitory interneurons. However, the original development of this enhancer by Gord Fishell and others did have solid expression in HVC (Dimidschstein et al., 2016, Nature Neuroscience), and the enhancer was used to successfully target inhibitory neurons in nearby nidopallium NCM (Spool et al., 2022, Curr Biol). Citing these two studies would improve the standing of this viral approach. Nevertheless, the specific construct used here is not the same as the published studies mentioned above (AAV9-dlx-TeNT). The authors therefore need to show expression of the virus using some histological confirmation to cement the idea that they are indeed targeting inhibitory interneurons with this manipulation. The methods statement "a single injection (~100 nL) in the center of HVC was sufficient to label enough cells" is not convincing in the absence of quantified photomicrographs.

The authors present no physiological confirmation of TeNT on E/I balance directly, and so we don't have a clear picture of how/whether HVC interneurons are physiologically altered by this manipulation. That said, the Npix recordings show that there was a tremendous increase in gamma power following TeNT manipulation, which subsides as the protracted song recovery unfolds. This finding is somewhat counterintuitive, given that gamma oscillations are typically driven by inhibitory neurons in many systems (including songbird pallium) while the TeNT manipulation is purported to cause *reductions* in inhibitory neurotransmitter release within HVC. Some interpretation of these incongruent results would be useful in the Discussion.

The degradation and recovery of song is based mainly on the measures of duration of syllables and inter-syllable intervals, but HVC is also a key locus for song syllable sequence coding. The supplementary figures show some changes in sequences. It would improve the interpretation of both the degradation and recovery of the song to know whether syllable sequences (iiiABCCDDEF) truly recovered or were morphed in some way (e.g., iiiCDDDBEF). The PCA analyses (that the authors conducted) for these two potential outcomes would likely be very similar, but the actual songs would differ greatly under these two scenarios in terms of syllable sequence. From the representative spectrograms, it appears that the song syllable sequence does indeed recover well in these examples (perhaps less so in Supplementary Figure 3). A simple Markov-chain analysis of the syllable sequences across birds in the study would provide important confirmation of these insights.

Reviewer #2 (Public review):

This article addresses the question of how complex behavior is maintained despite perturbations in underlying motor circuits. Using zebra finch song production as a model system, the authors employ a genetic approach to perturb activity in GABAergic neurons within the vocal control nucleus HVC. Specifically, they use AAV to deliver the tetanus toxin light chain (TeNT) under the interneuron-specific DLX promoter, with the goal of silencing interneurons. This manipulation causes rapid degradation of song, followed by recovery over several weeks.

The authors characterize the recovery using a combination of transcriptomic analysis, electrophysiology, and lesion studies. Notably, the recovery does not require the lMAN, which is typically considered critical for vocal learning and plasticity. The authors speculate that homeostatic mechanisms within the motor pathway - potentially involving microglial remodeling -may mediate this recovery.

The strength of the study lies in the striking behavioral effects - both degradation and recovery - resulting from a specific circuit perturbation, and the use of complementary approaches (gene expression, neurophysiology, behavior, and lesions) to link circuit changes to behavior. The approach is creative, and the findings are intriguing. More detailed comments are provided below that may help enhance the manuscript's value to the community.

(1) In Figure 1b, the authors show changes in the relative abundance of cell types following TeNT expression in HVC. The most prominent change, as noted by the authors, is an increase in microglia. However, there are also apparent changes in the proportions of other cell types-particularly decreases in neurons and radial glia. How do the authors interpret the observed reductions in GABAergic and glutamatergic cells, as well as radial glia? Are these decreases statistically significant? Given the magnitude of these changes, could they reflect sampling differences (e.g., inclusion of tissue outside HVC) or neuronal cell death? Alternatively, is it possible that the absolute number of mature neurons remains constant, and increases in other cell types shift the relative proportions? The authors should clarify how to interpret the Y-axis of this plot. It appears to reflect relative abundance rather than absolute cell numbers, which has important implications for interpretation.

(2) The authors appear to define their own cell type clusters and labels, rather than using standard classifications (e.g., Colquitt et al. 2021; Colquitt et al. 2023). This makes cross-study comparisons difficult. For example, Colquitt describes four classes of putative immature neurons (pre2-pre4, GABA-pre). In contrast, the authors refer to "neuroblasts" in Figure 1b. Are these equivalent to pre2-pre4 and/or to "GABA-pre"? What about "migrating neuroblasts" in Supplementary Figure 11? The authors could consider using the standard nomenclature, or if they disagree with that classification, explain why an alternative scheme is warranted.

(3) The transcriptomic data are underexplored. Many genes appear differentially expressed (e.g., in Figure 1c), however, the main text contains little discussion of differential gene expression beyond MHC I and B2M. It would be useful to discuss whether transcriptomic data support or rule out any other specific mechanistic hypotheses for recovery.

(4) The authors attribute increased microglial markers to interneuron silencing rather than inflammation from viral injection, based on control virus results (lines 143-146). However, is it plausible that TeNT expression itself, or batch variability, could drive differences in inflammation? The authors could address these alternatives with additional evidence or discussion.

Reviewer #3 (Public review):

Summary:

This manuscript investigates at behavioral and mechanistic levels the recovery of zebra finch song production after a genetically targeted insult to HVC, a vocal premotor nucleus known to generate stereotyped neural sequences that drive the correspondingly stereotyped song. This study is a close follow up to past work, published in Nature Neuroscience last year (Wang et al, 2024), in which custom lentiviruses were used to deliver a persistently active sodium channel, NacBAC or TeNT to block synaptic release, specifically to the excitatory projection neurons in HVC. In this past work, these manipulations resulted in rapid degradation of song, followed by a slow recovery that, remarkably, did not require practice. Song recovery was associated with synaptic remodeling that appeared to homeostatically bring the affected neurons back to a normal firing regime. This past paper was important because it clearly demonstrated behaviorally and mechanistically how neural plasticity can restore a learned behavior without practice, showing that dominant reinforcement learning models of birdsong are not the full story.

This past work sets the context for the current paper, which instead targets the inhibitory neuronal population in HVC for silencing via viral-mediated expression of TeNT. Again, this sophisticated targeting of HVC interneurons resulted in rapid degradation of song, followed by a much slower but seemingly full recovery.

Strengths:

Overall, this paper has several strengths. First, it provides yet another convincing example of non-canonical vocal learning in the zebra finch because LMAN (a nucleus required for trial and error song learning) is not required for song recovery. Second, its targeting of interneurons clarifies the extent to which inhibition in HVC is essential for vocal patterning (not surprising but important to show). Third, by using RNAseq of HVC at the time of peak song disruption, it zeroes in on specific genetic/cellular activations associated with a lack of inhibition (e.g., microglial activation and MHC1 expression), opening up new avenues for future study. Using in vivo electrophysiology it also characterizes some gross circuit-level abnormalities in HVC-RA transmission and during sleep.

Weaknesses:

Yet the paper also has several areas for improvement, primarily:

Main issues

(1) Narrative-level confusion, a mix of results, many hanging threads

The arc of this paper is very hard to follow, new experiments arise without a clear setup or connection to past ones. Concepts jump around unpredictably. The reading experience would be dramatically improved if there were a clear single line of logic going through the entire paper, which could be accomplished by inserting a paragraph at the end of the intro section that walks the reader step-by-step through what they are going to see. I don't recommend this for all papers - but this paper requires it, in my opinion, because we have such an unusual combination of experimental approaches, outcomes, and data formats (behavior, RNA seq, targeted tests of microglial activation in the setting of adult impairment and song development, electrophysiology during sleep. It's very difficult for me to tie this all together into a crisp narrative that sticks with me days after reading the paper. Instead, it feels like some disconnected factoids. Examples:<br /> a) Characterization of degradation and slow recovery (much slower than targeting of projection neurons form past work (Wang et al, 2024).<br /> b) Activation of microglia and MHC1 during the degraded period; microglia return to normal at recovery.<br /> c) Developmenta profile of microglia expression.<br /> e) Sleep replay in HVC is perturbed during the degraded state. Mostly returns to normal following recovery, but *some* aspects are still abnormal.<br /> f) Detailed ephys analysis of HVC excitability and RA suppression, invoking ideas that HVC drives RA inhibition.<br /> g) LMAN lesions do not block degradation or recovery.

There are at least three threads of this paper - it therefore reads like three different papers stitched together into one - united only by the method of HVC interneuron targeting. In my view, a pretty major overhaul is required, even if it means cutting out specific details and figures that distract from the paper's message (for example there is a whole sub-section analyzing HVC impact on RA that vaguely invokes ideas of HVC engagement of RA

(2) Interpretation of microglia is confusing and unresolved

Microglia activation is measured at peak song disruption, and returns to normal following recovery. To test if this phenomenon is associated with learning or degradation, the authors measure microglia during development.

"The increased inhibitory tone in HVC and the number of microglia could induce synaptic changes that contribute to degraded song production. Alternatively, the rise in microglia could be part of the recovery response to produce synaptic changes needed to regain the song following perturbation."

This is a great if/then statement on how to interpret the microglial activation at the core of the paper. But it remains unresolved. Is there a causal experiment that could distinguish these possibilities?

(3) The quantification of song dynamics during the recovery process in LMAN lesioned birds is required to support claims. Perhaps the most interesting claim of the paper - that recovery happens without LMAN, is not sufficiently supported by data analyses. This is a major problem.

The same analysis used in the LMAN-intact degradation/recovery dataset should be used for the LMAN dataset. At present, there are no quantification, only example spectrograms. Also, Supplementary Figure 4 and Supplementary Figure 5 are identical, suggesting a lack of proofreading in this part of the manuscript. For example the reader cannot even ascertain if the key aspect of song degradation - the production of exceedingly long syllables - is occurring in the LMAN lesioned animals.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors Eapen, et al. investigated the peptide inhibitors of Cdc20. They applied a rational design approach, substituting residues found in the D-box consensus sequences to better align the peptides with the Cdc20-degron interface. In the process, the authors designed and tested a series of more potent binders, including ones that contain unnatural amino acids, and verified binding modes by elucidating the Cdc-20-peptide structures. The authors further showed that these peptides can engage with Cdc20 in the cellular context, and can inhibit APC/CCdc20 ubiquitination activity. Finally, the authors demonstrated that these peptides could be used as portable degron motifs that drive the degradation of a fused fluorescent protein.

Strengths:

This manuscript is clear and straightforward to follow. The investigation of different peptide variations was comprehensive and well-executed. This work provided the groundwork for the development of peptide drug modalities to inhibit degradation or applying peptides as portable motifs to achieve targeted degradation. Both of which are impactful. The additional points provided by the authors in response to reviewers further strengthened the manuscript and enhanced its clarity.

Weaknesses:

None, the authors have addressed all my comments, and I have no additional suggestions.

Reviewer #3 (Public review):

Summary:

Eapen and coworkers use a rational design approach to generate new peptide-inspired ligands at the D-box interface of cdc20. These new peptides serve as new starting points for blocking APC/C in the context of cancer, as well as manipulating APC/C for targeted protein degradation therapeutic approaches.

Strengths:

The characterization of new peptide-like ligands is generally solid and multifaceted, including binding assays, thermal stability enhancement in vitro and in cells, X-ray crystallography, and degradation assays.

Comments on revisions:

I am satisfied with the changes in response to the first round of review.

Reviewer #1 (Public review):

Summary:

The study dissects distinct pools of diacylglycerol (DAG), continuing a line of research on the central concept that there is a major lipid metabolism DAG pool in cells, but also a smaller signaling DAG pool. It tests the hypothesis that the second pool is regulated by Dip2, which influences Pkc1 signaling. The group shows that stressed yeast increase specific DAG species C36:0 and 36:1, and propose this promotes Pkc1 activation via Pck1 binding 36:0. The study also examines how perturbing the lipid metabolism DAG pool via various deletions such as lro1, dga1, and pah1 deletion impacts DAG and stress signaling. Overall this is an interesting study that adds new data to how different DAG pools influence cellular signaling.

Strengths:

The study nicely combined lipidomic profiling with stress signaling biochemistry and yeast growth assays.

Weaknesses:

One suggestion to improve the study is to examine the spatial organization of Dip2 within cells, and how this impacts its ability to modulate DAG pools. Dip2 has previously been proposed to function at mitochondria-vacuole contacts (Mondal 2022). Examining how Dip2 localization is impacted when different DAG pools are manipulated such as by deletion Pah1 (also suggested to work at yeast contact sites such as the nucleus-vacuole junction), or with Lro1 or Dga1 deletion would broaden the scope of the study.

Comments on revisions:

The revision addresses several of the concerns raised previously. Most importantly, it softens several conclusions that more clearly delineates limitations of the study. The study has yet to address how Dip2 and Pkc1 crosstalk, but new text addresses this limitation. There is also more analysis of Dip2 localization in other conditions where cell DAG pools are elevated (ie a LRO1 and DGA1 double KO, as well as PAH1 KO). Loss of these proteins elevates ER DAG, but Dip2 remains mitochondrially associated. This may imply DAG specificity, or that changes to DAG pools globally does not impact Dip2 import into mitochondria.

Reviewer #2 (Public review):

Summary:

The authors use yeast genetics, lipidomic and biochemical approaches to demonstrate the DAG isoforms (36:0 and 36:1) can specifically activate PKC. Further, these DAG isoforms originate from PI and PI(4,5)P2. The authors propose that the Psi1-Plc1-Dip2 functions to maintain a normal level of specific DAG species to modulate PKC signalling.

Strengths:

Data from yeast genetics are clear and strong. The concept is potentially interesting and novel.

Weaknesses: More evidence is needed to support the central hypothesis. The authors may consider the following:

(1) Figure 2: the authors should show/examine C36:1 DAG. Also, some structural evidence would be highly useful here. What is the structural basis for the assertion that the PKC C1 domain can only be activated by C36:0/1 DAG but not other DAGs? This is a critical conclusion of this work and clear evidence is needed.

(2) Does Dip2 colocalize with Plc1 or Pkc1? Does Dip2 reach the plasma membrane upon Plc activation?

Comments on revisions:

The authors have addressed my concerns.

Reviewer #1 (Public review):

Summary:

This manuscript addresses the challenge of understanding and capturing the similarity among large numbers of visual images. The authors show that an automated approach using artificial neural networks that focuses upon the embedding of similarity through behaviorally relevant dimensions can predict human similarity data up to a certain level of granularity.

Strengths:

The manuscript starts with a very useful introduction that sets the stage with an insightful Figure 1. The methods are state of the art and well thought off, and the data are compelling. The authors demonstrate the added value of their approach in several directions, resulting in a manuscript that is highly relevant for different domains. The authors also explore its limitations (e.g., granularity).

Weaknesses:

Although this manuscript and the work it describes are already of high quality, I see several ways in which it could be further improved. Below I rank these suggestions tentatively in order of importance.

Predictions obtain correlations above 0.80, often close to correlations of 0.90. The performance of DimPred is not trivial, given how much better it performs relative to classic RSA and feature reweighting. Yet, the ceiling is not sufficiently characterized. What is the noise ceiling in the main and additional similarity sets that are used? If the noise ceiling is higher than the prediction correlations, then can the authors try to find the stimulus pairs for which the approach systematically fails to capture similarity? Or is the mismatch very distributed across the full stimulus set?

Also in the section on p. 8-p.9, it is crucial to provide information on the noise ceiling of the various datasets.

This consideration of noise ceiling brings me to another consideration. Arguments have been made that a focus on overall prediction accuracy might mask important differences in underlying processes that can be demonstrated in more specific, experimental situations (Bowers et al., 2023). Can the authors exclude the possibility that their automatic approach would fail dramatically in specifically engineered situations? Some examples can be found in the 2024 challenge of the BrainScore platform. How can future users of this approach know whether they are in such a situation or not?

The authors demonstrated one limitation of the DimPred approach to capture fine-grained similarity among highly similar stimuli. The implications of this finding were not clear to me from the Abstract etc, because it is not sufficiently highlighted in the summaries that in this case DimPred performs even worse, and much worse, than more simple approaches like feature reweighting and even than classic RSA. I would discuss this outcome more in detail. With hindsight, this problem might not be so surprising given that DimPred relies upon the embedding with a few tens dimensions that mostly capture between-category differences. To me, this seems like a more fundamental limitation than a mere problem of granularity or lack of data, as suggested in the abstract.

The DimPred approach is based on the dimensions of a similarity embedding derived from human behavior. What is important here is (i) that DimPred is based upon an approach that tries to capture latent dimensions; or (ii) that these dimensions are behaviorally relevant? There are a lot of dimension-focused approaches. Generic ones are PCA, MDS, etc. More domain-specific approaches in cogneuro include the following: (i) for two-dimensional shape representations, good results have been obtained with image-computable dimensions of various levels of complexity (Morgenstern et al., 2021, PLOS Comput. Biol.); (ii) another dimension-focused approach has focused upon identifying dimensions that are universal across networks & human representations (Chen & Bonner, 2024, arXiv). Would such generic or more specific approaches work as well as DimPred?

Reviewer #2 (Public review):

In this paper, the authors successfully incorporated the 49 dimensions found in a human similarity judgment task to better train DNNs to perform accurate human-like object similarity judgments. The results of the model performance are impressive but I am not totally convinced that the present modeling approach may bring new insights regarding the mental and neural representations of visual objects in the human brain. I have a few thoughts that I would like the authors to consider.

(1) Can the authors provide a detailed description of what these off-the-shelf DNNs are trained on? For models trained on visual images only, because semantic information was never present during training, it is not surprising they fail to capture such information, even with additional DimPred training. For the CLIP models, because visual-sematic associations were included during training, it again comes as no surprise that these models can do better even without DimPred training. Similarly, the results of homogenous image sets are not particularly surprising. In this regard, I am finding the paper reports many obvious results. Better motivations should be used to justify why particular models and analyses were performed, what predictions can be made, and how the results may be informative beyond what we already know.

(2) I am curious as to what DimPred training is doing exactly. If you create an arbitrary similarity structure (i.e., not the one derived from human similarity judgment) by, e.g., shuffling the values during training or creating 49 arbitrary dimensions, can the models be trained to follow this new arbitrary structure? In other words, do the models intrinsically contain a human-like structure, but we just have to find the right parameters to align them with the human structure or do we actually impose/force the human similarity structure onto the model with DimPred training?

Is it also an issue that you are including more parameters during DimPred training and that increased parameters alone can increase performance?

(3) There is very little information on how Figure 8 is generated. I couldn't find in the Methods any detailed descriptions of how the values were calculated. Are results from both the category-insensitive and category-sensitive embedding obtained from the same OpenCLIP-RN50x64? Figure 8 reports the relative improvement. What do the raw activation maps look like for the category-insensitive and category-sensitive embedding? I am surprised that the improvement is seen primarily in the early visual cortex (EVC) and higher visual areas but not more extensively in association areas sensitive to semantics. Why should EVC show such large improvements, given that category information is stored elsewhere?

Related to this point, how do other DNN models account for human brain fMRI responses in the present study? Many prior studies have documented the similarities and differences between DNN and human fMRI visual object representations. Do category-sensitive CLIP models outperform other DNN models? It is important to report the full results. Even though category-sensitive CLIP models outperform category-insensitive CLIP ones, if the overall model performance is low compared to the other DNNs, the results would not be very meaningful/impressive. I am just wondering if, in the process of achieving better human-like similarity judgment performance, these models lose some of the ability to account for visual object representations in the human ventral visual cortex.

(4) I am wondering how precisely the present results may yield new insights into the mental and neural representations of visual objects in the human brain. Prior human studies have already identified 49 dimensions that can capture human similarity judgment. Beyond predicting performance for new pairs of objects, how would the present modeling approach help us understand more about the human brain? The authors discussed this, but I am not sure the arguments are convincing.

Reviewer #3 (Public review):

Summary:

The authors compare how well their automatic dimension prediction approach (DimPred) can support similarity judgements and compare it to more standard RSA approaches. The authors show that the DimPred approach does better when assessing out-of-sample heterogeneous image sets, but worse for out-of-sample homogeneous image sets. DimPred also does better at predicting brain-behaviour correspondences compared to an alternative approach. The work appears to be well done, but I'm left unsure what conclusions the authors are drawing.

In the abstract, the authors write: "Together, our results demonstrate that current neural networks carry information sufficient for capturing broadly-sampled similarity scores, offering a pathway towards the automated collection of similarity scores for natural images". If that is the main claim, then they have done a reasonable job supporting this conclusion. However the importance of automating this process for broadly-sampled object categories is not made so clear.

But the authors also highlight the importance that similarity judgements have been for theories of cognition and brain, such as in the first paragraph of the paper they write: "Similarity judgments allow us to improve our understanding of a variety of cognitive processes, including object recognition, categorization, decision making, and semantic memory6-13. In addition, they offer a convenient means for relating mental representations to representations in the human brain14,15 and other domains16,17". The fact that the authors also assess how well a CLIP model using DimPred can predict brain activation suggests that their work is not just about automating similarity judgements, but highlighting how their approach reveals that ANNs are more similar to brains than previously assessed.

My main concern is with regards to the claim that DimPred is revealing better similarities between ANNs and brains (a claim that the authors may not be making, but this should be clarified). The fact that predictions are poor for homogenous images is problematic for this claim, and I expect their DimPred scores would be very poor under many conditions, such as when applied to line drawings of objects, or a variety of addition out-of-sample stimuli that are easily identified by humans. The fact that so many different models get such similar prediction scores (Fig 3) also raises questions as to the inferences you can make about ANN-brain similarity based on the results. Do the authors want to claim that CLIP models are more like brains?

With regards to the brain prediction results, why is the DimPred approach doing so much better in V1? I would not think the 49 interpretable categories are encoded in V1, and the ability to predict would likely reflect a confound rather than V1 encoding these categories (e.g., if a category was "things that are burning" then DNN might predict V1 activation based on the encoding of colour).

In addition, more information is needed on the baseline model, as it is hard to appreciate whether we should be impressed by the better performance of DimPred based on what is provided: "As a baseline, we fit a voxel encoding model of all 49 dimensions. Since dimension scores were available only for one image per category36, for the baseline model, we used the same value for each image of the same category and estimated predictive performance using cross-validation". Is it surprising that predictions are not good with one image per category? Is this a reasonable comparison?

Relatedly, what was the ability of the baseline model to predict? (I don't think that information was provided). Did the authors attempt to predict outside the visual brain areas? What would it mean if predictions were still better there?

Minor points:

The authors write: "Please note that, for simplicity, we refer to the similarity matrix derived from this embedding as "ground-truth", even though this is only a predicted similarity". Given this, it does not seem a good idea to use "ground truth" as this clarification will be lost in future work citing this article.

It would be good to have the 49 interpretable dimensions listed in the supplemental materials rather than having to go to the original paper.

Strengths:

The experiments seem well done.

Weaknesses:

It is not clear what claims are being made.

Joint Public Reviews:

In this study, the authors suggest that DuoHexaBody-CD37, a biparatopic CD37-targeting antibody, can induce direct cytotoxicity in diffuse large B-cell lymphoma (DLBCL) cells through antibody clustering and SHP-1 activation, independent of complement. They further propose that DuoHexaBody-CD37 inhibits cytokine-mediated pro-survival signalling, suggesting a broader role for CD37-directed therapy in disrupting tumour supportive signalling networks.

A strength of the study is the systematic in vitro characterisation of signalling responses to DuoHexaBody-CD37 across both malignant and normal B-cells. The inclusion of phosphoproteomic profiling and mutant constructs provides mechanistic detail, and the findings may be of interest to researchers working on antibody therapeutics in lymphoma.

However, the evidence supporting key mechanistic processes - particularly the role of SHP-1 in mediating cytotoxicity and the requirement for Fc receptor crosslinking - is incomplete and would benefit from further functional validation. While CD37 has been explored previously as a therapeutic target, this study does add mechanistic insight into direct cytotoxicity and cytokine modulation. Nevertheless, the exclusive reliance on in vitro systems makes the translational relevance unclear.

Overall, the study provides valuable insight into CD37-mediated signalling in lymphoma cells, but the evidence remains incomplete to support broader conclusions about therapeutic impact.

Reviewer #1 (Public review):

Summary:

The authors analyze transcription in single cells before and after 4000 rads of ionizing radiation. They use Seuratv5 for their analyses, which allows them to show that most of the genes cluster along the proximal-distal axis. Due to the high heterogeneity in the transcripts, they use the Herfindahl-Hirschman index (HHI) from Economics, which measures market concentration. Using the HHI, they find that genes involved in several processes (like cell death, response to ROS, DNA damage response (DDR)) are relatively similar across clusters. However, ligands activating the JAK/STAT, Pvr, and JNK pathways and transcription factors Ets21C and dysf are upregulated regionally. The JAK/STAT ligands Upd1,2,3 require p53 for their upregulation after irradiation, but the normal expression of Upd1 in unirradiated discs is p53-independent. This analysis also identified a cluster of cells that expressed tribbles, encoding a factor that downregulates mitosis-promoting String and Twine, that appears to be G2/M arrested and expressed numerous genes involved in apoptosis, DDR, the aforementioned ligands, and TFs. As such, the tribbles-high cluster contains much of the heterogeneity.

Strengths:

(1) The authors have used robust methods for rearing Drosophila larvae, irradiating wing discs, and analyzing the data with Seurat v5 and HHI.

(2) These data will be informative for the field.

(3) Most of the data is well-presented.

(4) The literature is appropriately cited.

Weaknesses:

(1) The data in Figure 1 are single-image representations. I assume that counting the number of nuclei that are positive for these markers is difficult, but it would be good to get a sense of how representative these images are and how many discs were analyzed for each condition in B-M.

(2) Some of the figures are unclear.

Reviewer #2 (Public review):

This manuscript investigates the question of cellular heterogeneity using the response of Drosophila wing imaginal discs to ionizing radiation as a model system. A key advance here is the focus on quantitatively expressing various measures of heterogeneity, leveraging single-cell RNAseq approaches. To achieve this goal, the manuscript creatively uses a metric from the social sciences called the HHI to quantify the spatial heterogeneity of expression of individual genes across the identified cell clusters. Inter- and intra-regional levels of heterogeneity are revealed. Some highlights include the identification of spatial heterogeneity in the expression of ligands and transcription factors after IR. Expression of some of these genes shows dependence on p53. An intriguing finding, made possible by using an alternative clustering method focusing on cell cycle progression, was the identification of a high-trbl subset of cells characterized by concordant expression of multiple apoptosis, DNA damage repair, ROS-related genes, certain ligands, and transcription factors, collectively representing HIX genes. This high-trbl set of cells may correspond to an IR-induced G2/M arrested cell state.

Overall, the data presented in the manuscript are of high quality but are largely descriptive. This study is therefore perceived as a resource that can serve as an inspiration for the field to carry out follow-up experiments.

Reviewer #3 (Public review):

Summary:

Cruz and colleagues report a single-cell RNA sequencing analysis of irradiated Drosophila larval wing discs. This is a pioneering study because prior analyses used bulk RNAseq analysis, so differences at single-cell resolution were not discernible. To quantify heterogeneity in gene expression, the authors make clever use of a metric used to study market concentration, the Herfindahl-Hirschman Index. They make several important observations, including region-specific gene expression coupled with heterogeneity within each region and the identification of a cell population (high Trbl) that seems disproportionately responsible for radiation-induced gene expression.

Strengths:

Overall, the manuscript makes a compelling case for heterogeneity in gene expression changes that occur in response to uniform induction of damage by X-rays in a single-layer epithelium. This is an important finding that would be of interest to researchers in the field of DNA damage responses, regeneration, and development.

Weaknesses:

This work would be more useful to the field if the authors could provide a more comprehensive discussion of both the impact and the limitations of their findings, as explained below.

Propidium iodide staining was used as a quality control step to exclude cells with a compromised cell membrane. But this would exclude dead/dying cells that result from irradiation. What fraction of the total do these cells represent? Based on the literature, including works cited by the authors, up to 85% of cells die at 4000R, but this likely happens over a longer period than 4 hours after irradiation. Even if only half of the 85% are PI-positive by 4 hr, this still removes about 40% of the cell population from analysis. The remaining cells that manage to stay alive (excluding PI) at 4 hours and included in the analysis may or may not be representative of the whole disc. More relevant time points that anticipate apoptosis at 4 hr may be 2 hr after irradiation, at which time pro-apoptotic gene expression peaks (Wichmann 2006). Can the authors rule out the possibility that there is heterogeneity in apoptosis gene expression, but cells with higher expression are dead by 4 hours, and what is left behind (and analyzed in this study) may be the ones with more uniform, lower expression? I am not asking the authors to redo the study with a shorter time point, but to incorporate the known schedule of events into their data interpretation.

If cluster 3 is G1/S, cluster 5 is late S/G2, and cluster 4 is G2/M, what are clusters 0, 1, and 2 that collectively account for more than half of the cells in the wing disc? Are the proportions of clusters 3, 4, and 5 in agreement with prior studies that used FACS to quantify wing disc cells according to cell cycle stage?

The EdU data in Figure 1 is very interesting, especially the persistence in the hinge. The authors speculate that this may be due to cells staying in S phase or performing a higher level of repair-related DNA synthesis. If so, wouldn't you expect 'High PCNA' cells to overlap with the hinge clusters in Figures 6G-G'? Again, no new experiments are needed. Just a more thorough discussion of the data.

Trbl/G2/M cluster shows Ets21C induction, while the pattern of Ets21C induction as detected by HCR in Figures 5H-I appears in localized clusters. I thought G2/M cells are not spatially confined. Are Ets21C+ cells in Figure 5 in G2/M? Can the overlap be confirmed, for example, by co-staining for Trbl or a G2/M marker with Ets21C?

Induction of dysf in some but not all discs is interesting. What were the proportions? Any possibility of a sex-linked induction that can be addressed by separating male and female larvae?

Reviewer #1 (Public review):

In the manuscript, Aldridge and colleagues investigate the role of IL-27 in regulating hematopoiesis during T. gondii infection. Using loss-of-function approaches, reporter mice, and the generation of serial chimeric mice, they elegantly demonstrate that IL-27 induction plays a critical role in modulating bone marrow myelopoiesis and monocyte generation to the infection site. The study is well-designed, with clear experimental approaches that effectively address the mechanisms by which IL-27 regulates bone marrow myelopoiesis and prevents HSC exhaustion.

Reviewer #2 (Public review):

Summary:

Aldridge et al. aim to demonstrate the role of IL27 in limiting emergency myelopoiesis in response to Toxoplasma gondii infection by acting directly at the level of early haematopoietic progenitors.

They used different mouse genetic models, such as HSC lineage tracing, IL27 and IL27R-deficient mice, to show that:

(1) HSCs actively participate in emergency myelopoiesis during Toxoplasma gondii infection.

(2) The absence of IL27 and IL27R increases monocyte progenitors and monocytes, mainly inflammatory monocytes CCR2hi.

(3) At steady state, loss of IL27 impairs HSC fitness as competitive transplantation shows long-term engraftment deficiency of IL27 BM cells. This impairment is exacerbated after infection.

(4) IL27 is produced by various BM and other tissue cells at steady state, and its expression increases with infection, mainly by increasing the number of monocytes producing it.

Although it is indisputable that IL27 has a role in emergency myelopoiesis by limiting the number of pro-inflammatory monocytes in response to infection, the authors' claim that it acts only on HSCs and not on more committed progenitors (CMP, GMP, MP) is not supported by the quality of the data presented here, as described below in the weakness section. In addition, this study highlights a role for IL27 during infection, but does not focus on trained immunity, which is the focus of the targeted elife issue.

Weaknesses:

(1) In Figure 4, MFI quantification is required. This figure also shows the expression level (FACS and RNA) in progenitors (GMP and CMP, GP, MP), which is quite similar to that of HSC at this level, so it is really surprising that CMP does not respond at all to IL27 (S5C).

(2) Total BM was used to test the direct effect of IL27 on HSC. There could be an indirect effect from other more mature BM cells, even if they show lower receptor expression than HSC. This should be done on a different sorted population to prove the direct effect of IL27 on HSC. The authors need to look more closely at some stat-dependent genes or stat itself in different sorted cell populations, not just irgm1. It is also known that Stat is associated with increased HSC proliferation in response to IFN, which is the opposite of what is observed here.

(3) The decrease in HSC fitness in IL27R KO at steady state could be an indirect effect of the increase in proinflammatory monocytes contributing to high levels of inflammatory cytokines in the BM and thus chronic HSC activation that is enhanced in response to infection. What is the pro-inflammatory cytokine profile of the BM of IL27 or IL27R deficient mice and of mixed chimera mice?

(4) Furthermore, the FACS profile of KI67/brdu of Figure 7 is doubtful, as it is shown in different literature that KSL are not predominantly quiescent as shown here, but about 50% are KI67-. This is also inconsistent with the increase of HSC observed in Figure 1. Quantification of total BruDU+ HSC and other progenitors is also important to quantify all cells that have proliferated during infection. As the repopulation of IL27-deficient BM is also lower in the absence of infection, the proliferation of HSC in IL27R KO mice in the absence of infection is also important.

(5) The immunofluorescence in Figure 3 shows a high level of background and it is difficult to see the GFP and tomato positive cells. In this sense, the number of HSCs quantified as Procr+ (more than 8000 on a single BM section) is inconsistent with the total number of HSCs that a BM can contain (i.e., around 6000 per BM as quantified in Figure 1).

(6) The addition of arrows to the figure will help to visualise positive cells. It is also not clear why the author normalised the GFP+ cells to the tomato+ cells in Figure 3D.

(7) Furthermore, even if monocytes represent a high proportion of IL27-producing cells, they are only 50% of the cells at 5dpi, as shown in Figure 3 and S4. Without other monocyte markers, line 307 is incorrect.

(8) How do the authors explain that in Figure 1, 5-10% of labelled precursors and monocytes can give 100% of monocytes? This would mean that only labelled HSC can differentiate into PEC monocytes.

Reviewer #1 (Public review):

Summary:

This study addresses the encoding of forelimb movement parameters using a reach-to-grasp task in mice. The authors use a modified version of the water-reaching paradigm developed by Galinanes and Huber. Two-photon calcium imaging was then performed with GCaMP6f to measure activity across both the contralateral caudal forelimb area (CFA) and the forelimb portion of primary somatosensory cortex (fS1) as mice perform the reaching behavior. Established methods were used to extract the activity of imaged neurons in layer 2/3, including methods for deconvolving the calcium indicator's response function from fluorescence time series. Video-based limb tracking was performed to track the positions of several sites on the forelimb during reaching and extract numerous low-level (joint angle) and high-level (reach direction) parameters. The authors find substantial encoding of parameters for both the proximal and distal parts of the limb across both CFA and fS1, with individual neurons showing heterogeneous parameter encoding. Limb movement can be decoded similarly well from both CFA and fS1, though CFA activity enables decoding of reach direction earlier and for a more extended duration than fS1 activity. Collectively, these results indicate involvement of a broadly distributed sensorimotor region in mouse cortex in determining low-level features of limb movement during reach-to-grasp.

Strengths:

The technical approach is of very high quality. In particular, the decoding methods are well designed and rigorous. The use of partial correlations to distinguish correlation between cortical activity and either proximal or distal limb parameters or either low- or high-level movement parameters was very nice. The limb tracking was also of extremely high quality, and critical here to revealing the richness of distal limb movement during task performance.

The task itself also reflects an important extension of the original work by Galinanes and Huber. The demonstration of a clear, trackable grasp component in a paradigm where mice will perform hundreds of trials per day expands the experimental opportunities for the field. This is an exciting development.

The findings here are important and the support for them is solid. The work represents an important step forward toward understanding the cortical origins of limb control signals. One can imagine numerous extensions of this work to address basic questions that have not been reachable in other model systems.

Collectively, these strengths made this manuscript a pleasure to read and review.

Weaknesses:

In the last section of the results, the authors purport to examine the representation of "higher-level target-related signals," using the decoding of reach direction. While I think the authors are careful in their phrasing here, I think they should be more explicit about what these signals could be reflecting. The "signals" here that are used to decode direction could relate to anything - low-level signals related to limb or postural muscles, or true high-level commands that dictate only what movement downstream motor centers should execute, rather than the muscle commands that dictate how. One could imagine using a partial correlation-type approach again here to extract a signal uncorrelated with all the measured low-level parameters, but there would still be all the unmeasured ones. Again, I think it is still ok to call these "high-level signals," but I think some explicit discussion of what these signals could reflect is necessary.

Related to this, I think the manuscript in general does not do an adequate job of explicitly raising the important caveats in interpreting parametric correlations in motor system signals, like those raised by Todorov, 2000. The authors do an expert job of handling the correlations, using PCA to extract uncorrelated components and using the partial correlation approach. However, more clarity about the range of possible signal types the recorded activity could reflect seems necessary.

The manuscript could also do a better job of clarifying relevant similarities and differences between the rodent and primate systems, especially given the claims about the rodent being a "first-class" system for examining the cellular and circuit basis of motor control, which I certainly agree with. Interspecies similarities and differences could be better addressed both in the Introduction, where results from both rodents and primates are intermixed (second paragraph), and in the Discussion, where more clarity on how results here agree and disagree with those from primates would be helpful. For example, the ratio of corticospinal projections targeting sensory and motor divisions of the spinal cord differs substantially between rodents and primates. As another example, the relatively high physical proximity between the typical neurons in mouse M1 and S1 compared to primates seems likely to yoke their activity together to a greater extent. There is also the relatively large extent of fS1 from which forelimb movements can be elicited through intracortical microstimulation at current levels similar to those for evoking movement from M1. All of these seem relevant in the context of findings that activity in mouse M1 and S1 are similar.

In addition, there are a number of other issues related to the interpretation of findings here that are not adequately addressed. These are described in the Recommendations for improvement.

Reviewer #2 (Public review):

Summary:

In this manuscript, Grier, Salimian, and Kaufman characterize the relationship between the activity of neurons in sensorimotor cortex and forelimb kinematics in mice performing a reach-to-grasp task. First, they train animals to reach to two cued targets to retrieve water reward, measure limb motion with high resolution, and characterize the stereotyped kinematics of the shoulder, elbow, wrist, and digits. Next, they find that inactivation of the caudal forelimb motor area severely impairs coordination of the limb and prevents successful performance of the task. They then use calcium imaging to measure the activity of neurons in motor and somatosensory cortex, and demonstrate that fine details of limb kinematics can be decoded with high fidelity from this activity. Finally, they show reach direction (left vs right target) can be decoded earlier in the trial from motor than from somatosensory cortex.

Strengths:

In my opinion, this manuscript is technically outstanding and really sets a new bar for motor systems neurophysiology in the mouse. The writing and figures are clear, and the claims are supported by the data. This study is timely, as there has been a recent trend towards recording large numbers of neurons across the brain in relatively uncontrolled tasks and inferring a widespread but coarse encoding of high-level task variables. The central finding here, that sensorimotor cortical activity reflects fine details of forelimb movement, argues against the resurgent idea of cortical equipotentiality, and in favor of a high degree of specificity in the responses of individual neurons and of the specialization of cortical areas.

Weaknesses:

It would be helpful for the authors to be more explicit about which models of mouse cortical function their results support or rule out, and how their findings break new conceptual ground.

Reviewer #1 (Public review):

Summary

Lysine acetoacetylation (Kacac) is a recently discovered histone post-translational modification (PTM) connected to ketone body metabolism. This research outlines a chemo-immunological method for detecting Kacac, eliminating the requirement for creating new antibodies. The study demonstrates that acetoacetate acts as the precursor for Kacac, which is catalyzed by the acyltransferases GCN5, p300, and PCAF, and removed by the deacetylase HDAC3. Acetoacetyl-CoA synthetase (AACS) is identified as a central regulator of Kacac levels in cells. A proteomic analysis revealed 139 Kacac sites across 85 human proteins, showing the modification's extensive influence on various cellular functions. Additional bioinformatics and RNA sequencing data suggest a relationship between Kacac and other PTMs, such as lysine β-hydroxybutyrylation (Kbhb), in regulating biological pathways. The findings underscore Kacac's role in histone and non-histone protein regulation, providing a foundation for future research into the roles of ketone bodies in metabolic regulation and disease processes.

Strengths

(1) The study developed an innovative method by using a novel chemo-immunological approach to the detection of lysine acetoacetylation. This provides a reliable method for the detection of specific Kacac using commercially available antibodies.

(2) The research has done a comprehensive proteome analysis to identify unique Kacac sites on 85 human proteins by using proteomic profiling. This detailed landscape of lysine acetoacetylation provides a possible role in cellular processes.

(3) The functional characterization of enzymes explores the activity of acetoacetyltransferase of key enzymes like GCN5, p300, and PCAF. This provides a deeper understanding of their function in cellular regulation and histone modifications.

(4) The impact of acetyl-CoA and acetoacetyl-CoA on histone acetylation provides the differential regulation of acylations in mammalian cells, which contributes to the understanding of metabolic-epigenetic crosstalk.

(5) The study examined acetoacetylation levels and patterns, which involve experiments using treatment with acetohydroxamic acid or lovastatin in combination with lithium acetoacetate, providing insights into the regulation of SCOT and HMGCR activities.

Weakness

(1) There is a limitation to functional validation, related to the work on the biological relevance of identified acetoacetylation sites. Hence, the study requires certain functional validation experiments to provide robust conclusions regarding the functional implications of these modifications on cellular processes and protein function. For example, functional implications of the identified acetoacetylation sites on histone proteins would aid the interpretation of the results.

(2) The authors could have studied acetoacetylation patterns between healthy cells and disease models like cancer cells to investigate potential dysregulation of acetoacetylation in pathological conditions, which could provide insights into their PTM function in disease progression and pathogenesis.

(3) The time-course experiments could be performed following acetoacetate treatment to understand temporal dynamics, which can capture the acetoacetylation kinetic change, thereby providing a mechanistic understanding of the PTM changes and their regulatory mechanisms.

(4) Though the discussion section indeed provides critical analysis of the results in the context of existing literature, further providing insights into acetoacetylation's broader implications in histone modification. However, the study could provide a discussion on the impact of the overlap of other post-translational modifications with Kacac sites with their implications on protein functions.

Impact

The authors successfully identified novel acetoacetylation sites on proteins, expanding the understanding of this post-translational modification. The authors conducted experiments to validate the functional significance of acetoacetylation by studying its impact on histone modifications and cellular functions.

Reviewer #2 (Public review):

In the manuscript by Fu et al., the authors developed a chemo-immunological method for the reliable detection of Kacac, a novel post-translational modification, and demonstrated that acetoacetate and AACS serve as key regulators of cellular Kacac levels. Furthermore, the authors identified the enzymatic addition of the Kacac mark by acyltransferases GCN5, p300, and PCAF, as well as its removal by deacetylase HDAC3. These findings indicate that AACS utilizes acetoacetate to generate acetoacetyl-CoA in the cytosol, which is subsequently transferred into the nucleus for histone Kacac modification. A comprehensive proteomic analysis has identified 139 Kacac sites on 85 human proteins. Bioinformatics analysis of Kacac substrates and RNA-seq data reveals the broad impacts of Kacac on diverse cellular processes and various pathophysiological conditions. This study provides valuable additional insights into the investigation of Kacac and would serve as a helpful resource for future physiological or pathological research.

The following concerns should be addressed:

(1) A detailed explanation is needed for selecting H2B (1-26) K25 sites over other acetylation sites when evaluating the feasibility of the chemo-immunological method.

(2) In Figure 2(B), the addition of acetoacetate and NaBH4 resulted in an increase in Kbhb levels. Specifically, please investigate whether acetoacetylation is primarily mediated by acetoacetyl-CoA and whether acetoacetate can be converted into a precursor of β-hydroxybutyryl (bhb-CoA) within cells. Additional experiments should be included to support these conclusions.

(3) In Figure 2(E), the amount of pan-Kbhb decreased upon acetoacetate treatment when SCOT or AACS was added, whereas this decrease was not observed with NaBH4 treatment. What could be the underlying reason for this phenomenon?

(4) The paper demonstrates that p300, PCAF, and GCN5 exhibit significant acetoacetyltransferase activity and discusses the predicted binding modes of HATs (primarily PCAF and GCN5) with acetoacetyl-CoA. To validate the accuracy of these predicted binding models, it is recommended that the authors design experiments such as constructing and expressing protein mutants, to assess changes in enzymatic activity through western blot analysis.

(5) HDAC3 shows strong de-acetoacetylation activity compared to its de-acetylation activity. Specific experiments should be added to verify the molecular docking results. The use of HPLC is recommended, in order to demonstrate that HDAC3 acts as an eraser of acetoacetylation and to support the above conclusions. If feasible, mutating critical amino acids on HDAC3 (e.g., His134, Cys145) and subsequently analyzing the HDAC3 mutants via HPLC and western blot can further substantiate the findings.

(6) The resolution of the figures needs to be addressed in order to ensure clarity and readability.

Reviewer #3 (Public review):

Summary:

This paper presents a timely and significant contribution to the study of lysine acetoacetylation (Kacac). The authors successfully demonstrate a novel and practical chemo-immunological method using the reducing reagent NaBH4 to transform Kacac into lysine β-hydroxybutyrylation (Kbhb).

Strengths:

This innovative approach enables simultaneous investigation of Kacac and Kbhb, showcasing their potential in advancing our understanding of post-translational modifications and their roles in cellular metabolism and disease.

Weaknesses:

The paper's main weaknesses are the lack of SDS-PAGE analysis to confirm HATs purity and loading consistency, and the absence of cellular validation for the in vitro findings through knockdown experiments. These gaps weaken the evidence supporting the conclusions.

Reviewer #1 (Public review):

Summary:

In this paper, authors investigated the role of RUNT-related transcription factor 2 (RUNX2) in oral squamous carcinoma (OSCC) growth and resistance to ferroptosis. They found that RUNX2 suppresses ferroptosis through transcriptional regulation of peroxiredoxin-2. They further explored the upstream positive regulator of RUNX2, HOXA10 and found that HOXA1/RNUX2/PRDX2 axis protects OSCC from ferroptosis.

Strengths:

The study is well designed and provides a novel mechanism of HOXA1/RNUX2/PRDX2 control of ferroptosis in OSCC.

Weaknesses:

According to the data presented in (Figure 2F, Figure 3F and G, Figure 5D and Figure 6E and F), apoptosis seems to be affected in the same amount as ferroptosis by HOXA1/RNUX2/PRDX2 axis, which raises a question on the authors' specific focus on ferroptosis in this study. Reasonably, authors should adapt the title and the abstract in a way that it recapitulates the whole data, which is HOXA1/RNUX2/PRDX2 axis control of cell death, including ferroptosis and apoptosis in OSCC.

Comments on revisions:

The revised manuscript has been well improved, and I'm satisfied with the authors' response to my comments.

Reviewer #1 (Public review):

Summary:

Epiney et al. use single-nuclei RNA sequencing (snRNA-seq) to characterize the lineage of Type-2 (T2) neuroblasts (NBs) in the adult Drosophila brain. To isolate cells born from T2 NBs, the authors used a genetic tool that specifically allows the permanent labeling of T2-derived cell types, which are then FAC-sorted for snRNA-seq. This effective labeling approach also allows them to compare the isolated T2 lineage cells with T1-derived cell types by a simple exclusion method. The authors begin by describing a transcriptomic atlas for all T1 and T2-derived neuronal and glia clusters, reporting that the T2-derived lineage comprises 161 neuronal clusters, in contrast to the T1 lineage which comprises 114 of them. The authors then use the expression of VAChT, VGlut, Gad1, Tbh, Ple, SerT, and Tdc2 to show that T2 neuroblasts generate all major neuron classes of fast-acting neurotransmitters. Strikingly, they show that a subset of glia and neuronal clusters have disproportionate enrichment in males or females, suggesting that T2 neuroblasts generate sex-biased cell types. The authors then proceed to characterize neuropeptide expression across T2-derived neuronal clusters and argue that the same neuropeptide can be expressed across different cell types, while similar cell types can express distinct neuropeptides. The functional implication of both observations, however, remains to be tested. Furthermore, the authors describe combinatorial transcription factor (TF) codes that are correlated with neuropeptide expression for T2-derived neurons along with an overall TF code for all T2-derived cell types, both of which will serve as an important starting point for future investigations. Finally, the authors map well-studied neuronal types of the central complex to the clusters of their T2-derived snRNA-seq dataset. They use known marker combinations, bulk RNA-seq data and highly specific split-GAL4 driver lines to annotate their T2-derived atlas, establishing a comprehensive transcriptomic atlas that would guide future studies in this field.

Strengths:

This study provides an in-depth transcriptomic characterization of neurons and glia derived from Type-2 neuroblast lineages. The results of this manuscript offer several future directions to investigate the mechanisms of diversifying neuronal identity. The datasets of T1-derived and T2-derived cells will pave the way for studies focused on the functional analysis of combinatorial TF codes specifying cell identity, sex-based differences in neurogenesis and gliogenesis, the relationship between neuropeptide (co)expression and cell identity, and the differential contributions of distinct progenitor populations to the same cell type.

Weaknesses:

The study presents several important observations based on the characterization of Type II neuroblast-derived lineages. However, a mechanistic insight is missing for most observations. The idea that there is a sex-specific bias to certain T2-derived neurons and glial clusters is quite interesting, however, the functional significance of this observation is not tested or discussed extensively. Finally, the authors do not show whether the combinatorial TF code is indeed necessary for neuropeptide expression or if this is just a correlation due to cell identity being defined by TFs. Functional knockdown of some candidate TFs for a subset of neuropeptide-expressing cells would have been helpful in this case.

Comments on revisions:

The authors have addressed my recommendations.

Reviewer #2 (Public review):

In this manuscript, Epiney et al., present a single-nucleus sequencing analysis of Drosophila adult central brain neurons and glia. By employing an ingenious permanent labeling technique, they trace the progeny of T2 neuroblasts, which play a key role in the formation of the central complex. This transcriptomic dataset is poised to become a valuable resource for future research on neurogenesis, neuron morphology, and behavior.

The authors further delve into this dataset with several analyses, including the characterization of neurotransmitter expression profiles in T2-derived neurons. While some of the bioinformatic analyses are preliminary, they would benefit from additional experimental validation in future studies.

Comments on revisions:

We appreciate the authors' efforts to address some of the comments. While these revisions have improved the clarity of certain sections, some of the larger concerns remain unaddressed. Specifically, the manuscript still lacks the additional analyses that would allow for more specific conclusions, rather than the general observations currently presented. Although the revisions have certainly made the text clearer, the core issue of needing more detailed analysis to draw more concrete conclusions still stands.

Reviewer #2 (Public review):

Summary:

The authors developed a novel tool, SCellBOW, to perform cell clustering and infer survival risks on individual cancer cell clusters from the single cell RNA seq dataset. The key ideas/techniques used in the tool include transfer learning, bag of words (BOW), and phenotype algebra which is similar to word algebra from natural language processing (NLP). Comparisons with existing methods demonstrated that SCellBOW provides superior clustering results and exhibits robust performance across a wide range of datasets. Importantly, a distinguishing feature of SCellBOW compared to other tools is its ability to assign risk scores to specific cancer cell clusters. Using SCellBOW, the authors identified a new group of prostate cancer cells characterized by a highly aggressive and dedifferentiated phenotype.

Strengths:

The application of natural language processing (NLP) to single-cell RNA sequencing (scRNA-seq) datasets is both smart and insightful. Encoding gene expression levels as word frequencies is a creative way to apply text analysis techniques to biological data. When combined with transfer learning, this approach enhances our ability to describe the heterogeneity of different cells, offering a novel method for understanding the biological behavior of individual cells and surpassing the capabilities of existing cell clustering methods. Moreover, the ability of the package to predict risk, particularly within cancer datasets, significantly expands the potential applications.

Weaknesses:

Given the promising nature of this tool, it would be beneficial for the authors to test the risk-stratification functionality on other types of tumors with high heterogeneity, such as liver and pancreatic cancers, which currently lack clinically relevant and well-recognized stratification methods. Additionally, it would be worthwhile to investigate how the tool could be applied to spatial transcriptomics by analyzing cell embeddings from different layers within these tissues.

Reviewer #1 (Public review):

Summary:

The authors investigated the population structure of the invasive weed Lantana camara from 36 localities in India using 19,008 genome-wide SNPs obtained through ddRAD sequencing.

Strengths:<br /> The manuscript is well-written, the analyses are sound, and the figures are of great quality.

Weaknesses:

The narrative almost completely ignores the fact that this plant is popular in horticultural trade and the different color morphs that form genetic populations are most likely the result of artificial selection by humans for certain colors for trade, and not the result of natural selfing. Although it may be possible that the genetic clustering of color morphs is maintained in the wild through selfing, there is no evidence in this study to support that. The high levels of homozygosity are more likely explained as a result of artificial selection in horticulture and relatively recent introductions in India. Therefore, the claim of the title that "the population structure.. is shaped by its mating system" is in part moot, because any population structure is in large part shaped by the mating system of the organism, but further misleading because it is much more likely artificial selection that caused the patterns observed.

Reviewer #2 (Public review):

Summary:

The authors performed a series of population genetic analyses in Lantana camara using 19,008 genome-wide SNPs data from 359 individuals in India. They found a clear population structure that did not show a geographical pattern, and that flower color was rather associated with population structure. Excess of homozygosity indicates a high selfing rate, which may lead to fixation of alleles in local populations and explain the presence of population structure without a clear geographic pattern. The authors also performed a forward simulation analysis, theoretically confirming that selfing promotes fixation of alleles (higher Fst) and reduction in genetic diversity (lower heterozygosity).

Strengths:

Biological invasion is a critical driver of biodiversity loss, and it is important to understand how invasive species adapt to novel environments despite limited genetic diversity (genetic paradox of biological invasion). Lantana camara is one of the hundred most invasive species in the world (IUCN 2000), and the authors collected 359 plants from a wide geographical range in India, where L. camara has invaded. The scale of the dataset and the importance of the target species are the strengths of the present study.

Weaknesses:

One of the most critical weaknesses of this study would be that the output modelling analysis is largely qualitative, which cannot be directly comparable to the empirical data. The main findings of the SLiM-based simulation were that selfing promotes the fixation of alleles and the reduction of genetic diversity. These are theoretically well-reported knowledge, and such findings themselves are not novel, although it may have become interesting these findings are quantitatively integrated with their empirical findings in the studied species. In that sense, a coalescent-based analysis such as an Approximate Bayesian Computation method (e.g. DIY-ABC) utilizing their SNPs data would be more interesting. For example, by ABC-based methods, authors can infer the split time between subpopulations identified in this study. If such split time is older than the recorded invasion date, the result supports the scenario that multiple introductions may have contributed to the population structure of this species. In the current form of the manuscript, multiple introductions were implicated but not formally tested.

I also have several concerns regarding the authors' population genetic analyses. First, the authors removed SNPs that were not in Hardy-Weinberg equilibrium (HWE), but the studied populations would not satisfy the assumption of HWE, i.e., random mating, because of a high level of inbreeding. Thus, the first screening of the SNPs would be biased strongly, which may have led to spurious outputs in a series of downstream analyses. Second, in the genetic simulation, it is not clear how a set of parameters such as mutation rate, recombination rate, and growth rate were determined and how they are appropriate. Importantly, while authors assume the selfing rate in the simulation, selfing can also strongly influence the effective mutation rate (e.g. Nordborg & Donnelly 1997 Genetics, Nordborg 2000 Genetics). It is not clear how this effect is incorporated in the simulation. Third, while the authors argue the association between flower color and population structure, their statistical associations were not formally tested. Also, it is not mentioned how flower color polymorphisms are defined. Could it be possible to distinguish many flower color morphs shown in Figure 1b objectively? I am concerned particularly because the authors also mentioned that flower color may change temporally and that a single inflorescence can have flowers of different colors (L160).

Reviewer #1 (Public review):

The authors present tviblindi, an algorithm to infer cell development trajectories from single-cell molecular data. The paper is well-written and the algorithm is conceptually interesting. However, the validation is incomplete as the comparison against existing trajectory inference methods is weak: although the lack of a proper benchmark was pointed out as the main weakness of the original version of the manuscript, the revised version still only contains qualitative comparisons against state-of-the-art methods.

Both me and Reviewer 2 pointed out that the lack of a proper benchmark against state-of-the-art methods on a wider variety of datasets (including scRNA-seq data) was a major weakness of the original version of the manuscript. In response to this criticism, the authors now did the following:

- They ran various competitor methods on the datasets that were used already for the previous version of the manuscript.<br /> - They ran tviblindi and two of the competitors on two public scRNA-seq datasets.<br /> - For all datasets, they qualitatively assessed the trajectories computed by tviblindi and its competitors and argued that tviblindi's trajectories better reflect the biological signal in the data.<br /> - The results of all of these additional analyses are reported in the supplement, which has now become very lengthy (88 pages).

In my opinion, this is insufficient to establish that tviblindi is comparable or even superior to the state of the art in the field. To show that this is the case, the authors would have to carry out a systematic benchmark study which relies on quantitative evaluation metrics rather than on qualitative intepretations of trajectories. As method developers, we are all susceptive to confirmation bias when comparing our new algorithms to the state of the art. To avoid this pitfall, reporting quantitative performance metrics is required. At the moment, the only quantitative metric reported by the authors is runtime, which is insufficient.

Moreover, the results of a benchmark study should be reported in the main manuscript, not in the supplement. When presenting a new algorithm in a field as crowded as trajectory inference, a benchmark against the state of the art serves to establish trust in the new algorithm and to provide the readers with a rationale to use it for their research. For this, the results of the benchmark have to be presented prominently and should not be hidden in the supplement.

A second major criticism raised in Reviewer 2's review of the original version of the manuscript is that tviblindi invites cherry picking due to its inherently interactive design. In response to this, the authors now argue at length that "the data-driven expert interpretation approach of tviblindi" (quote from Section 2.2.2) is a strength rather than a weakness. If we concede for the sake of the argument that tviblindi's "expert interpretation approach" is indeed a strength of the method (although I tend to agree with Reviewer 2 that it is rather a limitation), usability for biologists becomes critical. However, given the current implementation of tviblindi, its usability is far from optimal. The authors do not provide tviblindi as a web interface that is directly usable for domain experts without programming experience and not even as a package that is installable via some widely used package manager such as conda. Instead, they implemented tviblindi as an R package with a Shiny GUI that can either run in a Docker container or requires the installation of several dependencies. I therefore strongly doubt that many biologists will be able or willing to run tviblindi, which substantially limits the value of its "expert interpretation approach". Moreover, tviblindi does not support Apple silicon, which prevented also myself from testing the tool.

Reviewer #1 (Public review):

Summary:

In this work, Huang et al. revealed the complex regulatory functions and transcription network of 172 unknown transcriptional factors (TFs) in Pseudomonas aeruginosa PAO1. They have built a global TF-DNA binding landscape and elucidated binding preferences and functional roles of these TFs. More specifically, the authors established a hierarchical regulatory network and identified ternary regulatory motifs, and co-association modules. Since P. aeruginosa is a well known pathogen, the authors thus identified key TFs associated with virulence pathways (e.g., quorum sensing [QS], motility, biofilm formation), which could be potential drug targets for future development. The authors also explored the TF conservation and functional evolution through pan-genome and phylogenetic analyses. For the easy searching by other researchers, the authors developed a publicly accessible database (PATF_Net) integrating ChIP-seq and HT-SELEX data.

Strengths:

(1) The authors performed ChIP-seq analysis of 172 TFs (nearly half of the 373 predicted TFs in P. aeruginosa) and identified 81,009 significant binding peaks, representing one of the largest TF-DNA interaction studies in the field. Also, The integration of HT-SELEX, pan-genome, and phylogenetic analyses provided multi-dimensional insights into TF conservation and function.

(2) The authors provided informative analytical Framework for presenting the TFs, where a hierarchical network model based on the "hierarchy index (h)" classified TFs into top, middle, and bottom levels. They identified 13 ternary regulatory motifs and co-association clusters, which deepened our understanding of complex regulatory interactions.

(3) The PATF_Net database provides TF-target network visualization and data-sharing capabilities, offering practical utility for researchers especially for the P. aeruginosa field.

Weaknesses:

(1) There is very limited experimental validation for this study. Although 24 virulence-related master regulators (e.g., PA0815 regulating motility, biofilm, and QS) were identified, functional validation (e.g., gene knockout or phenotypic assays) is lacking, leaving some conclusions reliant on bioinformatic predictions. Another approach for validation is checking the mutations of these TFs from clinical strains of P. aeruginosa, where chronically adapted isolates often gain mutations in virulence regulators.

(2) ChIP-seq in bacteria may suffer from low-abundance TF signals and off-target effects. The functional implications of non-promoter binding peaks (e.g., coding regions) were not discussed.

(3) PATF_Net currently supports basic queries but lacks advanced tools (e.g., dynamic network modeling or cross-species comparisons). User experience and accessibility remain underevaluated. But this could be improved in the future.

Achievement of Aims and Support for Conclusions

(1) The authors successfully mapped global P. aeruginosa TF binding sites, constructed hierarchical networks and co-association modules, and identified virulence-related TFs, fulfilling the primary objectives. The database and pan-genome analysis provide foundational resources for future studies.

(2) The hierarchical model aligns with known virulence mechanisms (e.g., LasR and ExsA at the bottom level directly regulating virulence genes). Co-association findings (e.g., PA2417 and PA2718 co-regulating pqsH) resonate with prior studies, though experimental confirmation of synergy is needed.

Impact on the Field and Utility of Data/Methods

(1) This study fills critical gaps in TF functional annotation in P. aeruginosa, offering new insights into pathogenicity mechanisms (e.g., antibiotic resistance, host adaptation). The hierarchical and co-association frameworks are transferable to other pathogens, advancing comparative studies of bacterial regulatory networks.

(2) PATF_Net enables rapid exploration of TF-target interactions, accelerating candidate regulator discovery.

Reviewer #3 (Public review):

Summary:

The authors utilized ChIP-seq on strains containing tagged transcription factor (TF)-overexpression plasmids to identify binding sites for 172 transcription factors in P. aeruginosa. High-quality binding site data provides a rich resource for understanding regulation in this critical pathogen. These TFs were selected to fill gaps in prior studies measuring TF binding sites in P. aeruginosa. The authors further perform a structured analysis of the resulting transcriptional regulatory network, focusing on regulators of virulence and metabolism, in addition to performing a pangenomic analysis of the TFs. The resulting dataset has been made available through an online database. While the implemented approach to determining functional TF binding sites has limitations, the resulting dataset still has substantial value to P. aeruginosa research.

Strengths:

The generated TF binding site database fills an important gap in regulatory data in the key pathogen P. aeruginosa. Key analyses of this dataset presented include an analysis of TF interactions and regulators of virulence and metabolism, which should provide important context for future studies into these processes. The online database containing this data is well organized and easy to access. As a data resource, this work should be of significant value to the infectious disease community.

Weaknesses:

Drawbacks of the study include 1) challenges interpreting binding site data obtained from TF overexpression due to unknown activity state of the TFs on the measured conditions, 2) limited practical value of the presented TRN topological analysis, and 3) lack of independent experimental validation of the proposed master regulators of virulence and metabolism.

Reviewer #1 (Public review):

Summary:

In their manuscript, the authors provide compelling evidence that stimulus-frequency otoacoustic emission (SFOAE) phase-gradient delays predict the sharpness (quality factors) of auditory-nerve-fiber (ANF) frequency tuning curves in budgerigars. In contrast with mammals, neither SFOAE- nor ANF-based measures of cochlear tuning match the frequency dependence of behavioral tuning in this species of parakeet. Although the reason for the discrepant behavioral results (taken from previous studies) remains unexplained, the present data provide significant and important support for the utility of otoacoustic estimates of cochlear tuning, a methodology previously explored only in mammals.

Strengths:

* The OAE and ANF data appear solid and believable. (The behavioral data are taken from previous studies and the resulting limitations are discussed.)

* No other study in birds (and only a single previous study in mammals) has combined behavioral, auditory-nerve, and otoacoustic estimates of cochlear tuning in a single species.

* SFOAE-based estimates of cochlear tuning were obtained by assuming that the tuning ratio estimated in chicken applies also to the budgerigar. Possible complications arising from an avian apical-basal transition analogous to that found in mammals are discussed.

Reviewer #2 (Public review):

Summary:

Earlier behavioral data in the budgerigar have suggested frequency selectivity that was different from that in many other avian species, showing particularly good selectivity at around 3-4 kHz. It was unknown whether this unusual selectivity was determined in the inner ear, or whether it was a more central adaptation. The results using direct auditory-nerve tuning curves and less invasive stimulus-frequency otoacoustic emissions, suggest fairly normal-looking cochlear tuning in the budgerigar, implying that any behavioral/perceptual differences in frequency selectivity are likely more central in original.

Strengths:

- The study presents novel data in budgerigar, comparing the bandwidths of auditory-nerve tuning curves with the latencies of stimulus-frequency otoacoustic emissions (SFOAEs), which are thought to reflect the sharpness of cochlear tuning.<br /> - Using a conversion factor taken from previous data in the chicken to avoid circularity of reasoning, the study shows quite good correspondence between the non-invasive estimates obtained from SFOAEs and the tuning obtained from auditory-nerve fibers. Similarity between budgerigar and chicken are harder to ascertain with the way the data are presented.

Weaknesses:

- The comparison of SFOAEs and auditory-nerve tuning curves in the most interesting regions (beyond 3.5 kHz, where some perceptual anomalies seem to occur in some previous data), relies on an extrapolation of the data from the chicken.<br /> - No new behavioral data are presented, so the comparisons made in the paper are between studies separated by decades. None of the behavioral studies cited used the more current techniques that have been claimed to provide a behavioral estimate of cochlear tuning.

Reviewer #2 (Public review):

Summary:

By combining bioinformatical and experimental approaches, the authors address the question why several vertebrate lineages lack specific genes of the necroptosis pathway, or those that regulate the interplay between apoptosis and necroptosis. The lack of such genes was already known from previous publications, but the current manuscript provides a more in-depth analysis and also uses experiments in human cells to address the question of functionality of the remaining genes and pathways. A particular focus is placed on RIPK3/RIPK1 and their dual roles in inducing NFkB and/or necroptosis.

Strengths:

The well documented bioinformatical analyses provide a comprehensive data basis of the presence/absence of RIP-kinases, other RHIM proteins, apoptosis signaling proteins (FADD,CASP8,CASP10) and some other genes involved in these pathway. Several of these genes are known to be missing in certain animal lineages, which raises the question why their canonical binding partners are present in these species. By expressing several such proteins (both wildtype and mutants destroying particular interaction regions) in human cells, the authors succeed in establishing a general role of RIPK3 and RIPK1 in NFkB activation. This function appears to be better conserved and more universal than the necroptotic function of the RHIM proteins. The authors also scrutinize the importance of the kinase function and RHIM integrity for these separate functionalities.

Weaknesses:

A weakness of the presented study is the experimental restriction to human HEK293 cells. There are several situations where the functionality of proteins from distant organisms (like lampreys or even mussels) in human cells is not necessarily indicative of their function in native context. In some cases, these problems are addressed by co-expressing potential interaction partners, but not all of these experiments are really informative. However, I agree with the authors that it is not possible to perform all the experiments in native cells, and that comparing all proteins in the same (human) cell type allows for a better comparison.

The conclusions drawn by the authors are supported by convincing evidence. I have no doubts that this study will be very useful for future studies addressing the evolution of necroptosis and its regulation by NFkB and apoptosis.

Reviewer #3 (Public review):

In this study, the authors employ both computational and experimental methods to reveal functional conservation of RIP family kinases and associated proteins in animals, with particular focus on mammals and other major groups of vertebrates. The bionformatic part of the work involves genomic data from diverse animal groups, providing insightful data on loss and duplications patterns for RIP and other necroptosis-related genes, and positive selection signals for RIPK1/3 genes in certain mammalian clades. These findings are then extensively used for selecting species and RHIM tetrad candidates for further experiments, in which the authors demonstrate different modes of functional conservation for RIPK proteins in necroptosis and NF-kB signaling across vertebrate species.

As an only major drawback, I would mention several important findings which the authors make in the course of their research but do not pursue further in the experimental part of the paper. These include:

• An additional copy for RIPK2 (RIPK2B) found in monotremes and non-mammalian vertebrates and its functions;<br /> • The entire diversity of RHIM functional tetrad variants; of particular interest here are IQFG and IQLG tetrads specific for bats, which are known to harbor human-affecting viruses and were demonstrated to have their RIPK1/3 genes under positive selection in this study;<br /> • Functions and involvement of RIPK3 protein in NF-kB pathway in lampreys;<br /> • The mode of NF-kB activation in non-mammalian species retaining ZBP1 copies.

Further elucidation of some or all of these points in the experimental part would facilitate conceptualizing the paper's numerous findings, which otherwise might appear insufficiently scrutinized. On the other hand, I agree that at least some of them require separate studies to be elucidated in. Given the importance of the results presented in this paper, I believe these points will be further addressed in future works.

Reviewer #1 (Public review):

Summary:

The authors have provided a mechanism by which how presence of truncated P53 can inactivate function of full length P53 protein. The authors proposed this happens by sequestration of full length P53 by truncated P53. In the study, the performed experiments are well described.

Significance:

The work in significant, since it points out more mechanistic insight how wild type full length P53 could be inactivated in the presence of truncated isoforms, this might offer new opportunity to recover P53 function as treatment strategies against cancer.

Comments on latest version:

The authors have made significant effort to address my concerns using the system available to them. I find the justifications provided in the rebuttal letter and the revised figures satisfactory. My initial concerns regarding the overexpression system have been largely addressed. However, the experimental system used by the authors lacks the means to measure the effect on endogenous p53, which remains a limitation.

Reviewer #2 (Public review):

Summary:

The revised manuscript by Zhao and colleagues presents a novel and compelling investigation into the p53 isoforms, Δ133p53 and Δ160p53, which are implicated in aggressive cancer phenotypes. The primary goal of this study was to elucidate how these isoforms exert a dominant-negative impact on the activity of full-length p53 (FLp53). The authors demonstrate that the Δ133p53 and Δ160p53 isoforms display impaired binding to p53-regulated promoters. Their findings suggest that the dominant-negative effects observed are primarily due to the co-aggregation of FLp53 with Δ133p53 and Δ160p53.

Overall, the study is innovative, thoroughly executed, and supported by robust data analysis. The authors have effectively addressed the reviewers' criticisms and incorporated their suggestions in this revised manuscript.

Significance:

The manuscript by Zhao and colleagues presents a novel and compelling study on the p53 isoforms, Δ133p53 and Δ160p53, which are associated with aggressive cancer types. The main objective of the study was to understand how these isoforms exert a dominant negative effect on full-length p53 (FLp53). The authors discovered that the Δ133p53 and Δ160p53 proteins exhibit impaired binding to p53-regulated promoters. The data suggest that the predominant mechanism driving the dominant-negative effect is the co-aggregation of FLp53 with Δ133p53 and Δ160p53.

Reviewer #1 (Public review):

Summary:

The concept that trained immunity, as defined, can be beneficial to subsequent immune challenges is important in the broad context of health and disease. The significance of this manuscript is the finding that trained immunity is actually a two-edged sword, herein, detrimental in the context of LPS-induced Acute Lung Injury that is mediated by AMs.

Strengths:

Several lines of evidence in different mouse models support this conclusion. The postulation that differences in immune responses in individuals is linked to differences in the mycobiome and consequent B-glucan makeup is provocative.

Weaknesses:

However, the findings that the authors state are relevant to sepsis are actually confined to a specific lung injury model and not classically-defined sepsis, the ontogeny of the reprogrammed AMs is uncertain, and links in the proposed signaling pathways need to be strengthened.

Comments on the latest version:

The manuscript is improved with further clarifications and additional experimentation. My prior concerns are addressed.

Reviewer #2 (Public review):

Summary:

Prével et al. present an in vivo study in which they reveal an interesting aspect of β-glucan, a known inducer of enhanced immune responses termed trained immunity in sterile inflammation. The authors can show that β-glucan's can reprogram alveolar macrophages (AMs) in the lungs through neutrophils and IFNγ signaling and independent of Dectin1. This reprogramming occurs at both transcriptional and metabolic levels. After β-glucan training, LPS induced sterile inflammation exacerbated acute lung injury via enhanced immunopathology. These findings highlight a new aspect of β-glucan's role in trained immunity and its potential detrimental effects when enhanced pathogen clearance is not required.

Strengths:

- This manuscript is well-written and effectively conveys its message.

- The authors provide important evidence that β-glucan training is not solely beneficial but depending on the context can also enhance immunopathology. This will be important to the field for two reasons. It shows again that trained immunity can also be harmful. Jentho et al. 2021 had already provided further evidence for this aspect. And it highlights anew that LPS application is an insufficient infection model.

Original weaknesses noted:

- Only a little physiological data from the in vivo models is provided.

- Effects in histology appear to be rather weak.

Comments on latest version:

The authors have revised the new version according to my suggestions or responded in a sufficient manner to my requests, with one exception. I recommend to rename TNF as explained by Grimstad in JAMA Dermatol. 2016;152(5):557.

Reviewer #1 (Public review):

Summary:

This manuscript presents a study on expectation manipulation to induce placebo and nocebo effects in healthy participants. The study follows standard placebo experiment conventions with the use of TENS stimulation as the placebo manipulation. The authors were able to achieve their aims. A key finding is that placebo and nocebo effects were predicted by recent experience, which is a novel contribution to the literature. The findings provide insights into the differences between placebo and nocebo effects and the potential moderators of these effects.

Specifically, the study aimed to:

(1) assess the magnitude of placebo and nocebo effects immediately after induction through verbal instructions and conditioning<br /> (2) examine the persistence of these effects one week later, and<br /> (3) identify predictors of sustained placebo and nocebo responses over time.

Strengths:

An innovation was to use sham TENS stimulation as the expectation manipulation. This expectation manipulation was reinforced not only by the change in pain stimulus intensity, but also by delivery of non-painful electrical stimulation, labelled as TENS stimulation.

Questionnaire-based treatment expectation ratings were collected before conditioning and after conditioning, and after the test session, which provided an explicit measure of participants' expectations about the manipulation.

The finding that placebo and nocebo effects are influenced by recent experience provides a novel insight into a potential moderator of individual placebo effects.

Weaknesses:

There are a limited number of trials per test condition (10), which means that the trajectory of responses to the manipulation may not be adequately explored.

On day 8, one stimulus per stimulation intensity (i.e., VAS 40, 60, and 80) was applied before the start of the test session to re-familiarise participants with the thermal stimulation. There is a potential risk of revealing the manipulation to participants during the re-familiarization process, as they were not previously briefed to expect the painful stimulus intensity to vary without the application of sham TENS stimulation.

The differences between the nocebo and control conditions in pain ratings during conditioning could be explained by the differing physiological effects of the different stimulus intensities, so it is difficult to make any claims about expectation effects here.

A randomisation error meant that 25 participants received an unbalanced number of 448 trials per condition (i.e., 10 x VAS 40, 14 x VAS 60, 12 x VAS 80).

Reviewer #2 (Public review):

Summary:

Kunkel et al aim to answer a fundamental question: Do placebo and nocebo effects differ in magnitude or longevity? To address this question, they used a powerful within-participants design, with a very large sample size (n=104), in which they compared placebo and nocebo effects - within the same individuals - across verbal expectations, conditioning, testing phase, and a 1-week follow-up. With elegant analyses, they establish that different mechanisms underlie the learning of placebo vs nocebo effects, with the latter being acquired faster and extinguished slower. This is an important finding for both the basic understanding of learning mechanisms in humans and for potential clinical applications to improve human health.

Strengths:

Beyond the above - the paper is well-written and very clear. It lays out nicely the need for the current investigation and what implications it holds. The design is elegant, and the analyses are rich, thoughtful, and interesting. The sample size is large which is highly appreciated, considering the longitudinal, in-lab study design. The question is super important and well-investigated, and the entire manuscript is very thoughtful with analyses closely examining the underlying mechanisms of placebo versus nocebo effects.

Weaknesses:

There were two highly addressable weaknesses in my opinion:

(1) I could not find the preregistration - this is crucial to verify what analyses the authors have committed to prior to writing the manuscript. Please provide a link leading directly to the preregistration - searching for the specified number in the suggested website yielded no results.

(2) There is a recurring issue which is easy to address: because the Methods are located after the Results, many of the constructs used, analyses conducted, and even the main placebo and nocebo inductions are unclear, making it hard to appreciate the results in full. I recommend finding a way to detail at the beginning of the results section how placebo and nocebo effects have been induced. While my background means I am familiar with these methods, other readers will lack that knowledge. Even a short paragraph or a figure (like Figure 4) could help clarify the results substantially. For example, a significant portion of the results is devoted to the conditioning part of the experiment, while it is unknown which part was involved (e.g., were temperatures lowered/increased in all trials or only in the beginning).

Reviewer #1 (Public review):

Summary:

Previous studies have shown that the MSH6 family of mismatch repair proteins contains an unstructured N-terminal domain that contains either a PWWP domain, a Tudor domain or neither and that the interaction of the histone reader domains with the appropriate histone H3 modification enhances mismatch repair, and hence reduces mutation rates in coding regions to some extent. However, the elimination of the MSH6-histone modification probably does not completely eliminate mismatch repair, although the published papers on this point do not seem definitive.

In this study, the authors perform a details phylogenetic analysis of the presence of the PWWP and Tudor domains in MSH6 proteins across the tree of life. They observe that there are basically three classes of organisms that contain either a PWWP domain, a Tudor domain, or neither. On the basis of their analysis, they suggest that this represents convergent evolution of the independent acquisition of histone reader domains and that key amino acid residues in the reader domains are selected for.

Strengths:

The phylogenetic aspects of the work seem well done and the basic evolutionary conclusions of the work are well supported. The basic evolutionary conclusions are interesting and there is little to criticize from my perspective.

Weaknesses:

A major concern about this paper is that the authors fail to put their work into the proper context of what is already known about the N-terminus of MSH6. Further, their structural studies, which are really structural illustrations, are misleading, often incorrect, and not always helpful in addition to having been published before.

Reviewer #2 (Public review):

Summary:

In this work, Monroe JG and colleagues show a compelling case of convergent evolution in the fusion between an important mismatch repair protein (MSH6) and histone reader domains across the tree of life. These fused MSH6 readers have been shown to be important for the recruitment of MSH6 to exon-rich genome locations, therefore improving the efficiency of reducing mutation rates in coding regions.

Comparative genomic analyses here performed revealed independent instances of MSH6 fusion with histone readers in plants and metazoa with several instances of putative loss (or gain) across the phylogeny. The work also unveiled instances of MSH6 fusion putatively interesting domains in fungi which might be worth exploring in the future.

The authors also show potential signatures of purifying selection in functional amino acids MSH6 histone readers.

Overall the approach is adequate for the questions proposed to be answered, the analyses are rigorous and support the authors' claims.

DNA repair genes are essential to maintain genome stability and fidelity, and alterations in these pathways have been associated with hypermutation phenotypes in the context for instance of cancer in humans, with sometimes implications in treatment resistance. This is an important work that contributes to our understanding of the evolutionary consequences of the evolution of epigenome-targeted DNA repair.

Strengths:

The methods used are adequate for the questions and support the results. The search for MSH6 fusions was rigorous and conservative, which strengthens the significance of the claims on the evolutionary history of these fusion events.

Weaknesses:

I did not identify any major weaknesses, but please see my suggestions/recommendations.

Reviewer #3 (Public review):

Summary:

In the manuscript entitled "Convergent evolution of epigenome recruited DNA repair across the Tree of Life", Monroe et al. investigate bioinformatically how some important mechanisms of epigenome-targeted DNA repair evolved at the tree of life scale. They provide a clear example of convergent evolution of these mechanisms between animals and plants, investigating more than 4000 eukaryotic genomes, and uncovering a significant association between gain/retention of such mechanisms with genome size and high intron content, that at least partially explains the evolutionary patterns observed within major eukaryotic lineages.

Strengths:

The manuscript is well written, clear, and understandable, and has potentially broad interest. It provides a thorough analysis of the evolution of MSH6-related DNA repair mechanisms using more than 4000 eukaryotic genomes, a pretty impressive number allowing to identify both large-scale (i.e. kingdoms) as well as shorter-scale (i.e. phyla, orders) evolutionary patterns. Moreover, despite providing no experimental validation, it investigates with a sufficient degree of depth, a potential relationship between gain/retention of epigenome recruited DNA repair mediated by MSH6 and genomic, as well as life-history (population size, body mass, lifespan), traits. In particular, it provides convincing evidence for a causative effect between genome size/intron content and the presence/absence of this mechanism. Moreover, it stimulates further scientific investigation and biological questions to be addressed, such as the conservation of epigenomes across the tree of life, the existence of potential trade-offs in gain/retention vs. loss of such mechanisms, and the relationship between these processes, mutation rate heterogeneity, and evolvability.

Weaknesses:

Despite the interesting and necessary insights provided on (1) the evolution of DNA repair mechanisms, and (2) the convergent evolution of molecular mechanisms, this bioinformatic study emanates from studies in humans and Arabidopsis already showing signs of potential convergent evolution in aspects of epigenome-recruited DNA repair. For this, this study, although bioinformatically remarkably thorough, does not come as a surprise, potentially lowering its novelty.

What could have increased further its impact, interest, and novelty could have been a more comprehensive understanding of the causative processes leading to gain/retention vs. loss of MSH6-related epigenetic recruitment mechanisms. The authors provide interesting associations with life-history traits (yet not significant), and significant links with genome size and intron content only at the theoretical level. For the first aspect, the analyses could have expanded toward other life-history traits. For the second, maybe it could have been even possible to tackle experimentally some of the generated questions, functionally in some models, or deepened using specific case studies.

Reviewer #1 (Public review):

Summary:

Is peristimulus alpha (8-14 Hz) frequency and/or phase involved in shaping the length of visual and audiovisual temporal binding windows, as posited by the discrete sampling hypothesis? If so, to what extent and perceptual scenario are they functionally relevant? The authors addressed such questions by collecting EEG data during the completion of the widely-known 2-flash fusion paradigm, administered both in a standard (i.e., visual only, F2) and audiovisual (i.e., 2 flashes and 1 beep, F2B1) fashion. Instantaneous frequency estimation performed over parieto-occipital sensors revealed slower alpha rhythms right after stimulus onset in the F2B1 condition, as compared to the F2, a pattern found to correlate with the difference between modality-specific ISIs (F2B1-F2). Of note, peristimulus alpha frequency differed also between 1 vs 2 flashes reports, although in the visual modality only (i.e., faster alpha oscillations in 2 flash percept vs 1 flash). This pattern of results was reinvigorated in a causal manner via occipital tACS, which was capable of, respectively, narrowing down vs enlarging the temporal binding window of individuals undergoing 13 Hz vs 8 Hz stimulation in the F2 modality alone. To elucidate what the oscillatory signatures of crossmodal integration might be, the authors further focused on the phase of posterior alpha rhythms. Accordingly, the Phase Opposition Sum proved to significantly differ between modalities (F2B1 vs F2) during the prestimulus time window, suggesting that audiovisual signals undergo finer processing based on the ongoing phase of occipital alpha oscillations, rather than the speed at which these rhythms cycle. As a last bit of information, a computational model factoring in the electrophysiological assumptions of both the discrete sampling hypothesis and auditory-induced phase-resetting was devised. Analyses run on such synthetic data were partially able to reproduce the patterns witnessed in the empirical dataset. While faster frequency rates broadly provide a higher probability to detect 2 flashes instead of 1, the occurrence of a concurrent auditory signal in cross-modal trials should cause a transient elongation (i.e. slower frequency rate) of the ongoing alpha cycle due to phase-reset dynamics (as revealed via inter-trial phase clustering), prompting larger ISIs during F2B1 trials. Conversely, the model provides that alpha oscillatory phase might predict how well an observer dissociates sensory information from noise (i.e., perceptual clarity), with the second flash clearly perceived as such as long as it falls within specific phase windows along the alpha cycle.

Strengths:

The authors leveraged complementary approaches (EEG, tACS, and computational modelling), the results thereof not only integrate, but depict an overarching mechanistic scenario elegantly framing phase-resetting dynamics into the broader theoretical architecture posited by the discrete sampling hypothesis. Analyses on brain oscillations (either via frequency sliding and phase opposition sum) mostly appear to be methodologically sound, and very-well supported by tACS results. Under this perspective, the modelling approach serves as a convenient tool to reconcile and shed more light on the pieces of evidence gathered on empirical data, returning an appealing account on how cross-modal stimuli interplay with ongoing alpha rhythms and differentially affect multisensory processing in humans.

Weaknesses:

Some information relative to the task and the analyses is missing. For instance, it is not entirely clear from the text what the number of flashes actually displayed in explicit short trials is (1 or 2?). We believe it is always two, but it should be explicitly stated.

Moreover, the sample size might be an issue. As highlighted by a recent meta-analysis on the matter (Samaha & Romei, 2024), an underpowered sample size may very well drive null-findings relative to tACS data in F2B1 trials, in interplay with broad and un-individualized frequency targets.

Some criticality arises regarding the actual "bistability" of bistable trials, as the statistics relative to the main task (i.e., the actual means and SEMs are missing) broadly point toward a higher proclivity to report 2 instead of 1 flash in both F2B1 and F2 trials. This makes sense to some extent, given that 2 flashes have always been displayed (at least in bistable trials), yet tells about something botched during the pretest titration procedure.

Coming to the analyses on brain waves, one main concern relates to the phase-reset-induced slow-down of posterior alpha rhythms being of true oscillatory nature, rather than a mere evoked response (i.e., not sustained over time). Another question calling for some further scrutiny regards the overlooked pattern linking the temporal extent of the IAF differences between F2 and F2B1 trials with the ISIs across experimental conditions (explicit short, bistable, and explicit long). That is, the wider the ISI, the longer the temporal extent of the IAF difference between sensory modalities. Although neglected by the authors, such a trend speaks in favour of a rather nuanced scenario stemming from not only auditory-induced phase-reset alpha cycle elongation, but also some non-linear and perhaps super-additive contribution of flash-induced phase-resetting. This consideration introduces some of the issues about the computational simulation, which was modelled around the assumption of phase-resetting being triggered by acoustic stimuli alone. Given how appealing the model already is, I wonder whether the authors might refine the model accordingly and integrate the phase-resetting impact of visual stimuli upon synthetic alpha rhythms. Relatedly, I would also suggest the authors to throw in a few more simulations to explore the parameter space and assay, to which quantitative extent the model still holds (e.g. allowing alpha frequency to randomly change within a range between 8 and 13 Hz, or pivoting the phase delay around 10 or 50 ms). As a last remark, I would avoid, or at least tone down, concluding that the results hereby presented might reconcile and/or explain the null effects in Buergers & Noppeney, 2022; as the relationship between IAFs and audiovisual abilities still holds when examining other cross-modal paradigms such as the Sound-Induced Flash-Illusion (Noguchi, 2022), and the aforementioned patterns might be due to other factors, such as a too small sample size (Samaha & Romei, 2024).

Reviewer #2 (Public review):

Summary:

The authors used a visual flash discrimination task in which two flashes are presented one after another with different inter-stimulus intervals. Participants either perceive one flash or two flashes. The authors show that the simultaneous presence of an auditory input extends the temporal window of integration, meaning that two flashes presented shortly after one another are more likely to be perceived as a single flash. Auditory inputs are accompanied by a reduction in alpha frequency over visual areas. Prestimulus alpha frequency predicts perceptual outcomes in the absence of auditory stimuli, whereas prestimulus alpha phase becomes the dominant predictor when auditory input is present. A computational model based on phase-resetting theory supports these findings. Additionally, a transcranial stimulation experiment confirms the causal role of alpha frequency in unimodal visual perception but not in cross-modal contexts.

Strengths:

The authors elegantly combined several approaches-from behavior to computational modeling and EEG-to provide a comprehensive overview of the mechanisms involved in visual integration in the presence or absence of auditory input. The methods used are state-of-the-art, and the authors attempted to address possible pitfalls.

Weaknesses:

The use of Bayesian statistics could further strengthen the paper, especially given that a few p-values are close to the significance threshold (lines 162 & 258), but they are interpreted differently in different cases (absence of effect vs. trend).

Overall, these results provide new insights into the role of alpha oscillations in visual processing and offer an interesting perspective on the current debate regarding the roles of alpha phase and frequency in visual perception. More generally, they contribute to our understanding of the neural dynamics of multisensory integration.

Reviewer #3 (Public review):

Summary:

The authors investigated the impact of an auditory stimulus on visual integration at the behavioral, electrophysiological, and mechanistic levels. Although the role of alpha brain oscillations on visual perception has been widely studied, how the brain dynamics in the visual cortices are influenced by a cross-modal stimulus remains ill-defined. The authors demonstrated that auditory stimulation systematically induced a drop in visual alpha frequency, increasing the time window for audio-visual integration, while in the unimodal condition, visual integration was modulated by small variations within the alpha frequency range. In addition, they only found a role of the phase of alpha brain oscillations on visual perception in the cross-modal condition. Based on the perceptual cycles' theory framework, the authors developed a model allowing them to describe their results according to a phase resetting induced by the auditory stimulation. These results showed that the influence of well-known brain dynamics on one modality can be disrupted by another modality. They provided insights into the importance of investigating cross-modal brain dynamics, and an interesting model that extends the perceptual cycle framework.

Strengths:

The results are supported by a combination of various, established experimental and analysis approaches (e.g., two-flash fusion task, psychometric curves, phase opposition), ensuring strong methodological bases and allowing direct comparisons with related findings in the literature.

The model the authors proposed is an extension and an improvement of the perceptual cycle's framework. Interestingly, this model could then be tested in other experimental approaches.

Weaknesses:

There is an increasing number of studies in cognitive neuroscience showing the importance of considering inter-individual variability. The individual alpha frequency (IAF) varied from 8 to 13 Hz with a huge variability across participants, and studies have shown that the IAF influenced visual perception. Investigating inter-individual variations of the IAF in the reported results would be of great interest, especially for the model.

Although the use of non-invasive brain stimulation to infer causality is a method of great interest, the use of tACS in the presented work is not optimal. Instead of inducing alpha brain oscillations in visual cortices, the use of tACS to activate the auditory cortex instead of the actual auditory stimulation would have presented more interest.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors aimed to test the ability of bumblebees to use bird-view and ground-view for homing in cluttered landscapes. Using modelling and behavioural experiments, the authors showed that bumblebees rely most on ground-views for homing.

Strengths:

The behavioural experiments are well-designed, and the statistical analyses are appropriate for the data presented. 

Weaknesses:

Views of animals are from a rather small catchment area.

Missing a discussion on why image difference functions were sufficient to explain homing in wasps (Murray and Zeil 2017).

The artificial habitat is not really 'cluttered' since landmarks are quite uniform, making it difficult to infer ecological relevance.

Reviewer #2 (Public Review):

Summary:

In a 1.5m diameter, 0.8m high circular arena bumblebees were accustomed to exiting the entrance to their nest on the floor surrounded by an array of identical cylindrical landmarks and to forage in an adjacent compartment which they could reach through an exit tube in the arena wall at a height of 28cm. The movements of one group of bees were restricted to a height of 30cm, the height of the landmark array, while the other group was able to move up to heights of 80cm, thus being able to see the landmark array from above.

During one series of tests, the flights of bees returning from the foraging compartment were recorded as they tried to reach the nest entrance on the floor of the arena with the landmark array shifted to various positions away from the true nest entrance location. The results of these tests showed that the bees searched for the net entrance in the location that was defined by the landmark array.

In a second series of tests, access to the landmark array was prevented from the side, but not from the top, by a transparent screen surrounding the landmark array. These tests showed that the bees of both groups rarely entered the array from above, but kept trying to enter it from the side.<br /> The authors express surprise at this result because modelling the navigational information supplied by panoramic snapshots in this arena had indicated that the most robust information about the location of the nest entrance within the landmark array was supplied by views of the array from above, leading to the following strong conclusions:<br /> line 51: "Snapshot models perform best with bird's eye views";<br /> line 188: "Overall, our model analysis could show that snapshot models are not able to find home with views within a cluttered environment but only with views from above it.";<br /> line 231: "Our study underscores the limitations inherent in snapshot models, revealing their inability to provide precise positional estimates within densely cluttered environments, especially when compared to the navigational abilities of bees using frog's-eye views."

Strengths:

The experimental set-up allows for the recording of flight behaviour in bees, in great spatial and temporal detail. In principle, it also allows for the reconstruction of the visual information available to the bees throughout the arena.

Weaknesses:

Modelling:<br /> Modelling left out information potentially available to the bees from the arena wall and in particular from the top edge of the arena and cues such as cameras outside the arena. For instance, modelled IDF gradients within the landmark array degrade so rapidly in this environment, because distant visual features, which are available to bees, are lacking in the modelling. Modelling furthermore did not consider catchment volumes, but only horizontal slices through these volumes.

Behavioural analysis:<br /> The full potential of the set-up was not used to understand how the bees' navigation behaviour develops over time in this arena and what opportunities the bees have had to learn the location of the nest entrance during repeated learning flights and return flights.

Without a detailed analysis of the bees' behaviour during 'training', including learning flights and return flights, it is very hard to follow the authors' conclusions. The behaviour that is observed in the tests may be the result of the bees' extended experience shuttling between the nest and the entry to the foraging arena at 28cm height in the arena wall. For instance, it would have been important to see the return flights of bees following the learning flights shown in Figure 17.

Basically, both groups of bees (constrained to fly below the height of landmarks (F) or throughout the height of the arena (B)) had ample opportunities to learn that the nest entrance lies on the floor of the landmark array. The only reason why B-bees may not have entered the array from above when access from the side was prevented, may simply be that bumblebees, because they bumble, find it hard to perform a hovering descent into the array.

General:

The most serious weakness of the set-up is that it is spatially and visually constrained, in particular lacking a distant visual panorama, which under natural conditions is crucial for the range over which rotational image difference functions provide navigational guidance. In addition, the array of identical landmarks is not representative of natural clutter and, because it is visually repetitive, poses un-natural problems for view-based homing algorithms. This is the reason why the functions degrade so quickly from one position to the next (Figures 9-12), although it is not clear what these positions are (memory0-memory7).<br /> In conclusion, I do not feel that I have learnt anything useful from this experiment; it does suggest, however, that to fully appreciate and understand the homing abilities of insects, there is no alternative but to investigate these abilities in the natural conditions in which they have evolved.

Reviewer #1 (Public review):

Summary:

This paper tackles an important question: What drives the predictability of pre-stimulus brain activity? The authors challenge the claim that "pre-onset" encoding effects in naturalistic language data have to reflect the brain predicting the upcoming word. They lay out an alternative explanation: because language has statistical structure and dependencies, the "pre-onset" effect might arise from these dependencies, instead of active prediction. The authors analyze two MEG datasets with naturalistic data.

Strengths:

The paper proposes a very reasonable alternative hypothesis for claims in prior work. Two independent datasets are analyzed. The analyses with the most and least predictive words are clever, and nicely complement the more naturalistic analyses.

Weaknesses:

I have to admit that I have a hard time understanding one conceptual aspect of the work, and a few technical aspects of the analyses are unclear to me. Conceptually, I am not clear on why stimulus dependencies need to be different from those of prediction. Yes, it is true that actively predicting an upcoming word is different from just letting the regression model pick up on stimulus dependencies, but given that humans are statistical learners, we also just pick up on stimulus dependencies, and is that different from prediction? Isn't that in some way, the definition of prediction (sensitivity to stimulus dependencies, and anticipating the most likely upcoming input(s))?

This brings me to some of the technical points: If the encoding regression model is learning one set of regression weights, how can those reflect stimulus dependencies (or am I misunderstanding which weights are learned)? Would it help to fit regression models on for instance, every second word or something (that should get rid of stimulus dependencies, but still allow to test whether the model predicts brain activity associated with words)? Or does that miss the point? I am a bit unclear as to what the actual "problem" with the encoding model analyses is, and how the stimulus dependency bias would be evident. It would be very helpful if the authors could spell out, more explicitly, the precise predictions of how the bias would be present in the encoding model.

Reviewer #2 (Public review):

Summary:

At a high level, the reviewers demonstrate that there is an explanation for pre-word-onset predictivity in neural responses that does not invoke a theory of predictive coding or processing. The paper does this by demonstrating that this predictivity can be explained solely as a property of the local mutual information statistics of natural language. That is, the reason that pre-word onset predictivity exists could simply boil down to the common prevalence of redundant bigram or skip-gram information in natural language.

Strengths:

The paper addresses a problem of significance and uses methods from modern NeuroAI encoding model literature to do so. The arguments, both around stimulus dependencies and the problems of residualization, are compellingly motivated and point out major holes in the reasoning behind several influential papers in the field, most notably Goldstein et al. This result, together with other papers that have pointed out other serious problems in this body of work, should provoke a reconsideration of papers from encoding model literature that have promoted predictive coding. The paper also brings to the forefront issues in extremely common methods like residualization that are good to raise for those who might be tempted to use or interpret these methods incorrectly.

Weaknesses:

The authors don't completely settle the problem of whether pre-word onset predictivity is entirely explainable by stimulus dependencies, instead opting to show why naive attempts at resolving this problem (like residualization) don't work. The paper could certainly be better if the authors had managed to fully punch a hole in this.

Reviewer #3 (Public review):

Summary:

The study by Schönmann et al. presents compelling analyses based on two MEG datasets, offering strong evidence that the pre-onset response observed in a highly influential study (Goldstein et al., 2022) can be attributed to stimulus dependencies, specifically, the auto-correlation in the stimuli-rather than to predictive processing in the brain. Given that both the pre-onset response and the encoding model are central to the landmark study, and that similar approaches have been adopted in several influential works, this manuscript is likely to be of high interest to the field. Overall, this study encourages more cautious interpretation of pre-onset responses in neural data, and the paper is well written and clearly structured.

Strengths:

(1) The authors provide clear and convincing evidence that inherent dependencies in word embeddings can lead to pre-activation of upcoming words, previously interpreted as neural predictive processing in many influential studies.

(2) They demonstrate that dependencies across representational domains (word embeddings and acoustic features) can explain the pre-onset response, and that these effects are not eliminated by regressing out neighboring word embeddings - an approach used in prior work.

(3) The study is based on two large MEG datasets, showing that results previously observed in ECoG data can be replicated in MEG. Moreover, the stimulus dependencies appear to be consistent across the two datasets.

Weaknesses:

(1) To allow a more direct comparison with Goldstein et al., the authors could consider using their publicly available dataset.

(2) Goldstein et al. already addressed embedding dependencies and showed that their main results hold after regressing out the embedding dependencies. This may lessen the impact of the concerns about self-dependency raised here.

(3) While this study shows that stimulus dependency can account for pre-onset responses, it remains unclear whether this fully explains them, or whether predictive processing still plays a role. The more important question is whether pre-activation remains after accounting for these confounds.

Reviewer #1 (Public review):

Summary:

This fMRI study shows that two regions of the visual cortex (BA18 and BA19) of blind and sighted individuals carry information about the physical similarity of objects denoted by words. This effect was found for written words (Braille in blind, visual in sighted) but not spoken words. The evidence complements earlier studies reporting physical similarity effects in the occipitotemporal cortex of blind and sighted individuals (e.g., Peelen et al., 2014).

Strengths:

The study addresses an important question in the fields of neural plasticity and visual cortex organization. The study is generally well-conducted and the findings are clearly presented.

Weaknesses:

While the evidence is statistically strong, it is currently incomplete because of missing control analyses (see below). The framing of the results, as arguing against the pluripotent cortex account, is not entirely convincing as it was not clear that the study addressed the key predictions of that account.

Main comments:

(1) The study is framed as a test of Bedny's "cognitively pluripotent cortex" proposal (2017) that attributes the increased visual cortex response to linguistic stimuli in blind individuals to high-level cognitive functions. Key evidence for this account came from studies showing increased responses in blind visual cortex to certain grammatical manipulations and to solving mathematical equations. The current study did not include such manipulations. Instead, the current study focused on the representation of objects denoted by single words. Bedny's account did not make a strong argument that the physical similarity of word referents should be differently represented in blind and sighted individuals - if it did, please state this explicitly. Indeed, evidence that (some regions of) the visual cortex represent objects similarly in blind and sighted individuals does not seem incompatible with it.

(2) Throughout the manuscript (including the abstract) it was not clear what was meant with "visual cortex" or "visual areas"; whether this refers to early visual cortex (V1/BA17) or to visual cortex more generally (e.g., BA17-BA19, occipitotemporal cortex (MT, etc)). This is important for the theoretical arguments and for the interpretation of the results. If visual cortex = BA17, the current results point to potentially important differences between blind and sighted individuals, with the physical similarity of objects only observed in the visual cortex of the blind. If visual cortex is meant to include areas beyond BA17, the blind and sighted show similarities in the current study, although such similarities have been observed before using similar research approaches.

(3) Related to the point above, the abstract does not accurately describe the results, as it only describes the similarities between blind and sighted but not the differences. The study revealed differences between groups, particularly in BA17 - primary visual cortex. The differences between the groups are also illustrated by the strikingly different searchlight results in the two groups separately (Figure S6). These differences do not reach significance in a whole-brain-corrected contrast, but that likely reflects a lack of power (particularly for a between-group contrast).

(4) Results were found for written words but not spoken words (Figure S9). This is somewhat surprising considering that the visual cortex was more strongly activated for written words in the sighted, with this activation presumably not adding any information about the physical properties of word referents. Together with the widespread significance of clusters correlating with the physical similarity matrix (Figure 6), this raises the possibility of a confound. It would be good to ensure that this is not the case, e.g., you could create similarity matrices based on word length, word visual similarity (e.g., overlap in letters), and word frequency, and correlate these matrices with the physical similarity matrix to ensure that these correlations are not positive (or if they are, partial it out).

(5) The study included a task manipulation, with participants either judging physical or conceptual properties. This task manipulation is a central aspect of the design but does not feature anywhere in the results, and is also not discussed or introduced in the text. It would be interesting to know whether the results depend on the property (physical/conceptual) being task-relevant. But more importantly, a potential concern is that the responses in the task (given for each object using a two-response button box) correlate with physical or conceptual similarity and that this explains the fMRI findings. For example, two objects that are elongated would both receive a "yes" button press when participants answer the question "is this elongated"; these objects would also be rated as physically similar. This may apply more to physical than conceptual similarity. To exclude this possibility, the responses need to be analysed and included in the fMRI analyses, either as a regressor in the GLM or as another matrix to be partialed out at the final stage of analysis.

(4) Many of the blind participants had some residual vision (9/20 had light perception, 2/20 had contour perception); this could possibly have prevented the reorganization of visual cortex.

Reviewer #2 (Public review):

Summary:

The authors show, through rigorous and extensive analyses, that the visual cortex in both congenitally blind and sighted participants represented differences between individual words presented across sensory modalities. In both groups, the activation patterns for words in the visual cortex reflected physical, but not conceptual similarity between word referents. This suggests a similar representation for both groups of words, one derived from vision-oriented mechanisms, and does not reflect significant functional reorganization in blindness.

Strengths:

The theoretical question is sound, as is the analysis approach. The authors' literature discussion is thorough, and the writing is clear.

Weaknesses:

I have only minor concerns left open.

(1) In the representational connectivity analysis, what is the average value across the brain? The authors compare the representational correlation across brain regions to the average value, but the average itself is not reported.

(2) Can the authors add a map showing the representational connectivity values across the brain in addition to the bar plot? It would make it easier to see what networks show similar neural representation to the visual cortex.

(3) Are the participants in the behavioral experiment from which the physical and conceptual similarity between word referents were collected matching in age or education with the fMRI participants?

(4) Although there are no group differences in the correlation of the physical similarity, I think it is important to acknowledge that the effect is only significant at the searchlight level in the blind early visual cortex (Figure S6).

Reviewer #3 (Public review):

Summary:

This study examines semantic processing in the visual cortex of both congenitally blind and sighted individuals using fMRI and multivariate pattern analysis (MVPA). The key finding is that the visual cortex in both groups encodes the physical properties of word referents, rather than their conceptual similarities. These results suggest that the same representational mechanisms operate in both the blind and sighted brain.

Strengths:

(1) The findings contribute to a broader understanding of cortical reorganization and provide evidence for top-down processing of word referents, even in the absence of visual experience.

(2) The experiment incorporates both spoken and written word presentations (Braille for blind participants), ensuring that the results are not confounded by modality effects.

(3) The study employs a rigorous methodological approach, combining multivariate and univariate analyses to strengthen the validity of its findings.

(4) The paper is well-structured and clearly written, making it easy to follow.

Weaknesses:

(1) The word stimuli consists of only 20 nouns referring to concrete entities. However, in the behavioral experiment, participants rated the physical and conceptual similarity of only 30 word pairs, which represents just a subset of all possible word pair combinations. The average similarity ratings across subjects were then used to construct stimuli similarity matrices, which were correlated with the fMRI similarity matrices in the MVPA analysis. What is the rationale for presenting only a small subset of all possible word pair combinations to participants? Additionally, the instruction to rate the "conceptual similarity" of word pairs seems somewhat ambiguous. Would "conceptual similarity" correlate with "physical similarity"? Instead of subjective ratings, why not use cosine similarity scores from pretrained language models to construct the "conceptual similarity" matrices? This approach could provide a more objective and reproducible measure of conceptual similarity.

(2) There are only six questions each for assessing the physical and conceptual properties of the words in the fMRI experiment. Most of the physical property questions focus on shape-related attributes (e.g., round, angular, elongated, symmetrical), while the conceptual properties are limited to three pairs of antonyms (living/non-living, natural/manufactured, pleasant/unpleasant). These aspects seem insufficient to comprehensively characterize the physical and conceptual properties of the nouns. What was the rationale behind selecting only these six questions? Could this limited set of attributes introduce bias in how the neural representations in the visual cortex are interpreted?

(3) Two of the blind participants are right-handed, and two may have some form of contour vision. What was the rationale for including these participants? In addition, the sample size for blind participants is relatively small (N = 20). Does the sample size provide sufficient justification for the main conclusion that the visual cortex in both blind and sighted groups represents the physical properties of word referents? Additionally, could individual differences among blind participants impact the results, and were any analyses conducted to account for such variability?

(4) I appreciate the authors' effort to integrate both univariate and multivariate approaches in their analyses. However, the results appear somewhat contradictory: The MVPA results suggest similar neural representations of word referents in the visual cortex for both blind and sighted participants. However, the univariate analyses indicate higher activation in the visual cortex of blind participants. How can these two findings be reconciled? The authors attributed the increased activation in the visual cortex of blind participants to their "enhanced excitability", but what exactly does "excitability" mean in this context? Could this increased activation instead reflect an alternative neural strategy for processing semantic information in the blind brain? If so, how does this align with the claim that similar representational mechanisms exist in both blind and sighted individuals?

(5) The authors interpret their findings to suggest that the visual cortex can represent the physical properties of words even without visual experience, attributing this to top-down modulation from higher cognitive regions, which then backprojects to the visual cortex. However, it is unclear why only physical properties, and not conceptual properties, are backprojected. If higher cognitive regions modulate the visual cortex in a top-down manner, wouldn't both physical and conceptual attributes be expected to influence its activity? Could the authors clarify the mechanism that selectively supports physical property encoding over conceptual representation?

Reviewer #1 (Public review):

Summary:

It is known that the nrp operon is induced by copper deprivation and encodes the synthesis of chalkophores. The authors carried out a genetic analysis that revealed transcriptional differences for WT and Mtb∆nrp when exposed to the copper chelator tetrathiomolybdate (TTM). The authors found that copper chelation results in upregulation of genes in the chalkophore cluster as well as genes involved in the respiratory chain: including, components of the heme-dependent oxidase CytBD and subunits of the bcc:aa3 heme-copper oxidase. Utilizing several knockout variants and inhibitors, the authors showed that copper starvation survival requires chalkophore synthesis and that copper starvation results in dysfunctional bcc:aa3 oxidase. By monitoring oxygen consumption, they go on to show that copper deprivation inhibits respiration through the bcc:aa3 oxidase. Lastly, the authors compare virulence of WT Mtb, Mtb∆nrp and MtbΔnrpΔcydAB strains in mice spleen and lung. The Mtb∆nrp strain showed mild attenuation, but virulence in MtbΔnrpΔcydAB was severely attenuated and complementation with the chalkophore biosynthetic pathway restored Mtb virulence. These results suggest that chalkophore mediated protection of the respiratory chain is critical to Mtb virulence, and that redundant respiratory oxidases within Mtb provide respiratory chain flexibility that may promote host adaptation.

This new information about Mtb biology may be leveraged for drug discovery, highlighting that the Mtb respiratory pathway is a promising drug target, where one may target the Mtb chalkophore biosynthetic pathway in conjunction with CytBD, to obliterate Mtb.

Strengths: Overall, the paper is very clear and well written, with thorough and well-thought-out experimentation.

No weaknesses.

Comments on revisions:

The authors have addressed all the reviewers' comments.

Reviewer #2 (Public review):

Summary:

This is a well-written manuscript that clearly demonstrates that the nrp encoded diisonitrile chalkophore is necessary for function of the bcc-aa3 oxidase supercomplex under low copper conditions. In addition, the study demonstrates the chlakophore is important early during infection when copper sequestration is employed by the host as a method of nutritional immunity.

Strengths:

The authors use genetic approaches, including single and double mutants of chalkophore biosynthesis, and both the Mtb oxidases. Use a copper chelators to restrict copper in vitro. A strength of the work was the use of a synthesized a Mtb chalkophore analogue to show chemical complementation of the mutant nrp locus. Oxphos metabolic activity was measured by oxygen consumption and ATP levels. Importantly, the study demonstrated that chalkophore, especially in a strain lacking the secondary oxidase, was necessary for early infection and ruled out a role for adaptive immunity in the chalkophore lacking Mtb by use of SCID mice. It is interesting that after two weeks of infection and onset of adaptive immunity the chalkophore is not required, which is consistent with the host environment switching from a copper restricted to copper overload in phagosomes.

Weaknesses:

None noted

Reviewer #3 (Public review):

Summary:

In this manuscript, the group of Glickman expand on their previous studies on the function of chalkophores during growth of and infection by Mycobacterium tuberculosis. Previously, the group had shown that chalkophores, which are metallophores specific for the scavenging of copper, are induced by M. tuberculosis under copper deprivation conditions. Here, they show that chalkophores, under copper limiting conditions, are essential for the uptake of copper and maturation of a terminal oxidase, the heme-copper oxidase, cytochrome bcc:aa3. As M. tuberculosis has two redundant terminal oxidases, growth of and infection by M. tuberculosis is only moderated if both the chalkophores and the second terminal oxidase, cytochrome bd, are inhibited.

Strengths:

A strength of this work is that the lab-culture experiments are complemented with mice infection models, providing strong indications that host-inflicted copper deprivation is a condition that M. tuberculosis has adapted to for virulence.

Weaknesses:

Because the phenotype of M. tuberculosis lacking chalkophores is similar, if not identical, to using Q203, an inhibitor of cytochrome bcc:aa3, the authors propose that the copper-containing cytochrome bcc:aa3 is the only recipient of copper-uptake by chalkophores. A minor weakness of the work is that this latter conclusion is not verified under infection conditions and other copper-enzymes might still be functionally required during one or more stages of infection.

Comments on revisions:

I thank the authors for carefully addressing my suggestion to the original submission and congratulate them on their work.

Reviewer #1 (Public review):

In this paper, the authors had 2 aims:

(1) Measure macaques' aversion to sand and see if its' removal is intentional, as it likely in an unpleasurable sensation that causes tooth damage.

(2) Show that or see if monkeys engage in suboptimal behavior by cleaning foods beyond the point of diminishing returns, and see if this was related to individual traits such as sex and rank, and behavioral technique.

They attempted to achieve these aims through a combination of geochemical analysis of sand, field experiments, and comparing predictions to an analytical model.

The authors' conclusions were that they verified a long-standing assumption that monkeys have an aversion to sand as it contains many potentially damaging fine grained silicates, and that removing it via brushing or washing is intentional.

They also concluded that monkeys will clean food for longer than is necessary, i.e. beyond the point of diminishing returns, and that this is rank-dependent.

High and low-ranking monkeys tended not to wash their food, but instead over-brushed it, potentially to minimize handling time and maximize caloric intake, despite the long-term cumulative costs of sand.

This was interpreted through the *disposable soma hypothesis*, where dominants maximize immediate needs to maintain rank and increase reproductive success at the potential expense of long-term health and survival.

Strengths:

The field experiment seemed well designed, and their quantification of the physical and mineral properties of quartz particles (relative to human detection thresholds) seemed good relative to their feret diameter and particle circularity (to a reviewer that is not an expert in sand). The *Rank Determination* and *Measuring Sand* sections were clear.

In achieving Aim 1, the authors validated a commonly interpreted, but unmeasured function, of macaque and primate behavior-- a key study/finding in primate food processing and cultural transmission research.

I commend their approach in trying to develop a quantitative model to generate predictions to compare to empirical data for their second aim.<br /> This is something others should strive for.

I really appreciated the historical context of this paper in the introduction and found it very enjoyable and easy to read.

I do think that interpreting these results in the context of the *disposable soma hypothesis* and the potential implications in the *paleolithic matters* section about interpreting dental wear in the fossil record are worthwhile.

Reviewer #1 (Public review):

Summary:

In this manuscript, Muramoto and colleagues have examined a mechanism by which the executioner caspase Drice is activated in a non-lethal context in Drosophila. The authors have comprehensively examined this in the Drosophila olfactory receptor neurons using sophisticated techniques. In particular, they had to engineer a new reporter by which non-lethal caspase activation could be detected. The authors conducted a proximity labeling experiment and identified Fasciclin 3 as a key protein in this context. While removal of Fascilin 3 did not block non-lethal caspase activation (likely because of redundant mechanisms), its overexpression was sufficient to activate non-lethal caspase activation.

Strengths:

While non-lethal functions of caspases have been reported in several contexts, far less is known about the mechanisms by which caspases are activated in these non-lethal contexts. So, the topic is very timely. The overall detail of this work is impressive and the results, for the most part, are well controlled and justified.

Weaknesses:

The behavioral results shown in Fig. 6 need more explanation and clarification (more details below). As currently shown, the results of Fig. 6 seem uninterpretable. Also, overall presentation of the Figures and description in legends can be improved.

Comments on revisions:

The authors have adequately addressed my comments.

Reviewer #2 (Public review):

In this revised version of the study, the authors investigate the role of caspases in neuronal modulation through non-lethal activation. They analyze proximal proteins of executioner caspases using a variety of techniques, including TurboID and a newly developed monitoring system based on Gal4 manipulation, called MASCaT. They demonstrate that overexpression of Fas3G promotes the non-lethal activation of caspase Dronc in olfactory receptor neurons. In addition, they investigate the regulatory mechanisms of non-lethal function of caspase by performing a comprehensive analysis of proximal proteins of executioner caspase Drice. It is important to point out that the authors use an array of techniques from western blot to behavioral experiments and also that the generated several reagents, from fly lines to antibodies. In this revised version of the manuscript the authors addressed the concerns raised by this reviewer in a very thorough way. This is an interesting work that would appeal to readers of multiple disciplines. As a whole these findings suggest that overexpression of Fas3G enhances a non-lethal caspase activation in ORNs, providing a novel experimental model that will allow for exploration of molecular processes that facilitate caspase activation without leading to cell death.

Comments on revisions:

I would like to thank the authors for fully addressing my concerns.

Reviewer #2 (Public review):

Summary:

In a 1.5m diameter, 0.8m high circular arena bumblebees were accustomed to exit the entrance to their nest on the floor surrounded by an array of identical cylindrical landmarks and to forage in an adjacent compartment which they could reach through an exit tube in the arena wall at a height of 28cm. The movements of one group of bees were restricted to a height of 30cm, the height of the landmark array, while the other group was able to move up to heights of 80cm, thus being able to see the landmark array from above.

During one series of tests, the flights of bees returning from the foraging compartment were recorded as they tried to reach the nest entrance on the floor of the arena with the landmark array shifted to various positions away from the true nest entrance location. The results of these tests showed that the bees searched for the net entrance in the location that was defined by the landmark array.

In a second series of tests, access to the landmark array was prevented from the side, but not from top, by a transparent screen surrounding the landmark array. These tests showed that the bees of both groups rarely entered the array from above, but kept trying to enter it from the side.

The authors express surprise at this result because modelling the navigational information supplied by panoramic snapshots in this arena had indicated that the most robust information to the location of the nest entrance within the landmark array was supplied by views of the array from above, leading to the following strong conclusions:

line 51: "Snapshot models perform best with bird's eye views";<br /> line 188: "Overall, our model analysis could show that snapshot models are not able to find home with views within a cluttered environment but only with views from above it.";<br /> line 231: "Our study underscores the limitations inherent in snapshot models, revealing their inability to provide precise positional estimates within densely cluttered environments, especially when compared to the navigational abilities of bees using frog's-eye views."

Strengths:

The experimental set-up allows to record the flight behaviour of bees in great spatial and temporal detail and in principle also to reconstruct the visual information available to the bees throughout the arena.

Modelling: The revised manuscript now presents the results of modelling that includes information potentially available to the bees from the arena wall and in particular from the top edge of the arena.

As I predicted, this increases the width of rotational image difference functions and therefore provides directional guidance over a larger range of misalignments. However, the authors dismiss the modelling results based on such reconstructed views which more realistically describe the information available to the bumblebees, because (line 291ff): 'Further simulations with a rendered arena wall led to worse results because the agent was mainly led to the centre of the arena (Fig. S17, Fig. S18-21)".

What the modelling in Fig. 17 actually shows is that the agent is led more or less exactly to the 'entry points' to the arena chosen by the real bees (Fig. 4). The authors ignore this and in their rebuttal state that 'We hypothesised that the arena wall and object location created ambiguity'. The problem here is that you don't remove potential 'ambiguity' for real bees by ignoring information they are unlikely to ignore.

Behavioural analysis: The full potential of the set-up was not used to understand how the bees' navigation behaviour develops over time in this arena and what opportunities the bees have had to learn the location of the nest entrance during repeated learning flights and return flights.

Without a detailed analysis of the bees' behaviour during 'training', including learning flights and return flights, it is very hard to follow the authors' conclusions. The behaviour that is observed in the tests may be the result of the bees' extended experience shuttling between the nest and the entry to the foraging arena at 28cm height in the arena wall. For instance, it would have been important to see the return flights of bees following the learning flights shown in Fig. 17.

Basically both groups of bees (constrained to fly below the height of landmarks (F) or throughout the height of the arena (B)) had ample opportunities to learn that the nest entrance lies on the floor of the landmark array. The only reason why B-bees may not have entered the array from above when access from the side was prevented may simply be that bumblebees, because they bumble, find it hard to perform a hovering descent into the array.

The revised manuscript does not address my concerns. The rebuttal states that a detailed analysis of learning and return flights was 'outside the scope of this particular study', that their experimental design 'does not require the entire history of the bee's trajectory to be tested', that 'the entire flight history...will require...effort...conceptually' and that it would be 'difficult to test a hypothesis'.

These responses clarify the frustrating problem with this study: The authors are more concerned with testing hypotheses than with trying to understand how bumblebees learn to cope with a situation which constrains their learning choreography and confronts them with the one fundamental problem view-based homing has: repetitive scene elements.

Homing is an experience-dependent process and to understand what cues the bees used to navigate this set-up requires an analysis of the whole learning process. For instance, it may well be that the B+G+ bees initially did enter the array from above, but subsequently learnt a more efficient route into the array, by simply entering it from the side, followed by 'unguided' searching.

General: The most serious weakness of the set-up is that it is spatially and visually constrained, in particular lacking a distant visual panorama, which under natural conditions is crucial for the range over which rotational image difference functions provide navigational guidance. In addition, the array of identical landmarks is not representative of natural clutter and, because it is visually repetitive, poses unnatural problems for view-based homing algorithms. This is the reason why the functions degrade so quickly from one position to the next (Fig. 9-12) when more distant scene elements are excluded.

In conclusion, I do not feel that I have learnt anything useful from this experiment; it does suggest, however, that to fully appreciate and understand the homing abilities of insects, there is no alternative but to investigate these abilities in the natural conditions in which they have evolved. A nice start would be to build camera-based 3D models of natural bumblebee nest entrance environments and analyse whether there are any particularly unusual challenges for the visual localization of the nest entrance.

Reviewer #1 (Public review):

Summary and Strengths:

The study focuses on PIM1 and 2 in CD8 T cell activation and differentiation. These two serine/threonine kinases belong to a large network of Serine/Threonine kinases that acts following engagement of the TCR and of cytokine receptors and phosphorylates proteins that control transcriptional, translational and metabolic programs that result in effector and memory T cell differentiation. The expression of PIM1 and PIM2 is induced by the T-cell receptor and several cytokine receptors. The present study capitalized on high-resolution quantitative analysis of the proteomes and transcriptomes of Pim1/Pim2-deficient CD8 T cells to decipher how the PIM1/2 kinases control TCR-driven activation and IL-2/IL-15-driven proliferation, and differentiation into effector T cells.

Quantitative mass spectrometry-based proteomics analysis of naïve OT1 CD8 T cell stimulated with their cognate peptide showed that the PIM1 protein was induced within 3 hours of TCR engagement and its expression was sustained at least up to 24 hours. The kinetics of PIM2 expression was protracted as compared to that of PIM1. Such TCR-dependent expression of PIM1/2 correlated with the analysis of both Pim1 and Pim2 mRNA. In contrast, Pim3 mRNA was only expressed at very low levels and the PIM3 protein not detected by mass spectrometry. Therefore, PIM1 and 2 are the major PIM kinases in recently activated T cells. Pim1/Pim2 double knockout (Pim dKO) mice were generated on a B6 background and found to express lower number of splenocytes. No difference in TCR/CD28-driven proliferation was observed between WT and Pim dKO T cells over 3 days in culture. Quantitative proteomics of >7000 proteins further revealed no substantial quantitative or qualitative differences in protein content or proteome composition. Therefore, other signaling pathways can compensate for the lack of PIM kinases downstream of TCR activation.

Considering that PIM1 and PIM2 kinase expression is regulated by IL-2 and IL-15, antigen-primed CD8 T cells were expanded in IL-15 to generate memory phenotype CD8 T cells or expanded in IL-2 to generate effector cytotoxic T lymphocytes (CTL). Analysis of the survival, proliferation, proteome, and transcriptome of Pim dKO CD8 T cells kept for 6 days in IL-15 showed that PIM1 and PIM2 are dispensable to drive the IL-15-mediated metabolic or differentiation programs of antigen-primed CD8 T cells. Moreover, Pim1/Pim2-deficiency had no impact on the ability of IL-2 to maintain CD8 T cell viability and proliferation. However, WT CTL downregulated expression of CD62L whereas the Pim dKO CTL sustained higher CD62L expression. Pim dKO CTL were also smaller and less granular than WT CTL. Comparison of the proteome of day 6 IL-2 cultured WT and Pim dKO CTL showed that the latter expressed lower levels of the glucose transporters, SLC2A1 and SLC2A3, of a number of proteins involved in fatty acid and cholesterol biosynthesis, and CTL effector proteins such as granzymes, perforin, IFNg and TNFa. Parallel transcriptomics analysis showed that the reduced expression of perforin and some granzymes correlated with a decrease in their mRNA whereas the decreased protein levels of granzymes B and A, and of the glucose transporters SLC2A1 and SLC2A3 did not correspond with decreased mRNA expression. Therefore, PIM kinases are likely required for IL-2 to maximally control protein synthesis in CD8 CTL. Along that line, the translational repressor PDCD4 was increased in Pim dKO CTL and pan-PIM kinase inhibitors caused a reduction in protein synthesis rates in IL-2 expanded CTL. Finally, the differences between Pim dKO and WT CTL in terms of CD62L expression resulted in that Pim dKO CTL but not WT CTL retained the capacity to home to secondary lymphoid organs. In conclusion, this thorough and solid study showed that the PIM1/2 kinases shape the effector CD8 T cell proteomes rather than transcriptomes and are important mediators of IL2-signalling and CD8 T cell trafficking.

Weaknesses: None

Comments on revisions:

The authors have been able to provide in their rebuttal letter fair answers to most of the queries primarily raised by Reviewer 2 and they have incorporated the corresponding results in the revised text. It makes the paper stronger.

Reviewer #2 (Public review):

Summary:

Using a suite of techniques (e.g., RNA seq, proteomics, and functional experiments ex vivo) this paper extensively focuses on the role of PIM1/2 kinases during CD8 T-cell activation and cytokine-driven (i.e., IL-2 or IL-15) differentiation. The authors key finding is that PIM1/2 enhance protein synthesis in response to IL-2 stimulation, but not IL-15, in CD8+ T cells. Loss of PIM1/2 made T cells less 'effector-like', with lower granzyme and cytokine production, and a surface profile that maintained homing towards secondary lymphoid tissue. The cytokines the authors focus on are IL-15 and Il-2, which drive naïve CD8 T cells towards memory or effector states, respectively. Although PIM1/2 are upregulated in response to T-cell activation and cytokine stimulation (e.g., IL-15, and to a greater extent, IL-2), using T cells isolated from a global mouse genetic knockout background of PIM1/2, the authors find that PIM1/2 did not significantly influence T-cell activation, proliferation, or expression of anything in the proteome under anti-CD3/CD28 driven activation with/without cytokine (i.e., IL-15) stimulation ex vivo. This is perhaps somewhat surprising given PIM1/2 are upregulated, albeit to a small degree, in response to IL-15, and yet PIM1/2 did not seem to influence CD8+ T cell differentiation towards a memory state. Even more surprising is that IL-15 was previously shown to influence the metabolic programming of intestinal intraepithelial lymphocytes, suggesting cell-type specific effects from PIM kinases. What the authors went on to show, however, is that PIM1/2 KO altered CD8 T cell proteomes in response to IL-2. Using proteomics, they saw increased expression of homing receptors (i.e., L-selectin, CCR7), but reduced expression of metabolism-related proteins (e.g., GLUT1/3 & cholesterol biosynthesis) and effector-function related proteins (e.g., IFNy and granzymes). Rather neatly, by performing both RNA-seq and proteomics on the same IL-2 stimulated WT vs. PIM1/2 KO cells, the authors found that changes at the proteome level were not corroborated by differences in RNA uncovering that PIM1/2 predominantly influence protein synthesis/translation. Effectively, PIM1/2 knockout reduced the differentiation of CD8+ T cells towards an effector state. In vivo adoptive transfer experiments showed that PIM1/2KO cells homed better to secondary lymphoid tissue, presumably owing to their heightened L-selectin expression (although this was not directly examined).

Strengths:

Overall, I think the paper is scientifically good, and I have no major qualms with the paper. The paper as it stands is solid, and while the experimental aim of this paper was quite specific/niche, it is overall a nice addition to our understanding of how serine/threonine kinases impact T cell state, tissue homing, and functionality. Of note, they hint towards a more general finding that kinases may have distinct behaviour in different T-cell subtypes/states. I particularly liked their use of matched RNA-seq and proteomics to first suggest that PIM1/2 kinases may predominantly influence translation (then going on to verify this via their protein translation experiment - although I must add this was only done using PIM kinase inhibitors not the PIM1/2KO cells). I also liked that they used small molecule inhibitors to acutely reduce PIM1/2 activity, which corroborated some of their mouse knockout findings - this experiment helps resolve any findings resulting from potential adaptation issues from the PIM1/2 global knockout in mice but also gives it a more translational link given the potential use of PIM kinase inhibitors in the clinic. The proteomics and RNA seq dataset may be of general use to the community, particularly for analysis of IL-15 or IL-2 stimulated CD8+ T cells.

Weaknesses:

None. My comments here have been addressed in the previous review.

Reviewer #2 (Public review):

Summary:

Malaria transmission in the Gambia is highly seasonal, whereby periods of intense transmission at the beginning of the rainy season are interspersed by long periods of low to no transmission. This raises several questions about how this transmission pattern impacts the spatiotemporal distribution of circulating parasite strains, how parasites persist during the dry season, and how asymptomatic infections contribute to maintaining transmission during the low/no transmission season.

Combining a molecular barcode genotyping using 101 bi-allelic SNPs and SNPs from Whole Genome Sequence (WGS) in a "consensus barcode", the authors aimed at measuring the relatedness between parasites at different spatial (i.e., individual, household, village, and region) and temporal (i.e., high, low, and the corresponding the transitions) levels by assessing the fraction of the genome having a common ancestry (i.e. Identity-by-Descent (IBD)).

By measuring the Complexity of Infection (COI) and parasite relatedness by IBD the authors show that a large fraction of infections is polygenomic and stable over time, resulting in a high recombinational diversity. Moreover, they show that transmission intensity increases during the transition from the dry to wet seasons. However, they find that there is a higher probability of finding similar genotypes within the same household, but this similarity rapidly disappears over time and is not observed between different villages. If there is no drug selection during the dry season, and if resistance results in a fitness cost, alleles associated with drug resistance may change in frequency. The authors looked at the frequencies of six drug-resistance haplotypes (aat1, crt, dhfr, dhps, kelch13, and mdr1), and found no evidence of changes in allele frequencies associated with seasonality. They also find chronic infections lasting from one month to one and a half years with no dependence on age or gender.

This work makes use of genomic information and IBD analytic tools to show parasite relatedness from asymptomatic infections at different spatial and temporal scales, thus providing a better understanding of the transmission dynamics of malaria in highly seasonal environments.

Strength:

The authors use a combination of high-quality barcodes (425 barcodes representing 101 bi-allelic SNPs) and 199 high-quality genome sequences to infer the fraction of the genome with shared Identity by Descent (IBD) (i.e. a metric of recombination rate) over several time points covering two years. The barcode and whole genome sequence combination allows full use of a large dataset, to confidently infer the relatedness of parasite isolates at various spatiotemporal scales and show the advantage of using genomic information for understanding malaria transmission dynamics.

The authors aimed to establish how seasonal transmission cycles shape the spatiotemporal parasite population structure using metrics such as parasite genetic diversity, genetic relatedness, and frequency of drug resistance alleles, as well as the contribution of asymptomatic chronic carriers to sustained transmission. The results support their conclusions.

Using a combination of molecular barcodes and available whole genome sequence datasets opens new opportunities to understand malaria transmission dynamics in different transmission settings. This allows for data analysis at different spatiotemporal granularities, having a practical utility for identifying malaria control targets and acquiring metrics to evaluate malaria control programs. The development of molecular barcodes using similar SNPs by different malaria control programs would be of great utility to compare and understand malaria transmission dynamics in different settings worldwide.

Reviewer #3 (Public review):

This study aimed to examine the impact of seasonality on the population genetics of malaria parasites. To achieve this, the researchers conducted a longitudinal study in a region with seasonal malaria transmission. Over a 2.5-year period, blood samples were collected from 1,516 participants across four villages in the Upper River Region of The Gambia. These samples were tested for malaria parasite infection, and the parasites from positive samples were genotyped using a genetic barcode and/or whole genome sequencing. Genetic relatedness analysis was then performed to explore the findings

The study identified three key findings:

(1) The malaria parasite population undergoes continuous recombination, with no single genotype predominating, in contrast to viral populations;

(2) Parasite relatedness is influenced by both spatial and temporal factors; and

(3) The lowest genetic relatedness among parasites occurs during the transition from the low to high transmission seasons, which the authors linked to increased recombination during sexual reproduction in mosquitoes.

The results section is well-structured, and the figures are clear and self-explanatory. The methods are adequately described, providing a solid foundation for the findings. While there are no unexpected results, it is reassuring to see the anticipated outcomes supported by actual data. The conclusions are generally well-supported and the recommendation to target asymptomatic infections is logical and relevant.

Reviewer #1 (Public review):

Summary:

This paper examines changes in relaxation time (T1 and T2) and magnetization transfer parameters that occur in a model system and in vivo when cells or tissue are depolarized using an equimolar extracellular solution with different concentrations of the depolarizing ion K+. The motivation has been revised to state that the results suggest a potential approach to non-invasively detect changes in membrane potential using MRI.

Strengths:

The authors argue that the use of various concentrations of KCL in the extracellular fluid depolarize or hyperpolarize the cell pellets used, and that this change in membrane potential is the driving force for the T2 (and T1-supplementary material) changes observed. In particular, they report an increase in T2 with increasing KCL concentration in the extracellular fluid (ECF) of pellets of SH-SY5Y cells. To offset the increasing osmolarity of the ECF due to the increase in KCL, the NaCL molarity of the ECF is proportionally reduced. The authors measure the intracellular voltage using patch clamp recordings, which is a gold standard. With 80 mM of KCL in the ECF, a change in T2 of the cell pellets of ~10 ms is observed with the intracellular potential recorded as about -6 mv. A very large T1 increase of ~90 ms is reported under the same conditions. The PSR (ratio of hydrogen protons on macromolecules to free water) decreases by about 10% at this 80 mM KCL concentration. Similar results are seen in a Jurkat cell line and similar, but far smaller changes are observed in vivo, for a variety of reasons discussed. As a final control, T1 and T2 values are measured in the various equimolar KCL solutions. As expected, no significant changes in T1 and T2 of the ECF were observed for these concentrations.

Weaknesses:

While the concepts presented are interesting, and the actual experimental methods seem to be nicely executed, the conclusions are not supported by the data for a number of reasons. This is not to say that the data isn't consistent with the conclusions, but there are other controls not included that would be necessary to draw the conclusion that it is membrane potential that is driving these T1 and T2 changes. The results are consistent with Stroman et al. Magn. Reson. in Med. 59:700-706 (increased T2 with KCL) as well as some other cited work. However all those authors emphasize that cell swelling is the mechanism, not cell membrane potentials.

It is well established that cells swell/shrink upon depolarization/hyperpolarization. Cell swelling is accompanied by increased light transmittance in vivo, and this should be true in the pellet system as well. In a beautiful series of experiments, Stroman et al. (2008) showed in perfused brain slices that the cells swell upon equimolar KCL depolarization and the light transmittance increases. The time course of these changes is quite slow, of the order of many minutes, both for the T2-weighted MRI signal and for the light transmittance. Stroman et al. also show that hypoosmotic changes produce the exact same timecourse as the KCL depolarization changes (and vice versa for the hyperosmotic changes - which cause cell shrinkage). Their conclusion therefore, was that cell swelling (not membrane potential) was the cause of the T2-weighted changes observed, and that these were relatively slow (on the scale of many minutes).

What are the implications for the current study? Well, for one, the authors cannot exclude cell swelling as the mechanism for T2 changes, as they have not measured that. It is however well established that cell swelling occurs during depolarization, so this is not in question. Water in the pelletized cells is in slow/intermediate exchange with the ECF, and the solutions for the two compartment relaxation model for this are well established (see Menon and Allen, Magn. Reson. in Med. 20:214-227 (1991). The T2 relaxation times should be multiexponential (see point (3) further below). The current work cannot exclude cell swelling as the mechanism for T2 changes (it is mentioned in the paper, but not dealt with). Water entering cells dilutes the protein structures, changes rotational correlation times of the proteins in the cell and is known to increase T2. The PSR confirms that this is indeed happening, so the data in this work is completely consistent with the Stroman work and completely consistent with cell swelling associated with depolarization. The authors should have performed light scattering studies to demonstrate the degree cell swelling or shrinkage. Measuring intracellular potential is not enough to clarify the mechanism.

So why does it matter whether the mechanism is cell swelling or membrane potential? The reason is response time. Cell swelling due to depolarization is a slow process, slower than hemodynamic responses that characterize BOLD. And in fact, cell swelling under normal homeostatic conditions in vivo is virtually non-existent. Only sustained depolarization events typically associated with non-naturalistic stimuli or brain dysfunction produce cell swelling. Membrane potential changes associated with neural activity, on the other hand, are very fast. In this manuscript, the authors have convincingly shown a signal change that is virtually the same as what was seem in the Stroman publication, but they have not shown that there is a response that can be detected with anything approaching the timescale of an action potential. So one cannot definitely say that the changes observed are due to membrane potential. One can only say they are consistent with cell swelling, regardless of what causes the cell swelling. The First line of the discussion still claims that T2 relaxation time and pool size ratio (PSR) can detect responses to membrane potential changes modulated by ionic solutions. However, in the absence of cell swelling controls, this cannot be stated.

For this mechanism to be relevant to measuring neuronal activity directly or explaining techniques such DIANA, one needs to show that the cell swelling changes occur within a millisecond, which has never been reported. If one knows the populations of ECF and pellet, the T2s of the ECF and pellet and the volume change of the cells in the pellet, one can model any expected T2 changes due to neuronal activity. I think one would find that these are minuscule within the context of an action potential, or even bulk action potentials.

Comments on revisions:

The manuscript is well written and my previous methodological concerns have been clarified as well. There are no flaws in the experiments, but the interpretation really depends on simultaneous measurements of cell volume and membrane potential, which have yet to be done.

Reviewer #2 (Public review):

Summary:

Min et al. attempt to demonstrate a mechanism whereby magnetic resonance imaging (MRI) can reflect changes in neuronal membrane potentials. They approach this goal by studying how MRI contrast and cellular potentials together respond to treatment of cultured cells with ionic solutions that are known to depolarize or hyperpolarize excitable cells. The authors specifically examine two MRI-based measurements: (A) the transverse (T2) relaxation rate, which reflects microscopic magnetic fields caused by solutes and biological structures; and (B) the fraction or "pool size ratio" (PSR) of water molecules estimated to be bound to macromolecules, using an MRI technique called magnetization transfer (MT) imaging. They see that depolarizing K+ and Ba2+ concentrations lead to T2 increases and PSR decreases that vary approximately linearly with parallel measurements of voltage in a neuroblastoma cell line and that change similarly in a second cell type. They also show that depolarizing potassium concentrations evoke T2 increases in rat brains, and that these changes are reversed when potassium is renormalized. Min et al. argue that their results suggest a basis for noninvasive functional imaging of cellular voltage signals. If this were true, it would help validate a recent paper published by some of the authors (Toi et al., Science 378:160-8, 2022), in which they claimed to be able to detect millisecond-scale neuronal responses by MRI.

Strengths:

The discovery of a mechanism for relating cellular membrane potential to MRI contrast could yield an important means for studying functions of the nervous system. Achieving this has been a longstanding goal in the MRI community, but previous strategies have proven insufficient for neuroscientific or clinical applications. The current paper suggests that one of the simplest and most widely used MRI contrast mechanisms-T2 weighted imaging-may indicate correlates of membrane potential if measured in the absence of the hemodynamic signals that most functional MRI (fMRI) experiments rely on. The authors make their case using quantitative tests that include some controls for ion and cell type-specificity of their in vitro results and reversibility of MRI changes observed in vivo.

Weaknesses:

The major weakness of the paper is that it uses only slow correlational experiments to probe the relationship between MRI contrast and membrane potential. The authors do not examine effects on the subsecond time scale that is of greatest interest, and they do not adequately consider how biophysical factors with only loose relationship to electrophysiological variables could explain their imaging results. Notably, depolarizing ionic solutions that perturb membrane potential can also induce changes in cellular volume and tissue structure that in turn alter MRI contrast properties similarly to the results shown here. For example, a study by Stroman et al. (Magn Reson Med 59:700-6, 2008) reported reversible potassium-dependent T2 increases in neural tissue that correlate closely with light scattering-based indications of cell swelling. Phi Van et al. (Sci Adv 10:eadl2034, 2024) showed that potassium addition to one of the cell lines used here likewise leads to cell size increases and T2 increases. In their revised manuscript, the authors acknowledge that cell swelling might contribute to the MRI signals they report, but they do nothing to probe the contributions or characteristics of such effects. If cell swelling accounted for the author's MRI results, it would likely operate on a time scale far too slow to yield useful indications of membrane potential. Given these considerations and the absence of data demonstrating correspondence of electrophysiological measures with MRI readouts on a fast time scale, the paper fails to provide evidence that membrane potential changes can be meaningfully detected by MRI.

Reviewer #2 (Public review):

Summary:

The authors conduct a causal analysis of years of secondary education on brain structure in late life. They use a regression discontinuity anlaysis to measure the impact of a UK law change in 1972 that increased the years of mandatory education by 1 year. Using brain imaging data from the UK Biobank, they find essentially no evidence for 1 additional year of education altering brain structure in adulthood.

Strengths:

The authors pre-registered the study and the regression discontinuity was very carefully described and conducted. They completed a large number of diagnostic and alternate analyses to allow for different possible features in the data. (Unlike a positive finding, a negative finding is only bolstered by additional alternative anlayses).

Weaknesses:

While the work is of high quality for the precise question asked, ultimately the exposure (1 additional year of education) is a very modest manipulation and the outcome measured long after the intervention. Thus a null finding here is completely consistent educational attainement (EA) in fact having an impact on brain structure, where EA may reflect elements of training after second education (e.g. university, post-graduate qualifications, etc) and not just stopping education at 16 yrs yes/no.

Reviewer #3 (Public review):

Summary:

This study investigates evidence for a hypothesised, causal relationship between education, specifically the number of years spent in school, and brain structure as measured by common brain phenotypes such as surface area, cortical thickness, total volume and diffusivity.

To test their hypothesis, the authors rely on a "natural" intervention, that is, the 1972 ROSLA act that mandated an extra year of education for all 15-year olds. The study's aim is to determine potential discontinuities in the outcomes of interest at the time of the policy change, which would indicate a causal dependence. Naturalistic experiments of this kind are akin to randomised controlled trials, the gold standard for answering questions of causality.

Using two complementary, regression-based approaches, the authors find no discernible effect of spending an extra year in primary education on brain structure. The authors further demonstrate that observational studies showing an effect between education and brain structure may be confounded and thus unreliable when assessing causal relationships.

Strengths:

- A clear strength of this study is the large sample size totalling up to 30k participants from the UK Biobank. Although sample sizes for individual analyses are an order of magnitude smaller, most neuroimaging studies usually have to rely on much smaller samples.<br /> - This study has been preregistered in advance, detailing the authors' scientific question, planned method of inquiry and intended analyses, with only minor, justifiable changes in the final analysis.<br /> - The analyses look at both global and local brain measures used as outcomes, thereby assessing a diverse range of brain phenotypes that could be implicated in a causal relationship with a person's level of education.<br /> - The authors use multiple methodological approaches, including validation and sensitivity analyses, to investigate the robustness of their findings and, in the case of correlational analysis, highlight differences with related work by others.<br /> - The extensive discussion of findings and how they relate to the existing, somewhat contradictory literature gives a comprehensive overview of the current state of research in this area.

Weaknesses:

- This study investigates a well-posed but necessarily narrow question in a specific setting: 15-year old British students born around 1957 who also participate in the UKB imaging study roughly 60 years later. Thus conclusions about the existence or absence of any general effect of the number of years of education on the brain's structure are limited to this specific scenario.<br /> - The modelling approach used in this study requires that all covariates of no interest are equal before and after the cut-off, something that is impossible to test. However, other studies have not found specific issues that would invalidate ROSLA as a natural experiment.

Reviewer #2 (Public review):

Summary:

The goal of this work is to define the functions of T-box transcription factors Tbx3 and Tbx5 in the adult mouse ventricular cardiac conduction system (VCS) using a novel conditional mouse allele in which both genes are targeted in cis. A series of studies over the past 2 decades by this group and others have shown that Tbx3 is a transcriptional repressor that patterns the conduction system by repressing genes associated with working myocardium, while Tbx5 is a potent transcriptional activator of "fast" conduction system genes in the VCS. In a previous work, the authors of the present study further demonstrated that Tbx3 and Tbx5 exhibit an epistatic relationship whereby the relief of Tbx3-mediated repression through VCS conditional haploinsufficiency allows better toleration of Tbx5 VCS haploinsufficiency. Conversely, excess Tbx3-mediated repression through overexpression results in disruption of the fast-conduction gene network despite normal levels of Tbx5. Based on these data the authors proposed a model in which repressive functions of Tbx3 drive adoption of conduction system fate, followed by segregation into a fast-conducting VCS and slow-conduction AVN through modulation of the Tbx5/Tbx3 ratio in these respective tissue compartments.

The question motivating the present work is: If Tbx5/Tbx3 ratio is important for slow versus fast VCS identity, what happens when both genes are completely deleted from the VCS? Is conduction system identity completely lost without both factors and if so, does the VCS network transform into a working myocardium-like state? To address this question, the authors have generated a novel mouse line in which both Tbx5 and Tbx3 are floxed on the same allele, allowing complete conditional deletion of both factors using the VCS-specific MinK-CreERT2 line, convincingly validated in previous work. The goal is to use these double conditional knockout mice to further explore the model of Tbx3/Tbx5 co-dependent gene networks and VCS patterning. First the authors demonstrate that the double conditional knockout allele results in the expected loss of Tbx3 and Tbx5 specifically in the VCS when crossed with Mink-CreERT2 and induced with tamoxifen. The double conditional knockout also results in premature mortality. Detailed electrophysiological phenotyping demonstrated prolonged PR and QRS intervals, inducible ventricular tachycardia, and evidence of abnormal impulse propagation along the septal aspect of the right ventricle. In addition, the mutants exhibit downregulation of VCS genes responsible for both fast conduction AND slow conduction phenotypes with upregulation of 2 working myocardial genes including connexin-43. The authors conclude that loss of both Tbx3 and Tbx5 results in "reversion" or "transformation" of the VCS network to a working myocardial phenotype, which they further claim is a prediction of their model and establishes that Tbx3 and Tbx5 "coordinate" transcriptional control of VCS identity.

Overall Appraisal:

As noted above, the present study does not further explore the Tbx5/Tbx3 ratio concept since both genes are completely knocked out in the VCS. Instead, the main claims are that absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function. However, only limited data are presented to support the claim of transcriptional reprogramming since the knockout cells are not directly compared to working myocardial cells at the transcriptional level and only a small number of key genes are assessed (versus genome-wide assessment). In addition, the optical mapping dataset has alternative interpretations that are not excluded or thoroughly discussed.

In sum, while this study adds an elegantly constructed genetic model to the field, the data presented mostly fit within the existing paradigm of established functions of Tbx3 and Tbx5. The authors present some evidence to support the claim that VCS cells adopt a working myocardial phenotype in the absence of Tbx3 and Tbx5, but some key experiments that could more definitively test this model were not performed, reducing the degree to which the data support the conclusions.

Strengths:

(1) Successful generation of a novel Tbx3-Tbx5 double conditional mouse model<br /> (2) Successful VCS-specific deletion of Tbx3 and Tbx5 using a VCS-specific inducible Cre driver line<br /> (3) Well-powered and convincing assessments of mortality and physiological phenotypes<br /> (4) Isolation of genetically modified VCS cells using flow.

Weaknesses:

(1) In general, the data is consistent with a long-standing and well-supported model in which Tbx3 represses working myocardial genes and Tbx5 activates expression of VCS genes, which seem like distinct roles in VCS patterning.<br /> (2) More direct quantitative comparison of Tbx5 Adult VCS KO with Tbx5/Tbx3 Adult VCS double KO would be helpful to ascertain whether deletion of Tbx3 on top of Tbx5 deletion changes the underlying phenotype in some discernable way beyond mRNA expression of a few genes. Superficially, the phenotypes look quite similar at the EKG and arrhythmia inducibility level and no optical mapping data from single Tbx5 KO is presented for comparison to the double KO. I understand that single Tbx5 VCS KO mutants have been evaluated in previous publications but I think in order to evaluate the claims presented here, it would be important to do a direct comparison using the same assays and conditions.<br /> (3) The authors claim that double knockout VCS cells transform to working myocardial fate, but there is no comparison of gene expression levels between actual working myocardial cells and the Tbx3/Tbx5 DKO VCS cells so it's hard to know if the data reflect an actual cell state change or a more non-specific phenomenon with global dysregulation of gene expression or perhaps dedifferentiation. I understand that the upregulation of Gja1 and Smpx is intended to address this, but it's only two genes and it seems relevant to understand their degree of expression relative to actual working myocardium. In addition, the gene panel is somewhat limited and does not include other key transcriptional regulators in the VCS such as Irx3 and Nkx2-5. RNA-seq in these populations would provide a clearer comparison among the groups.<br /> (4) From the optical mapping data, it is difficult to distinguish between the presence of (1) a focal proximal right bundle branch block due to dysregulation of gene expression in the VCS but overall preservation of the right bundle and its distal ramifications; from (2) actual loss of the VCS with reversion of VCS cells to a working myocardial fate. Related to this, the authors claim that this experiment allows for direct visualization of His bundle activation, but can the authors confirm or provide evidence that the tissue penetration of their imaging modality allows for imaging of a deep structure like the AV bundle as opposed to the right bundle branch which is more superficial? Does the timing of the separation of the sharp deflection from the subsequent local activation suggest visualization of more distal components of the VCS rather than the AV bundle itself? Additional clarification would be helpful.

impact:

The present study contributes a novel and elegantly constructed mouse model to the field. The data presented generally corroborate existing models of transcriptional regulation in the VCS. Acknowledging that the present work is strong start, some additional studies not included in the present manuscript will be needed for this new mouse model to decisively advance the field of VCS transcriptional biology.

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterised the cardiac phenotype associated to cardiac conduction system (CCS)-specific combined deletion of Tbx5 and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9 weeks old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterising VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analysed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutant and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lays in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Linage tracing analysis using the reporter lines included in the crosses described in the study, will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot will demonstrate that indeed the cells that were specified as VCS in the adult heart become working myocardium in the absence of Tbx3 and Tbx5 function.

Comments on revisions:

I would like to thank the authors for their revised manuscript and for their corrections based on the suggestions from the 3 reviewers. Although I would have preferred to see some of the additional experiments suggested by any of the reviewers to improve the robustness and depth of the study integrated in the revised version of the manuscript, I acknowledge that the authors may prefer to develop them as follow-up studies. So, looking forward to seeing the follow-up study unravelling the detailed molecular regulation controlled by Tbx3/Tbx5 during the formation and maintenance of the ventricular cardiac conduction system.

Joint Public Review:

In this manuscript, the authors aim to evaluate the robustness of stable asymmetric polarization patterns by analyzing both a minimal 2-node network and a more biologically realistic 5-node network based on the C. elegans polarization system. They introduce a computational pipeline for systematically exploring reaction-diffusion network dynamics. Their study highlights the limitations of the widely used 2-node antagonistic network, demonstrating its susceptibility to simple modifications that disrupt polarization. However, they show that polarization stability can be restored by combining multiple regulatory mechanisms, and that spatially varying kinetic parameters can fine-tune the interface position. The authors further investigate the 5-node network of C. elegans, identifying key parameters that enhance its robustness against perturbations. Their findings provide novel insights into the mechanisms that ensure stable polarization in biological systems.

The major strengths of this work lie in its rigorous computational approach and the clarity of its findings. The authors demonstrate that the widely used 2-node antagonistic network is highly sensitive to parameter changes, requiring precise fine-tuning to maintain stable polarization. However, they show that stability can be restored through compensatory modifications, which expand the range of parameter sets supporting polarization. By further exploring spatial parameter variations, the authors reveal how compensatory adjustments can stabilize polarization patterns, offering insights into potential biological mechanisms regulating interface localization.

Extending their analysis to the C. elegans polarization network, the authors construct a 5-node model grounded in an extensive literature review. Their computational pipeline identifies key parameters that enhance robustness, and their model successfully replicates experimental observations, even in mutant conditions. Notably, among 34 possible network structures, only the naturally evolved 5-node network with mutual inhibition between specific components maintains stable polarization, highlighting its evolutionary optimization. This work significantly advances our understanding of polarization maintenance and provides a valuable framework for future in silico experiments.

Despite its strengths, the study has some limitations related to simplifying assumptions. The model neglects cortical flows and the role of actomyosin dynamics, which are known to be crucial during the establishment phase of polarization in the C. elegans zygote. While the authors focus on the maintenance phase, the absence of these biomechanical effects may limit the model's applicability to the full polarization process. Additionally, the assumption of infinitely fast cytoplasmic diffusion disregards potential effects of cytoplasmic flows on the stability of molecular distributions. Experimental measurements suggest that cytoplasmic diffusion coefficients are only an order of magnitude higher than membrane diffusion coefficients, meaning that finite diffusion combined with cytoplasmic flows could influence polarization stability. Although the authors acknowledge and discuss these limitations, incorporating these effects in future models could provide a more complete picture of the polarization dynamics in C. elegans embryos.

Reviewer #1 (Public review):

Summary:

The manuscript by Egawa and colleagues investigates differences in nodal spacing in an avian auditory brain stem circuit. The results are clearly presented and data are of very high quality. The authors make two main conclusions:

(1) Node spacing, i.e. internodal length, is intrinsically specified by the oligodendrocytes in the region they are found in, rather than axonal properties (branching or diameter).

(2) Activity is necessary (we don't know what kind of signaling) for normal numbers of oligodendrocytes and therefore the extent of myelination.

These are interesting observations, albeit phenomenon. I have only a few criticisms that should be addressed:

(1) The use of the term 'distribution' when describing the location of nodes is confusing. I think the authors mean rather than the patterns of nodal distribution, the pattern of nodal spacing. They have investigated spacing along the axon. I encourage the authors to substitute node spacing or internodal length for node distribution.

(2) In Seidl et al. (J Neurosci 2010) it was reported that axon diameter and internodal length (nodal spacing) were different for regions of the circuit. Can the authors help me better understand the difference between the Seidl results and those presented here?

(3) The authors looked only in very young animals - are the results reported here applicable only to development, or does additional refinement take place with aging?

(4) The fact that internodal length is specified by the oligodendrocyte suggests that activity may not modify the location of nodes of Ranvier - although again, the authors have only looked during early development. This is quite different than this reviewer's original thoughts - that activity altered internodal length and axon diameter. Thus, the results here argue against node plasticity. The authors may choose to highlight this point or argue for or against it based on results in adult birds?:

Significance:

This paper may argue against node plasticity as a mechanism for tuning of neural circuits. Myelin plasticity is a very hot topic right now and node plasticity reflects myelin plasticity. this seems to be a circuit where perhaps plasticity is NOT occurring. That would be interesting to test directly. One limitation is that this is limited to development.

Reviewer #2 (Public review):

Summary:

Egawa et al describe the developmental timeline of the assembly of nodes of Ranvier in the chick brainstem auditory circuit. In this unique system, the spacing between nodes varies significantly in different regions of the same axon from early stages, which the authors suggest is critical for accurate sound localization. Egawa et al set out to determine which factors regulate this differential node spacing. They do this by using immunohistological analyses to test the correlation of node spacing with morphological properties of the axons, and properties of oligodendrocytes, glial cells that wrap axons with the myelin sheaths that flank the nodes of Ranvier. They find that axonal structure does not vary significantly, but that oligodendrocyte density and morphology varies in the different regions traversed by these axons, which suggests this is a key determinant of the region-specific differences in node density and myelin sheath length. They also find that differential oligodendrocyte density is partly determined by secreted neuronal signals, as (presumed) blockage of vesicle fusion with tetanus toxin reduced oligodendrocyte density in the region where it is normally higher. Based on these findings, the authors propose that oligodendrocyte morphology, myelin sheath length, and consequently nodal distribution are primarily determined by intrinsic oligodendrocyte properties rather than neuronal factors such as activity.

Major comments:

(1) It is essential that the authors validate the efficiency of TeNT to prove that vesicular release is indeed inhibited, to be able to make any claims about the effect of vesicular release on oligodendrogenesis/myelination.

(2) Related to 1, can the authors clarify if their TeNT expression system results in the whole tract being silenced? It appears from Fig. 6 that their approach leads to sparse expression of TeNT in individual neurons, which enables them to measure myelination parameters. Can the authors discuss how silencing a single axon can lead to a regional effect in oligodendrocyte number?

(3) The authors need to fully revise their statistical analyses throughout and supply additional information that is needed to assess if their analyses are adequate:<br /> (3.1) the authors use a variety of statistical tests and it is not always obvious why they chose a particular test. For example, in Fig. 2G they chose a Kruskal-Wallis test instead of a two-way ANOVA or Mann-Whitney U test, which are much more common in the field. What is the rationale for the test choice?<br /> (3.2) in some cases, the choice of test appears wholly inappropriate. For example, in Fig. 3H-K, an unpaired t-test is inappropriate if the two regions were analysed in the same samples. In Fig. 5, was a t-test used for comparisons between multiple groups in the same dataset? If so, an ANOVA may be more appropriate.<br /> (3.3) in some cases, the authors do not mention which test was used (Fig 3: E-G no test indicated, despite asterisks; G/L/M - which regression test that was used? What does r indicate?)<br /> (3.4) more concerningly, throughout the results, data may have been pseudo-replicated. t-tests and ANOVAs assume that each observation in a dataset is independent of the other observations. In figures 1-4 and 6 there is a very large "n" number, but the authors do not indicate what this corresponds to. This leaves it open to interpretation, and the large values suggest that the number of nodes, internodal segments, or cells may have been used. These are not independent experimental units, and should be averaged per independent biological replicate - i.e. per animal (N).<br /> (3.5) related to the pseudo-replication issue, can the authors include individual datapoints in graphs for full transparency, per biological replicates, in addition or in alternative to bar-graphs (e.g. Fig. 5 and 6).

(4) The main finding of the study is that the density of nodes differs between two regions of the chicken auditory circuit, probably due to morphological differences in the respective oligodendrocytes. Can the authors discuss if this finding is likely to be specific to the bird auditory circuit?

(5) Provided the authors amend their statistical analyses, and assuming significant differences remain as shown, the study shows a correlation (but not causation) between node spacing and oligodendrocyte density, but the authors did not manipulate oligodendrocyte density per se (i.e. cell-autonomously). Therefore, the authors should either include such experiments, or revise some of their phrasing to soften their claims and conclusions. For example, the word "determine" in the title could be replaced by "correlate with" for a more accurate representation of the work. Similar sentences throughout the main text should be amended.

(6) The authors fail to introduce, or discuss, very pertinent prior studies, in particular to contextualize their findings with:<br /> (6.1) known neuron-autonomous modes of node formation prior to myelination, e.g. Zonta et al (PMID 18573915); Vagionitis et al (PMID 35172135); Freeman et al (PMID 25561543)<br /> (6.2) known effects of vesicular fusion directly on myelinating capacity and oligodendrogenesis, e.g. Mensch et al (PMID 25849985)<br /> (6.3) known correlation of myelin length and thickness with axonal diameter, e.g. Murray & Blakemore (PMID 7012280); Ibrahim et al (PMID 8583214); Hildebrand et al (PMID 8441812).<br /> (6.4) regional heterogeneity in the oligodendrocyte transcriptome (page 9, studies summarized in PMID 36313617)

Significance:

In our view the study tackles a fundamental question likely to be of interest to a specialized audience of cellular neuroscientists. This descriptive study is suggestive that in the studied system, oligodendrocyte density determines the spacing between nodes of Ranvier, but further manipulations of oligodendrocyte density per se are needed to test this convincingly.

Reviewer #3 (Public review):

Summary:

The authors have investigated the myelination pattern along the axons of chick avian cochlear nucleus. It has already been shown that there are regional differences in the internodal length of axons in the nucleus magnocellularis. In the tract region across the midline, internodes are longer than in the nucleus laminaris region. Here the authors suggest that the difference in internodal length is attributed to heterogeneity of oligodendrocytes. In the tract region oligodendrocytes would contribute longer myelin internodes, while oligodendrocytes in the nucleus laminaris region would synthesize shorter myelin internodes. Not only length of myelin internodes differs, but also along the same axon unmyelinated areas between two internodes may vary. This is an interesting contribution since all these differences contribute to differential conduction velocity regulating ipsilateral and contralateral innervation of coincidence detector neurons. However, the demonstration falls rather short of being convincing.

Major comments:

(1) The authors neglect the possibility that nodal cluster may be formed prior to myelin deposition. They have investigated stages E12 (no nodal clusters) and E15 (nodal cluster plus MAG+ myelin). Fig. 1D is of dubious quality. It would be important to investigate stages between E12 and E15 to observe the formation of pre-nodes, i.e., clustering of nodal components prior to myelin deposition.

(2) The claim that axonal diameter is constant along the axonal length need to be demonstrated at the EM level. This would also allow to measure possible regional differences in the thickness of the myelin sheath and number of myelin wraps.

(3) The observation that internodal length differs is explain by heterogeneity of sources of oligodendrocyte is not convincing. Oligodendrocytes a priori from the same origin remyelinate shorter internode after a demyelination event.

Significance:

The authors suggest that the difference in internodal length is attributed to heterogeneity of oligodendrocytes. In the tract region oligodendrocytes would contribute longer myelin internodes, while oligodendrocytes in the nucleus laminaris region would synthesize shorter myelin internodes. Not only length of myelin internodes differs, but also along the same axon unmyelinated areas between two internodes may vary. This is an interesting contribution since all these differences contribute to differential conduction velocity regulating ipsilateral and contralateral innervation of coincidence detector neurons.

Reviewer #1 (Public review):

Summary:

Ma & Yang et al. report a new investigation aimed at elucidating one of the key nutrients S. Typhimurium (STM) utilizes with the nutrient-poor intracellular niche within macrophage, focusing on the amino acid beta-alanine. From these data, the authors report that beta-alanine plays important roles in mediating STM infection and virulence. The authors employ a multidisciplinary approach that includes some mouse studies, and ultimately propose a mechanism by which panD, involved in B-Ala synthesis, mediates regulation of zinc homeostatisis in Salmonella.

Strengths and weaknesses:

The results and model are adequately supported by the authors' data. Further work will need to be performed to learn whether the Zn2+ functions as proposed in their mechanism. By performing a small set of confirmatory experiments in S. Typhi, the authors provide some evidence of relevance to human infections.

Impact:

This work adds to the body of literature on the metabolic flexibility of Salmonella during infection that enable pathogenesis.

Reviewer #3 (Public review):

Summary:

Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focussed on beta-alanine. It is also known that Salmonella requires beta-alanine from many other studies. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which is required for pathogenesis.

Strengths:

Made a couple of knockouts in Salmonella and did transcriptomic to understand the global gene expression pattern

Weaknesses:

Transport of Beta-alanine to SCV is not yet elucidated. Is it possible to determine whether the Zn transporter is involved in B-alanine transport?

Beta-alanine can also be shuttled to form carnosine along with histidine. If beta-alanine is channelled to make more carnosine, then the virulence phenotypes may be very different.

Some amino acid transporters can be knocked out to see if beta-alanine uptake is perturbed. Like ArgT transport Arginine, and its mutation perturbs the uptake of beta-alanine. What is the beta-alanine concentration in the SCV? SCVS can be purified at different time points, and the Beta-alanine concentration can be measured

Reviewer #2 (Public Review):

Summary:

This paper describes a new approach to detecting directed causal interactions between two genes without directly perturbing either gene. To check whether gene X influences gene Z, a reporter gene (Y) is engineered into the cell in such a way that (1) Y is under the same transcriptional control as X, and (2) Y does not influence Z. Then, under the null hypothesis that X does not affect Z, the authors derive an equation that describes the relationship between the covariance of X and Z and the covariance of Y and Z. Violation of this relationship can then be used to detect causality.

The authors benchmark their approach experimentally in several synthetic circuits. In 4 positive control circuits, X is a TetR-YFP fusion protein that represses Z, which is an RFP reporter. The proposed approach detected the repression interaction in 2 of the 4 positive control circuits. The authors constructed 16 negative control circuit designs in which X was again TetR-YFP, but where Z was either a constitutively expressed reporter, or simply the cellular growth rate. The proposed method detected a causal effect in two of the 16 negative controls, which the authors argue is perhaps not a false positive, but due to an unexpected causal effect. Overall, the data support the potential value of the proposed approach.

Strengths:

The idea of a "no-causality control" in the context of detected directed gene interactions is a valuable conceptual advance that could potentially see play in a variety of settings where perturbation-based causality detection experiments are made difficult by practical considerations.

By proving their mathematical result in the context of a continuous-time Markov chain, the authors use a more realistic model of the cell than, for instance, a set of deterministic ordinary differential equations.

The authors have improved the clarity and completeness of their proof compared to a previous version of the manuscript.

Limitations:

The authors themselves clearly outline the primary limitations of the study: The experimental benchmark is a proof of principle, and limited to synthetic circuits involving a handful of genes expressed on plasmids in E. coli. As acknowledged in the Discussion, negative controls were chosen based on the absence of known interactions, rather than perturbation experiments. Further work is needed to establish that this technique applies to other organisms and to biological networks involving a wider variety of genes and cellular functions. It seems to me that this paper's objective is not to delineate the technique's practical domain of validity, but rather to motivate this future work, and I think it succeeds in that.

Might your new "Proposed additional tests" subsection be better housed under Discussion rather than Results?

I may have missed this, but it doesn't look like you ran simulation benchmarks of your bootstrap-based test for checking whether the normalized covariances are equal. It would be useful to see in simulations how the true and false positive rates of that test vary with the usual suspects like sample size and noise strengths.

It looks like you estimated the uncertainty for eta_xz and eta_yz separately. Can you get the joint distribution? If you can do that, my intuition is you might be able to improve the power of the test (and maybe detect positive control #3?). For instance, if you can get your bootstraps for eta_xz and eta_yz together, could you just use a paired t-test to check for equality of means?

The proof is a lot better, and it's great that you nailed down the requirement on the decay of beta, but the proof is still confusing in some places:

- On pg 29, it says "That is, dividing the right equation in Eq. 5.8 with alpha, we write the ..." but the next equation doesn't obviously have anything to do with Eq. 5.8, and instead (I think) it comes from Eq 5.5. This could be clarified.

- Later on page 29, you write "We now evoke the requirement that the averages xt and yt are stationary", but then you just repeat Eq. 5.11 and set it to zero. Clearly you needed the limit condition to set Eq. 5.11 to zero, but it's not clear what you're using stationarity for. I mean, if you needed stationarity for 5.11 presumably you would have referenced it at that step.

It could be helpful for readers if you could spell out the practical implications of the theorem's assumptions (other than the no-causality requirement) by discussing examples of setups where it would or wouldn't hold.

Reviewer #1 (Public review):

The manuscript by Rios et al. investigates the potential of GSK3 inhibition to reprogram human macrophages, exploring its therapeutic implications in conditions like severe COVID-19. The authors present convincing evidence that GSK3 inhibition shifts macrophage phenotypes from pro-inflammatory to anti-inflammatory states, thus highlighting the GSK3-MAFB axis as a potential therapeutic target. Using both GM-CSF- and M-CSF-dependent monocyte-derived macrophages as model systems, the study provides extensive transcriptional, phenotypic, and functional characterizations of these reprogrammed cells. The authors further extend their findings to human alveolar macrophages derived from patient samples, demonstrating the clinical relevance of GSK3 inhibition in macrophage biology.

The experimental design is sound, leveraging techniques such as RNA-seq, flow cytometry, and bioenergetic profiling to generate a comprehensive dataset. The study's integration of multiple model systems and human samples strengthens its impact and relevance. The findings not only offer insights into macrophage plasticity but also propose novel therapeutic strategies for macrophage reprogramming in inflammatory diseases.

Strengths:

(1) Robust Experimental Design: The use of both in vitro and ex vivo models adds depth to the findings, making the conclusions applicable to both experimental and clinical settings.

(2) Thorough Data Analysis: The extensive use of RNA-seq and gene set enrichment analysis (GSEA) provides a clear transcriptional signature of the reprogrammed macrophages.

(3) Relevance to Severe COVID-19: The study's focus on macrophage reprogramming in the context of severe COVID-19 adds clinical significance, especially given the relevance of macrophage-driven inflammation in this disease.

Weaknesses:

There are no significant weaknesses in the study.

Reviewer #1 (Public review):

Summary:

The topic of tumor-immune co-evolution is an important, understudied topic with, as the authors noted, a general dearth of good models in this space. The authors have made important progress on the topic by introducing a stochastic branching process model of antigenicity/immunogenicity and measuring the proportion of simulated tumors that go extinct. The model is extensively explored, and the authors provide some nice theoretical results in addition to simulated results.

Major comments

The text in lines 183-191 is intuitively and nicely explained. However, I am not sure all of it follows from the figure panels in Figure 2. For example, the authors refer to a mutation that has a large immunogenicity, but it's not shown how many mutations, or the relative size of the mutations in Figure 2. The same comment holds true for the claim that spikes also arise for mutations with low antigenicity.

Reviewer #2 (Public review):

Summary:

In this work, the authors developed a model of tumour-immune dynamics, incorporating stochastic antigenic mutation accumulation and escape within the cancer cell population. They then used this model to investigate how tumour-immune interactions influence tumour outcome and summary statistics of sequencing data.

Strengths:

This novel modeling framework addresses an important and timely topic. The authors consider the useful question of how bulk and single-cell sequencing may provide insights into the tumour-immune interactions and selection processes.

Weaknesses:

One set of conclusions presented in the paper is the presence of cyclic dynamics between effector/cancer cells, antigenicity, and immunogenicity. However, these conclusions are supported in the manuscript by two sample trajectories of stochastic simulations, and these provide mixed support for the conclusions (i.e. the phasing asynchrony described in the text does not seem to apply to Figure 2C). Similarly, the authors also find immune selection effects on the shape of the mutational burden in Figure 5 D/H using a qualitative comparison between the distributions and theoretical predictions in the absence of immune response. However the discrepancy appears quite small in panel D, and there are no quantitative comparisons provided to evaluate the significance. An analysis of the robustness of all the conclusions to parameter variation is missing. Lastly, the role of the Appendix results in the main messages of the paper is unclear.

Reviewer #1 (Public review):

Processing in the primary visual cortex (V1) of mice is not only based on sensory inputs but also strongly modulated by locomotion. In this study, Meier et al. ask whether neurons that are modulated by locomotion form clusters in V1. Their work is based on previous studies from their lab establishing a modularity in the organization of primary visual cortex based on M2-muscarinic-acetylcholine-receptor-positive patches and interpatches (Ji et al. 2015, D'Souza et al. 2019). In these studies, they have highlighted the clustering of specific visual pathways and inhibition. In the current study, they extend this modularity to motor inputs, confirming a clustering of locomotion modulated neurons but also show that these clusters overlap with the M2-negative interpatches of layer 1. Finally, they establish a blueprint for visual processing streams in V1, segregating projections to and from lateral visual areas (LM, AL, and RL) from projections to and from the lateral areas, including the visual area PM, the retrosplenial cortex (RSP), and the secondary motor area (MOs).

Conceptually, this study provides an important finding in the organization of locomotion-related signaling in primary visual cortex, which clearly has substantial implications for sensory processing in visual cortex. While the anatomical data are solid, the link to physiology is incomplete. In conclusion, there are numerous issues that leave the main findings in some doubt, so the authors have some work to do before I find this story convincing.

Major issues:

(1) The major results in this study rely on proper quantification of neuronal responses during resting and running. Recently, it has been reported that hemodynamic occlusion can strongly influence measurements of fluorescent changes using two-photon imaging (Yogesh et al. 2025, doi.org/10.1101/2024.10.29.620650). Since it is unclear whether there is an inherent bias in vasculature and hemodynamic occlusion in M2 patches and interpatches, a quantification of the effect of hemodynamic occlusion would be necessary. This control would ideally be done using mice with GFP expression to test if there is still a clustering of locomotion-modulated neurons that overlaps with M2-negative interpatches. Alternatively, the authors should at the very least quantify the vascularization in M2 patches and interpatches.

(2) To assess the effects, the authors use a correlation analysis for many of their findings (e.g., Figures 2b,c, 4j,k, ...). This, however, is inappropriate to assess the significance of the results. I suggest redoing all statistics with hierarchical bootstrap sampling (Saravanan et al. 2020, PMID: 33644783) or similar.

(3) The authors use two different measures to assess whether and to what extent a neuron is locomotion sensitive, the LMI and "locomotion-responsive". While the LMI is defined based on recording in the light and dark (Figure 2), the "locomotion-responsiveness" is defined only in the dark (Figure 3a,c,d). The link between the two measures should be clarified.

a) Additionally, Figure 2b shows higher average LMI for interpatches, but the locomotion-responsive fraction is similar in interpatches and patches (relative number of pairs in Figure 3c and Figure 3d). How do the authors explain this discrepancy?

b) How is the LMI calculated - based on the average or the maximum response over stimuli? One particular stimulus? If the LMI is defined for each stimulus separately, what is plotted in Figure 2b?

(4) In the last panels of Figures 4-7, the authors analyze the alignment of cell bodies with the M2 patches. While in superficial layers it might be straightforward to align the cell body locations with the M2 patches and interpatches in layer 1, this alignment does not appear to be trivial for deeper layers. The authors should provide additional material to convince the reader of the proper alignment.

(5) Related to point 4 above - Given the importance of a proper alignment of M2 patches with the in vivo imaging, the in vivo - ex vivo alignment should be more convincing than Figure 1 C-E. Measuring M2 patches in vivo (as the authors have tried to do) would have provided more solid evidence. Have the authors tried to remove the dura for their in vivo imaging to increase signal-to-noise? In any case, more examples of proper alignment are necessary.

(6) The authors state that locomotion selectively affects M2-/M2- pairs based on Figure 3c. However, to make this claim, there should be a significant difference between the correlation of stimulus-driven noise of M2-/M2- locomotion-responsive pairs and M2-/M2- locomotion-unresponsive pairs, AND no significant difference in the same analysis for M2+/M2+ pairs (i.e., testing the differences between the bars in Figure 3c and Figure 3d).

Reviewer #2 (Public review):

Summary:

Meier et al. explore the variability of locomotion-related modulations in mouse area V1. They present 4 major findings: V1 L2/3 neurons beneath M2- interpatches are more strongly locomotion-modulated than those beneath M2+ patches, while V1 L2/3 neurons are more strongly orientation tuned. They then use viral tracing to examine the relationship of M2- interpatches and M2+ patches with inputs from and outputs to HVOs, MO, RSP, and LP, and find evidence for different closed-loop subnetworks within L1; these relationships, however, are more complicated for cell bodies in L2/3. Finally, they also describe an overlap between M2- interpatches and SOM+ dendrites/axons.

Strengths:

The strength of the manuscript is the detailed anatomical quantification of closed-loop connectivity, and the description of the organizing principles of M2- interpatches and M2+ patches.

Weaknesses:

The major weakness of the manuscript is the lack of a direct connection between the functional and the anatomical data, and the somewhat puzzling effects observed in the analysis of noise correlations. The former issue might be alleviated by modelling, where the authors could explore the space of possibilities that could explain the functional data based on the anatomical connectivity. Some control analyses could be done, for the comparison of noise correlations.

Reviewer #3 (Public review):

The authors build on the large body of their previous research, which showed that the mouse primary visual cortex is organised into two types of clusters, M2+ and M2-, which exhibit distinct input patterns from thalamus and higher visual cortical areas and distinct visual tuning preferences. The current study reveals that a like-to-like projection from within-cluster neurons to the areas that provide feedback projections and, furthermore, that neurons in the M2- clusters are more strongly affected by non-visual signals about the locomotion of the animal.

The study adds fundamental insights to our understanding of the principles of cortical organisation and computation, specifically how the cortex integrates sensory and action-related signals.

While the tracing data are very convincing, data analysis should be strengthened to support the claims:

(1) The locomotion modulation index (LMI) compares the mean activity during running and not running but does not seem to account for differences between visual stimuli, so that the LMI could be influenced by the neuron's visual tuning rather than its sensitivity to locomotion, e.g. if the mouse was running more when the neuron's preferred stimulus was presented. Trials should first be averaged per stimulus, and then across stimuli. Alternatively, only the preferred stimulus could be considered.

The significance test (unpaired t-test) suffers from the same flaw. Instead an ANOVA (with stimulus parameter as factor) would resolve the problem, or testing whether fitting the data with two tuning curves (one per locomotion state) or a single curve results in a lower error (using cross-validation).

Given that there is evidence that specific visual stimuli can induce more or less running in mice, this issue is very important to account for behavioural differences across stimuli.

(2) All bars in Figure 2b show a lower LMI than the reported mean LMI of 0.19. This should be checked.

(3) Correlation tests: Pearson correlation is only meaningful when applied to continuous data. A more suitable test for discrete data like the M2 patch quantile is a rank test like Kendall's coefficient of rank correlation. This applies to data in Figure 2b,c, 4j,k, Figure 2 - Supplement 2,1a, etc.

(4) How OSI was determined should be clarified. Specifically, were R_pref and R_ortho the mean responses to the two opposite movement directions? Similarly, how was the half-width at half-maximum of orientation determined? From the fits in Figure 2a, it looks like the widths of both Gaussians can be different.

(5) The correlation measures in Figure 3 would greatly benefit from additional analyses to help interpretation of the results.

a) Correlations between neurons typically increase with increasing firing rates (e.g., de la Rocha J, Doiron B, Shea-Brown E, Josić K, Reyes A. 2007. Correlation between neural spike trains increases with firing rate. Nature 448:802-6. doi:10.1038/nature06028). Could the higher correlations in M2+ pairs (Figure 3a) be explained by higher firing rates in M2+ compared to M2- neurons?

b) To determine correlations in Figure 3a, trials during locomotion and stationarity were pooled. As locomotion impacts the firing rate of the neurons, it would be helpful to separate correlations between the two states, locomotion vs stationarity, so the measures reflect something closer to "noise correlations" rather than tuning to locomotion.

c) Similarly, in Figure 3b, I wonder whether the large correlations in M2- pairs are driven by locomotion rather than functional connectivity. As suggested in b, a better test of noise correlations would be to account for locomotion, i.e., separate trials by stimulus identity and locomotion state. To prevent conditions with few trials from having greater weight in the overall noise correlations, I suggest the authors first z-score responses per condition, then determine noise correlations across all trials (as explained in Renart et al., 2010).

d) Correlations in Figure 3a,b should be tested with an ANOVA and a control for multiple tests.

(6) In plots like Figure 4j-l, it would be very informative to show individual measures (per ROI and mouse) in addition to mean +- SEM. As the counts are low (<10) it wouldn't obstruct the plot.

(7) The caption of Figure 4l says that most retrogradely labelled cells are located in L2/3. However, the plot only shows data from L2/3 and a single section of L4, so one cannot compare it to other layers. Can the authors corroborate the claim with data from other layers?

(8) Methods:<br /> The authors should provide more details on the visual stimuli: What was the background on which gratings were presented? How long was the inter-stimulus interval? What was presented during the inter-stimulus interval? How large were gratings used to map tuning to SF, TF, and orientation?

Reviewer #1 (Public review):

Summary:

The authors use a sophisticated task design and Bayesian computational modeling to test their hypothesis that information generalization (operationalized as a combination of self-insertion and social contagion) in social situations is disrupted in Borderline Personality Disorder. Their main finding relates to the observation that two different models best fit the two tested groups: While the model assuming both self-insertion and social contagion to be present when estimating others' social value preferences fit the control group best, a model assuming neither of these processes provided the best fit to BPD participants.

Strengths:

The revisions have substantially strengthened the paper and the manuscript is much clearer and easier to follow now. The strengths of the presented work lie in the sophisticated task design and the thorough investigation of their theory by use of mechanistic computational models to elucidate social decision-making and learning processes in BPD.

Weaknesses:

Some critical concerns remain after the first revision, particularly regarding the use of causal language and the clarity of the hypotheses and results, specified in the points below.

(1) The authors frequently refer to their predictions and theory as being causal, both in the manuscript and in their response to reviewers. However, causal inference requires careful experimental design, not just statistical prediction. For example, the claim that "algorithmic differences between those with BPD and matched healthy controls" are "causal" in my opinion is not warranted by the data, as the study does not employ experimental manipulations or interventions which might predictably affect parameter values. Even if model parameters can be seen as valid proxies to latent mechanisms, this does not automatically mean that such mechanisms cause the clinical distinction between BPD and CON, they could plausibly also refer to the effects of therapy or medication. I recommend that such causal language, also implicit to expressions like "parameter influences on explicit intentional attributions", is toned down throughout the manuscript.

(2) Although the authors have now much clearer outlined the stuy's aims, there still is a lack of clarity with respect to the authors' specific hypotheses. I understand that their primary predictions about disruptions to self-other generalisation processes underlying BPD are embedded in the four main models that are tested, but it is still unclear what specific hypotheses the authors had about group differences with respect to the tested models. I recommend the authors specify this in the introduction rather than refering to prior work where the same hypotheses may have been mentioned.

(3) Caveats should also be added about the exploratory nature of the many parameter group comparisons. If there are any predictions about group differences that can be made based on prior literature, the authors should make such links clear.

(4) I'm not sure I understand why the authors, after adding multiple comparison correction, now list two kinds of p-values. To me, this is misleading and precludes the point of multiple comparison corrections, I therefore recommend they report the FDR-adjusted p-values only. Likewise, if a corrected p-value is greater than 0.05 this should not be interpreted as a result.

(5) Can the authors please elaborate why the algorithm proposed to be employed by BPD is more 'entropic', especially given both their self-priors and posteriors about partners' preferences tended to be more precise than the ones used by CON? As far as I understand, there's nothing in the data to suggest BPD predictions should be more uncertain. In fact, this leads me to wonder, similarly to what another reviewer has already suggested, whether BPD participants generate self-referential priors over others in the same way CON participants do, they are just less favourable (i.e., in relation to oneself, but always less prosocial) - I think there is currently no model that would incorporate this possibility? It should at least be possible to explore this by checking if there is any statistical relationship between the estimated θ_ppt^m and 〖p(θ〗_par |D^0).

"To note, social contagion under M3 was highly correlated with contagion under M1 (see Fig S11). This provides some preliminary evidence that trauma impacts beliefs about individualism directly, whereas trauma and persecutory beliefs impact beliefs about prosociality through impaired trait mentalising" - I don't understand what the authors mean by this, can they please elaborate and add some explanation to the main text?

Reviewer #2 (Public review):

Summary:

The paper investigates social-decision making, and how this changes after observing the behaviour of other people, in borderline personality disorder. The paper employs a task including three phases, the first where participants make decision on how to allocate rewards to oneself and to a virtual partner, the second where they observe the same task performed by someone else, and a third phase equivalent to phase one, but with a new partner. Using sophisticated computational modelling to analyse choice data, the study reports that borderline participants (versus controls) are more certain about their preferences in phase one, used more neutral priors and are less flexible during phase two, and are less influenced by partners in phase three.

Strengths:

The topic is interesting and important, and the findings are potentially intriguing. The computational methods employed is clever and sophisticated, at the cutting edge of research in the field.

Weaknesses:

The paper is not based on specific empirical hypotheses formulated at the outset, but, rather, it uses an exploratory approach. Indeed, the task is not chosen in order to tackle specific empirical hypotheses. This, in my view, is a limitation since the introduction reads a bit vague and it is not always clear which gaps in the literature the paper aims to fill. As a further consequence, it is not always clear how the findings speak to previous theories on the topic.

Reviewer #3 (Public review):

In this paper, the authors use a three-phase economic game to examine the tendency to engage in prosocial versus competitive exchanges with three anonymous partners. In particular, they consider individual differences in the tendency to infer about others' tendencies based on one's preferences and to update one's preferences based on observations of others' behavior. The study includes a sample of individuals diagnosed with borderline personality disorder and a matched sample of psychiatrically healthy control participants.

On the whole, the experimental design is well-suited to the questions and the computational model analyses are thorough, including modern model-fitting procedures. I particularly appreciated the clear exposition regarding model parameterization and the descriptive Table 2 for qualitative model comparison. In the revised manuscript, the authors now provide a more thorough treatment of examining group differences in computational parameters given that the best-fitting model differed by group. They also examine the connection of their task and findings to related research focusing on self-other representation and mentalization (e.g., Story et al., 2024).

The authors note that the task does not encourage competition and instead captures individual differences in the motivation to allocate rewards to oneself and others in an interdependent setting. The paper could have been strengthened by clarifying how the Social Value Orientation framework can be used to interpret the motivations and behavior of BPD versus CON participants on the task. Although the authors note that their approach makes "clear and transparent a priori predictions," the paper could be improved by providing a clear and consolidated statement of these predictions so that the results could be interpreted vis-a-vis any a priori hypotheses.

Finally, the authors have amended their individual difference analyses to examine psychometric measures such as the CTQ alongside computational model parameter estimate differences. I appreciate that these analyses are described as exploratory. The approach of using a partial correlation network with bootstrapping (and permutation) was interesting, but the logic of the analysis was not clearly stated. In particular, there are large group (Table 1: CON vs. BPD) differences in the measures introduced into this network. As a result, it is hard to understand whether any partial correlations are driven primarily by mean differences in severity (correlations tend to be inflated in extreme groups designs due to the absence of observation in middle of scales forming each bivariate distribution). I would have found these exploratory analyses more revealing if group membership was controlled for.

Reviewer #2 (Public review):

Summary:

Demonstrate the breadth of IgA response as determined by isolating individual antigen-specific B cells and generating mAbs in mice following intranasal immunization of mice with SARS-CoV2 Spike protein. The findings show that some IgA mAb can neutralize the virus, but many do not. Notable immunization with Wuhan S protein generates a weak response to the omicron variant.

Strengths:

Detailed analysis characterizing individual B cells with the generation of mAbs demonstrates the response's breadth and diversity of IgA responses and the ability to generate systemic immune responses.

Comments on Revision:

I have re-reviewed the paper and responses to my and other reviewers' comments. I feel the authors have adequately addressed my and other reviewer's comments.

Reviewer #1 (Public review):

Summary:

Goal: Find downstream targets of cmk-1 phosphorylation, identify one that also seems to act in thermosensory habituation, test for genetic interactions between cmk-1 and this gene and assess where these genes are acting in the thermosensory circuit during thermosensory habituation.

Methods: Two in vitro analyses of cmk-1 phosphorylation of C. elegans proteins. Thermosensory habituation of cmk-1 and tax-6 mutants and double mutants was assessed by measuring rate of heat evoked reversals (reversal probability) of C. elegans before and after 20s ISI repeated heat pulses over 60 minutes.

Conclusions: cmk-1 and tax-6 act in separate habituation processes primarily in AFD, that interact complexly, but both serve to habituate the thermosensory reversal response. They found that cmk-1 primarily acts in AFD and tax-6 primarily acts in RIM (and FLP for naïve responses). They also identified hundreds of potential cmk-1 phosphorylation substrates in vitro.

Strengths:

The effects size in the genetic data is quite strong and a large number of genetic interaction experiments between cmk-1 and tax-1 demonstrate a complex interaction.

A major concern concerning this manuscript was the assumption that the process they are observing is habituation. The two previously cited papers using this (or a very similar) protocol, Lia and Glauser 2020 and Jordan and Glauser 2023, both use the word 'adaptation' to describe the observed behavioral decrement. Jordan and Glauser 2023 does occasionally use the words 'habituation' or 'habituation-like' 10 times, however it uses 'adaptation' over 100 times. It is critical to distinguish habituation from sensory adaptation (or fatigue) in this thermal reversal protocol. These processes are often confused/conflated, however they are very different; sensory adaptation is a process that decreases how much the nervous system is activated by a repeated stimulus, therefore it can even occur outside of the nervous system. Habituation is a learning process where the nervous system responds less to a repeated stimulus, despite (at least part of the nervous system) the nervous system still being similarly activated by the stimulus. Habituation is considered an attentional process, while adaptation is due to fatigue of sensory transduction machinery. Control experiments such as tests for dishabituation (where application of a different stimulus causes recovery of the decremented response) or rate of spontaneous recovery (more rapid recovery after short inter-stimulus intervals) are required to determine if habituation or sensory adaptation are occurring. These experiments will allow the results to be interpreted with clarity; without them, it isn't actually clear what biological process is actually being studied. The authors have accepted this distinction and now correctly call the process adaptation.

While there was originally some discrepancy between the two in vitro phosphorylation experiments and the in silico predictions, the revision has cleared up the issues.<br /> Figure 3 -S1: This model has been adjusted to more closely fit the data.

The authors have expanded the discussion about the significance of the sites of cmk-1 and tax-6 function in the neural circuit.

Reviewer #2 (Public review):

Summary:

The reduction in a response to a specific stimuli after repeated exposures is called habituation. Alterations in habituation to noxious stimuli are associated with chronic pain in humans, however the underlying molecular mechanisms involved are not clear. This study uses the nematode C. elegans to study genes and mechanisms that underlie adaptation to a form of noxious stimuli based on heat, termed thermo-noxious stimuli. The authors previously showed that the Calcium/Calmodulin-dependent protein kinase (CMK-1) regulates thermo-nociceptive adaptation in the nematode C. elegans. Although CMK-1 is a kinase with many known substrates, the downstream targets relevant for thermo-nociceptive adaptation are not known. In this study, the authors use two different kinase screens to identify phosphorylation targets of CMK-1. One of the targets they identify is Calcineurin (TAX-6). The authors show that CMK-1 phosphorylates a regulatory domain of Calcineurin at a highly conserved site (S443). In a series of elegant experiments, the authors use genetic and pharmacological approaches to increase or decrease CMK-1 and Calcineurin signaling to study their effects on thermo-nociceptive adaptation in C. elegans. They also combine these various approaches to study the interactions between these two signaling proteins. The authors use specific promoters to determine in which neurons CMK-1 and Calcineurin function to regulate thermo-nociceptive adaptation. The authors propose a model based on their findings, illustrating that CMK-1 and Calcineurin act mostly in different neurons to antagonistically regulate adaptation to thermo-nociceptive stimuli in a complex manner.

Strengths:

- Given the conservation of adaptation across phylogeny, identifying genes and mechanisms that underlie nociceptive adaptation in C. elegans may be relevant for understanding chronic pain in humans.<br /> - The identification of canonical CaM Kinase phosphorylation motifs in the substrates identified in the CMK-1 substrate screen validates the screen.<br /> - The use of loss and gain of function approaches to study the effects of CMK-1 and Calcineurin on thermo-nociceptive responses and adaptation is elegant.<br /> - The ability to determine the cellular place of action of CMK-1 and Calcineurin using neuron specific promoters in the nematode is a clear strength of the genetic model system.

Weaknesses:

- The manuscript begins by identifying Calcineurin as a direct substrate of CMK-1 but ends by showing that CMK-1 and Calcineurin mostly act in different neurons to regulate nociceptive adaptation, thus the physiological relevance of CMK-1 phosphorylation of Calcineurin is not clear.

Reviewer #1 (Public review):

Summary:

This manuscript described a structure-guided approach to graft important antigenic loops of the neuraminidase to a homotypic but heterologous NA. This approach allows the generation of well-expressed and thermostable recombinant proteins with antigenic epitopes of choice to some extent. The loop-grafted NA was designated hybrid.

Strengths:

The hybrid NA appeared to be more structurally stable than the loop-donor protein while acquiring its antigenicity. This approach is of value when developing a subunit NA vaccine which is difficult to express. So that antigenic loops could be potentially grafted to a stable NA scaffold to transfer strain-specific antigenicity.

Reviewer #2 (Public review):

In their manuscript, Rijal and colleagues describe a 'loop grafting' strategy to enhance expression levels and stability of recombinant neuraminidase. The work is interesting and important.

Major points from first round of review:

(1) The authors overstress the importance of the epitopes covered by the loops they use and play down the importance of antibodies binding to the side, the edges, or the underside of the NA. A number of papers describing those mAbs are also not included.

(2) The rationale regarding the PR8 hybrid is not well described and should be described better.

(3) Figure 3B and 6C: This should be given as numbers (quantified), not as '+'.

(4) Figure 5A and 7A: Negative controls are missing.

(5) The authors claim that they generate stable tetramers. Judging from SDS-PAGE provided in Supplementary Figure 3B (BS3-crosslined), many different species are present including monomers, dimers, tetramers, and degradation products of tetramers. In line 7 for example there are at least 5 bands.

[Editors' note: the authors have appropriately responded to and addressed these points.]

Reviewer #1 (Public review):

Summary:

This manuscript by Kremer et al. characterizes the tissue-specific responses to changes in TFAM levels and mtDNA copy number in prematurely aging mice (polg mutator model). The authors find that overexpression of TFAM can have beneficial or detrimental effects depending on the tissue type. For instance, increased TFAM levels increase mtDNA copy number in the spleen and improve spleen homeostasis but do not elevate mtDNA copy number in the liver and impair mtDNA expression. Similarly, the consequences of reduced TFAM expression are tissue-specific. Reduced TFAM levels improve brown adipocyte tissue function while other tissues are unaffected. The authors conclude that these tissue-specific responses to altered TFAM levels demonstrate that there are tissue-specific endogenous compensatory mechanisms in response to the continuous mutagenesis produced in the prematurely aging mice model, including upregulation of TFAM expression, elevated mtDNA copy number, and altered mtDNA gene expression. Thus, the impact of genetically manipulating global TFAM expression is limited and there must be other determinants of mtDNA copy number under pathological conditions beyond TFAM.

Strengths:

Overall, this is an interesting study. It does a good job of demonstrating that given the multi-functional role of TFAM, the outcome of manipulating its activity is complex.

Weaknesses:

No major weaknesses noted. The authors have adopted all our suggestions to improve the clarity of the manuscript.

Reviewer #2 (Public review):

Summary:

This study by Kremer et al. investigates the impact of modulation of expression of TFAM, a key protein involved in mitochondrial DNA (mtDNA) packaging and expression, in mtDNA mutator mice, which carry random mtDNA mutations. While previous research suggested that increasing TFAM could counteract the pathological effects of mtDNA mutations, this study reveals that the effects of TFAM modulation are tissue-specific. These findings highlight the complexity of mtDNA copy number regulation and gene expression, emphasizing that TFAM alone is not the sole determinant of mtDNA levels in contexts where oxidative phosphorylation is impaired. Other factors likely play a significant role, underscoring the need for nuanced approaches when targeting TFAM for therapeutic interventions.

Strengths:

The data presented in the manuscript are of high quality and support the major conclusions.

Comments on revisions:

The authors have thoroughly addressed all the points raised during the first round of review. Their revisions effectively clarify key aspects of the manuscript, and the additional data and explanations have significantly improved the overall quality of the work. I believe the manuscript is now well-prepared for publication.

Reviewer #1 (Public review):

Summary:

In this paper Kawasaki et al describe a regulatory role for the PIWI/piRNA pathway in rRNA regulation in Zebrafish. This regulatory role was uncovered through a screen for gonadogenesis defective mutants, which identified a mutation in the meioc gene, a coiled-coil germ granule protein. Loss of this gene leads to redistribution of Piwil1 from germ granules to the nucleolus, resulting in silencing of rRNA transcription.

Strengths:

Most of the experimental data provided in this paper is compelling. It is clear that in the absence of meioc, PiwiL1 translocates in to the nucleolus and results in down regulation of rRNA transcription. the genetic compensation of meioc mutant phenotypes (both organismal and molecular) through reduction in PiwiL1 levels are evidence for a direct role for PiwiL1 in mediating the phenotypes of meioc mutant.

Weaknesses:

Questions remain on the mechanistic details by which PiwiL1 mediated rRNA down regulation, and whether this is a function of Piwi in an unperturbed/wildtype setting. There is certainly some evidence provided in support of the a natural function for piwi in regulating rRNA transcription (figure 5A+5B). However, the de-enrichment of H3K9me3 in the heterozygous (Figure 6F) is very modest and in my opinion not convincingly different relative to the control provided. It is certainly possible that PiwiL1 is regulating levels through cleavage of nascent transcripts. Another aspect I found confounding here is the reduction in rRNA small RNAs in the meioc mutant; I would have assumed that the interaction of PiwiL1 with the rRNA is mediated through small RNAs but the reduction in numbers do not support this model. But perhaps it is simply a redistribution of small RNAs that is occurring. Finally, the ability to reduce PiwiL1 in the nucleolus through polI inhibition with actD and BMH-21 is surprising. What drives the accumulation of PiwiL1 in the nucleolus then if in the meioc mutant there is less transcription anyway?

Despite the weaknesses outlined, overall I find this paper to be solid and valuable, providing evidence for a consistent link between PIWI systems and ribosomal biogenesis. Their results are likely to be of interest to people in the community, and provide tools for further elucidating the reasons for this link.

Reviewer #2 (Public review):

Summary:

In this study, the authors report that Meioc is required to upregulate rRNA transcription and promote differentiation of spermatogonial stem cells in zebrafish. The authors show that upregulated protein synthesis is required to support spermatogonial stem cells' differentiation into multi-celled cysts of spermatogonia. Coiled coil protein Meioc is required for this upregulated protein synthesis and for increasing rRNA transcription, such that the Meioc knockout accumulates 1-2 cell spermatogonia and fails to produce cysts with more than 8 spermatogonia. The Meioc knockout exhibits continued transcriptional repression of rDNA. Meioc interacts with and sequesters Piwil1 to the cytoplasm. Loss of Meioc increases Piwil1 localization to the nucleolus, where Piwil1 interacts with transcriptional silencers that repress rRNA transcription.

Strengths:

This is fundamental study that expands our understanding of how ribosome biogenesis contributes to differentiation and demonstrates that zebrafish Meioc plays a role in this process during spermatogenesis. This work also expands our evolutionary understanding of Meioc and Ythdc2's molecular roles in germline differentiation. In mouse, the Meioc knockout phenocopies the Ythdc2 knockout, and studies thus far have indicated that Meioc and Ythdc2 act together to regulate germline differentiation. Here, in zebrafish, Meioc has acquired a Ythdc2-independent function. This study also identifies a new role for Piwil1 in directing transcriptional silencing of rDNA.

Comments on revisions:

Major and minor concerns were addressed in the revision.

Reviewer #3 (Public review):

Summary:

The paper describes the molecular pathway to regulate germ cell differentiation in zebrafish through ribosomal RNA biogenesis. Meioc sequesters Piwil1, a Piwi homolog, which suppresses the transcription of the 45S pre-rDNA by the formation of heterochromatin, to the perinuclear bodies.

Strong points:

The authors nicely provided the molecular evidence on the antagonism of Meioc to Piwil1 in the rRNA synthesis, which supported by the genetic evidence that the inability of the meioc mutant to enter meiosis is suppressed by the piwil1 heterozygosity. The authors nicely address my previous points.

Weak points:

Although the authors made an effort to revise the text. However, there are still some points that the authors need to check their text. Some of them are shown in "Minor points" below. I am sorry that some of them should have been pointed in my previous review.

Reviewer #1 (Public Review):

The study starts with the notion that in an AD-like disease model, ILC2s in the Rag1 knock-out were expanded and contained relatively more IL-5+ and IL-13+ ILC2s. This was confirmed in the Rag2 knock-out mouse model.

By using a chimeric mouse model in which wild-type knock-out splenocytes were injected into irradiated Rag1 knock-out mice, it was shown that even though the adaptive lymphocyte compartment was restored, there were increased AD-like symptoms and increased ILC2 expansion and activity. Moreover, in the reverse chimeric model, i.e. injecting a mix of wild-type and Rag1 knock-out splenocytes into irradiated wild-type animals, it was shown that the Rag1 knock-out ILC2s expanded more and were more active. Therefore, the authors could conclude that the RAG1 mediated effects were ILC2 cell-intrinsic.

Subsequent fate-mapping experiments using the Rag1Cre;reporter mouse model showed that there were indeed RAGnaïve and RAGexp ILC2 populations within naïve mice. Lastly, the authors performed multi-omic profiling, using single-cell RNA sequencing and ATAC-sequencing, in which a specific gene expression profile was associated with ILC2. These included well-known genes but the authors notably also found expression of Ccl1 and Ccr8 within the ILC2. The authors confirmed their earlier observations that in the RAGexp ILC2 population, the Th2 regulome was more suppressed, i.e. more closed, compared to the RAGnaïve population, indicative of the suppressive function of RAG on ILC2 activity. I do agree with the authors' notion that the main weakness was that this study lacks the mechanism by which RAG regulates these changes in ILC2s.

The manuscript is very well written and easy to follow, and the compelling conclusions are well supported by the data. The experiments are meticulously designed and presented. I wish to commend the authors for the study's quality.

Reviewer #2 (Public Review):

Summary:

The study by Ver Heul et al., investigates the consequences of RAG expression for type 2 innate lymphoid cell (ILC2) function. RAG expression is essential for the generation of the receptors expressed by B and T cells and their subsequent development. Innate lymphocytes, which arise from the same initial progenitor populations, are in part defined by their ability to develop in the absence of RAG expression. However, it has been described in multiple studies that a significant proportion of innate lymphocytes show a history of Rag expression. In compelling studies several years ago, members of this research team revealed that early Rag expression during the development of Natural Killer cells (Karo et al., Cell 2014), the first described innate lymphocyte, had functional consequences.

Here, the authors revisit this topic, a worthwhile endeavour given the broad history of Rag expression within all ILCs and the common use of RAG-deficient mice to specifically assess ILC function. Focusing on ILC2s and utilising state-of-the-art approaches, the authors sought to understand whether early expression of Rag during ILC2 development had consequences for activity, fitness, or function. Having identified cell-intrinsic effects in vivo, the authors investigated the causes of this, identifying epigenetic changes associated with the accessibility genes associated with core ILC2 functions.

The manuscript is well written and does an excellent job of supporting the reader through reasonably complex transcriptional and epigenetic analyses, with considerate use of explanatory diagrams. Overall I think that the conclusions are fair, the topic is thought-provoking, and the research is likely of broad immunological interest. I think that the extent of functional data and mechanistic insight is appropriate.

Strengths:

- The logical and stepwise use of mouse models to first demonstrate the impact on ILC2 function in vivo and a cell-intrinsic role. Initial analyses show enhanced cytokine production by ILC2 from RAG-deficient mice. Then through two different chimeric mice (including BM chimeras), the authors convincingly show this is cell intrinsic and not simply as a result of lymphopenia. This is important given other studies implicating enhanced ILC function in RAG-/- mice reflect altered competition for resources (e.g. cytokines).

- Use of Rag expression fate mapping to support analyses of how cells were impacted - this enables a robust platform supporting subsequent analyses of the consequences of Rag expression for ILC2.

- Use of snRNA-seq supports gene expression and chromatin accessibility studies - these reveal clear differences in the data sets consistent with altered ILC2 function.

- Convincing evidence of epigenetic changes associated with loci strongly linked to ILC2 function. This forms a detailed analysis that potentially helps explain some of the altered ILC2 functions observed in ex vivo stimulation assays.

- Provision of a wealth of expression data and bioinformatics analyses that can serve as valuable resources to the field.

Reviewer #1 (Public review):

Summary:

The authors aimed to classify hepatocellular carcinoma (HCC) patients into distinct subtypes using a comprehensive multi-omics approach. They employed an innovative consensus clustering method that integrates multiple omics data types, including mRNA, lncRNA, miRNA, DNA methylation, and somatic mutations. The study further sought to validate these subtypes by developing prognostic models using machine learning algorithms and extending the findings through single-cell RNA sequencing (scRNA-seq) to explore the cellular mechanisms driving subtype-specific prognostic differences.

Strengths:

(1) Comprehensive Data Integration: The study's integration of various omics data provides a well-rounded view of the molecular characteristics underlying HCC. This multi-omics approach is a significant strength, as it allows for a more accurate and detailed classification of cancer subtypes.

(2) Innovative Methodology: The use of a consensus clustering approach that combines results from 10 different clustering algorithms is a notable methodological advancement. This approach reduces the bias that can result from relying on a single clustering method, enhancing the robustness of the findings.

(3) Machine Learning-Based Prognostic Modeling: The authors rigorously apply a wide array of machine learning algorithms to develop and validate prognostic models, testing 101 different algorithm combinations. This comprehensive approach underscores the study's commitment to identifying the most predictive models, which is a considerable strength.

(4) Validation Across Multiple Cohorts: The external validation of findings in independent cohorts is a critical strength, as it increases the generalizability and reliability of the results. This step is essential for demonstrating the clinical relevance of the proposed subtypes and prognostic models.

Weaknesses:

(1) Inconsistent Storyline:<br /> Despite the extensive data mining and rigorous methodologies, the manuscript suffers from a lack of a coherent and consistent narrative. The transition between different sections, particularly from multi-omics data integration to single-cell validation, feels disjointed. A clearer articulation of how each analysis ties into the overall research question would improve the manuscript.

(2) Questionable Relevance of Immune Cell Activity Analysis:<br /> The evaluation of immune cell activities within the cancer cell model raises concerns about its meaningfulness. The methods used to assess immune function in the tumor microenvironment may not be fully appropriate, potentially limiting the insights gained from this part of the study.

(3) Incomplete Single-Cell RNA-Seq Validation:<br /> The validation of the findings using single-cell RNA-seq data appears insufficient to fully support the study's claims. While the authors make an effort to extend their findings to the single-cell level, the analysis lacks depth. A more comprehensive validation is necessary to substantiate the robustness of the identified subtypes.

(4) Figures and Visualizations:<br /> Several figures in the manuscript are missing necessary information, which affects the clarity of the results. For instance, the pathways in Figure 3A could be clustered to enhance interpretability, the blue bar in Figure 4A is unexplained, and Figure 4B is not discussed in the text. Additionally, the figure legend in Figure 7C lacks detail, and many figure descriptions merely repeat the captions without providing deeper insights.

(5) Appraisal of the Study's Aims and Results<br /> The authors have set out to achieve an ambitious goal of classifying HCC patients into distinct prognostic subtypes and validating these findings through both bulk and single-cell analyses. While the methodologies employed are innovative and the data integration comprehensive, the study falls short in fully achieving its aims due to inconsistencies in the narrative and incomplete validation. The results partially support the conclusions, but the lack of coherence and depth in certain areas limits the overall<br /> impact of the study.

(6) Impact on the Field<br /> If the identified weaknesses are addressed, this study has the potential to significantly impact the field of HCC research. The multi-omics approach combined with machine learning is a powerful framework that could set a new standard for cancer subtype classification. However, the current state of the manuscript leaves some uncertainty regarding the practical applicability of the findings, particularly in clinical settings.

(7) Additional Context<br /> For readers and researchers, this study offers a valuable look into the potential of integrating multi-omics data with machine learning to improve cancer classification and prognostication. However, readers should be aware of the noted weaknesses, particularly the need for more consistent narrative development and comprehensive validation of the methods. Addressing these issues could greatly enhance the study's utility and relevance to the community.

Comments on revisions:

The authors have addressed the reviewers' concerns effectively.

Reviewer #1 (Public review):

Summary:

The authors propose a new method to quantitatively assess morphogenetic processes during organismal development. They apply their method to ascidian morphogenesis and thus find that gastrulation is a two-step process.

The method applies to morphogenetic changes of surfaces. It consists of the following steps: first, surface deformations are quantified based on microscopy images without requiring cellular segmentation and tracking. This is achieved by mapping, at each time point, a polygonal mesh initially defined on a sphere to the surface of the embryo. The mapped vertices of this polygonal mesh then serve as (Lagrangian) markers for the embryonic surface. From these, one can infer the deformation of the surface, which can be expressed in terms of the strain tensor at each point of the surface. Changes in the strain tensor give the strain rate, which captures the morphogenetic processes. Second, at each time point, the strain rate field is decomposed in terms of spherical harmonics. Finally, the evolution of the weights of the various spherical harmonics in the decomposition is analysed via a wavelet analysis. The authors apply their workflow to ascidian development between 4 and 8.7 hpf. From their analysis they find clear indications for gastrulation and neurulation and identify two sub-phases of gastrulation, namely, endoderm invagination and 'blastophore closure'.

Strengths:

The combination of various tools allows the authors to obtain a quantitative description of the developing embryo without the necessity of identifying fiducial markers. Visual inspection shows that their method works well. Furthermore, this quantification then allows for an unbiased identification of different morphogenetic phases.

Weaknesses:

At times, the explanation of the method is hard to follow, unless the reader is already familiar with concepts like level-set methods or wavelet transforms. Furthermore, the software for performing the determination of Lagrangian markers or the subsequent spectral analysis does not seem to be available to the readers.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors proposed a method to quantitatively analyze 3D live imaging data of early developing embryos, using the ascidian development as an example. For this purpose, the previously proposed level set method was used to computationally track the temporal evolution of reference points introduced on the embryo surface. Then, from the obtained three-dimensional trajectories, the velocity field was obtained, from which the strain rate field was computed. The strain rate field was analyzed using spherical harmonics.

In this paper, the authors focused on the modes with lower order with real coefficients. The time evolution of these modes was analyzed using wavelet transforms. The results obtained by the pipeline reflected the developmental stages of ascidian embryos.

Strengths:

In this way, this manuscript proposes a pipeline of analyses combining various methods. The strength of this method lies in its ability to quantitatively analyze the deformation of the entire embryo without the requirement for cellular segmentation and tracking.

Weaknesses:

The mathematics behind this method is not straightforward to understand. The value of this method will be understood as analyses of real data using this method accumulate.

Comments on revised version:

I have reviewed the revised manuscript and the reply from the authors. All concerns have been addressed appropriately.

Reviewer #2 (Public review):

The revised manuscript by Genzoni et al. reports the striking discovery of a regulatory role for trophic eggs. Prior to this study, trophic eggs were widely assumed to play a nutritional role in the colony, but this study shows that trophic eggs can suppress queen development, and therefore, can play a role in regulating caste determination in specific social contexts. In this revised version of the manuscript, the authors have addressed many of the concerns raised in the first version regarding the lack of sufficient information and context in the Introduction and Discussion. I have several (mostly minor) comments I would like the authors to address:

Comments:

(1) The authors' experimental design is based on the comparison of a larva-only (control) versus larva+3 trophic eggs (treatment). The authors convincingly show that the larva plus 3 trophic eggs treatment has an inhibitory effect versus larva-only control. However, the authors should have also done a treatment composed of larva + 3 viable eggs to determine if the inhibitory effect observed on queens is specific to trophic eggs or whether it is an inhibitory effect of all eggs. This has had important mechanistic consequences, because if the inhibitory effect is specific to trophic eggs, it means there are specific inhibitory factors deposited in trophic eggs during oogenesis and the differences observed between trophic versus viable eggs are meaningful beyond just nutritional differences. If the inhibitory effect is a property of all eggs, then the inhibitory factor is dumped into all eggs and the differences observed between trophic and viable eggs are related to something else. In all cases, this reviewer is not necessarily asking that they perform this additional treatment, but the authors have to be clear in the text that they cannot claim that the inhibitory effect is specific to trophic eggs alone without doing this experiment.

(2) The other untested assumption the authors are making is that queen-laid trophic eggs would behave the same as worker-laid trophic eggs. This is apparent in the Discussion (line 422). They should instead highlight the interesting question of whether worker-laid trophic eggs would be similar in composition and have the same effect on caste as queen-laid eggs.

(3) To this reviewer, they are missing a crucial explanation in the discussion. As far as this reviewer knows, young queens produce a higher proportion of trophic eggs than older queens, meaning that trophic egg production decreases with age of the queen. This raises the possibility that trophic eggs may, in part, function to prevent the production of more virgin queens in young and immature colonies with small colony sizes. This would allow colonies to invest in producing more workers at a time when rapidly expanding the colony is crucial in young colonies' life. Production of trophic eggs, therefore, may have a dual function: one for nutrition and larval survival, and one in suppressing queen development in immature young colonies. It can be said then that trophic eggs can regulate / influence caste determination in specific social / life history contexts of the colony, rather than only proposing that trophic eggs are a constant attempt by the queen to manipulate her offspring. I prefer the superorganism explanation, but readers should at least hear explanations at the individual and superorganism scales as a way of explaining the authors' discovery that trophic eggs suppress further queen development.

(4) Why did the authors change the wording from caste "determination" to caste "differentiation." Determination is more appropriate because the trophic eggs do not affect morphogenesis of queens or workers, but rather the developmental switch between queens and workers.

(5) Khila and Abouheif (2008) is listed in the References but not cited in the text.

(6) On Line 70-81: "...may play a role in the regulation of body size" - I think the authors are trying to be broad in their language here since one study showed trophic eggs increased worker size but didn't induce queens, but this statement implies that the hypothesis is that trophic eggs act via body size to affect caste. Since the authors don't measure body size changes, only binary caste outcome, this is not the best way to set up the question. Could instead just conclude that previous work shows an effect on both caste and body size.

(7) Paragraph beginning line 432: this paragraph seems out of place, not well connected to previous parts of discussion. It introduces the term "egg cannibalism" without defining it - not clear if this is meant as a synonym for eating of trophic eggs, or broader (i.e., eating viable eggs also). Could either remove the paragraph, or better set up the context that egg-eating behaviour is common in ants, could have evolved for worker policing reasons and/or for nutritional exchange, trophic eggs (and potentially co-option of trophic eggs for caste determination functions) presumably evolved in this context of existing egg-eating behaviour.

(8) Line 41: Should read 'play an important part.

(9) Line 51: The food that was given is listed, but there is no information about the quantity of food given.

(10) Line 74: The paragraph states that queens were isolated for 16 hours per day. However, it lacks a clear reason for this specific duration. Why 16 hours? Could this isolation period have impacted egg quality or larval development?

(11) Line 76: The eggs were collected every 8 hours and then held for 10 days until hatching. This is a very long time for eggs to be held outside of the normal colony environment. This could have a large impact on the viability of the eggs, and the resulting larvae.

(12) Line 78: twice "that" in "suggested that that the larger castes"

(13) Lines 96-97: the following sentence is unclear: "The question mark indicates that it is unclear whether about the evidence for the production trophic eggs by queens and workers"

(14) Line 209: By simply stating "binomial GLMM," the authors are leaving out a crucial piece of information. Readers cannot fully understand how the model was fitted or how the coefficients should be interpreted without knowing the link function. Therefore, the critique is that for complete and replicable science, the link function must be reported.

Reviewer #1 (Public review):

Summary:

The authors' stated aim is to introduce so-called Minkowski tensors to characterize and quantify the shape of cells in tissues. The authors introduce Minkowski tensors and then define the p-atic order q_p, where p is an integer, as a cell shape measure. They also introduce a previously defined measure of p-atic order in the form of the parameter γ_p. The authors compute q_pp for data obtained by simulating an active vertex model and a multiphase field model, where they focus on p=2 and p=6 - nematic and hexatic order - as the two values of highest biological relevance. Based on their analysis, the authors claim that q₂ and q₆ are independent, that there is no crossover for the coarse-grained quantities, that the comparison of q_p for different values of p is not meaningful, and determine the dependence of the mean value of q₂ and q₆q₆ on cell activity and deformability. They then apply their method to data from MDCK monolayers and argue that the γ_p "fail to capture the nuances of irregular cell shapes".

Strength:

The work presents a set of parameters that are useful for analyzing cell shape.

Weaknesses:

The main weakness of the manuscript is that the points that the authors make are not sufficiently elaborated or supported by the data. Although they start out with Minkowski tensors, they eventually only consider the parameters q_p, which can be defined without any recourse to Minkowski tensors. Also, I dare to doubt that the average reader will benefit from the introduction to Minkowski tensors as it remains abstract and does not really go beyond repeating definitions. Eventually, for me, the work boils down to the statement that when you want to characterize (2d) cell shape, then it is better to take the whole cell contour instead of only the positions of the vertices of a polygon that approximates the full cell shape. By the way, for polygons, the q_p and γ_p should convey the same information as the vertex positions contain the whole geometric information.

Some statements made about the values of q_p are not supported by the data. For example, an independence of values of q₂ and q₆ cannot be inferred from Figure 7. Actually, Figure 8 points to some dependence between these values as the peaks of the pdfs move in the opposite direction as deformability and activity are changed. Figure 1 suggests that in general, larger cells have lower values of q_p for all p. Some more serious quantification should be obtained here.

The presented experimental data on MDCK cells is anecdotal.

Reviewer #2 (Public review):

Summary:

Orientational symmetries of cells and tissues play an important role in describing processes in development and disease, and the methods used to investigate them rely on the detection of cell shape. In this interesting and very timely manuscript by Lea Happel et al., Minkowski tensors are introduced to study the orientational symmetries of cells and set in comparison to existing shape descriptors, such as the shape function introduced by Armengol-Collado et al., which captures the orientational symmetry by the vertex positions of the polygonal shape of the cell. As an advantage, the Minkowski tensors consider the real cell shape with its arbitrary curvature of the cortex. Using computational models, such as the active vertex model and the multiphase field model, as well as experimental support with MDCK monolayers, the authors find that the orientational symmetries are independent of one another, as well as that they are dependent on the activity and deformability of the cells, resulting in a monotonic trend. A trend that has not been observed for the hexatic symmetry using the shape function. Together with the lack of hexatic-nematic crossover at the tissue scale, the authors suggest a reconsideration of findings from other shape descriptors. Taken together, the Minkowski tensors set a framework to investigate orientational symmetries at a single cell scale and how they may interplay in biological tissues.

Strengths:

The authors introduce the Minkowski tensors, which capture the p-atic orders of cells in tissues, considering their real shape instead of a polygonal approximation as reported for other shape descriptors in the literature. Thus, they do not depend on the vertex positions of the cells nor on the number of neighboring cells. The Minkowski tensors capture the dependence of the p-atic orders on the cell activity and deformability in a monotonic manner, which makes them a robust tool for quantifying p-atic orders at a single-cell scale, especially for rounded cells. The robustness has been tested by comparing the results of two computational model systems that simulate cell monolayers and whose results have been extended with experimental data. The Minkowski tensors have been used to explore the role of cell-cell adhesion and density in epithelial cells and have shown similar results to the shape function, a polygonal shape descriptor.

Weaknesses:

The authors point out the importance of studying the orientational order in biological systems. However, the current version of the manuscript lacks statistical information, a description of analysis methods, and experimental support. This support is needed to strengthen (i) the results of the two computational models and (ii) give weight to the authors' strong claim against other widely accepted shape descriptors capturing p-atic orders. The Minkowski tensors, which consider the real cell shapes, are reported to be a better method to investigate the p-atic orders of cells than the shape function introduced by Armengol-Collado et al. While there may be differences in the reported results coming from the two different approaches, both approaches show similar trends. As it stands, there is substantiated discussion as to why one method would be better than the other. The shape function, γ₆, may not be monotonic for great changes in cell activity and deformability, hinting at a potential weakness. In contrast to the shape function and results by Armengol-Collado et al. and Eckert et al., the coarse-grained Minkowski tensors do not capture the hexatic-nematic crossover at the tissue scale, applied here only to computational models. The cells simulated in the computational models have a similar size and the monolayer has a nearly regular pattern, which does not reflect the density variance in biological tissues. To strengthen the author's claim that there is no crossover at the tissue scale, experimental verification is essential. Further, the robustness of the Minkowski tensors seems to rely on determining the p-atic orders on the shape of individual cells in the tissue. However, when applying the shape descriptor to experimental systems, the p-atic orders are very low, perhaps too low for comparisons between different p-atic orders with meaningful conclusions.

Reviewer #3 (Public review):

Hapel et al. submit an article entitled “Quantifying the shape of cells - from Minkowski tensors to p-atic order”. The paper reports the p-actic quantitative method - established in physics - to extract cell shapes in experiments using phase contrast images of MDCK cells and simulations - vertex model and phase fields. The rationale of the quantification with adaptation of Minkowski tensors, as well as the detailed extraction of distributions of shapes and plots, distributions quantifying shapes are documented, with an emphasis on changes in cell shapes and their importance in epithelial dynamics.

Higher rank tensors are considered as well as representations with intuitive meanings and q_i orders and their potential correlations or absence of correlations. For example, q₂ and q₆, and statements about nematic and hexatic orders. A strong body of evidence is already reported in the papers of Armengol et al., quoted substantially in the paper, and the authors insist on an improvement thanks to the Minkowski tensors approach to challenge the former crossovers correlations statements.

Although the approach seems to present advantages, the paper does not appear sufficiently novel. Beyond the Armengol et al. paper, the advantages of this approach compared to the shear decomposition (from MPI-PKS Dresden) or the links joining centroids and its neighbours approach (MSC/Curie Paris) for example.

Reviewer #1 (Public review):

Summary:

In recent years, it has become increasingly evident how beautifully intricate IAC are at the nanoscale. Studies like the one presented here that shed light on the precise inner organisation of IAC are thus quite important and relevant in order to obtain a better in-depth understanding of IAC functioning and the contribution of different integrin subtypes to cell adhesive and mechanotransductive processes.

Interestingly, the authors found a distinct localisation of α5β1 and αVβ3 integrin nanoclusters within focal adhesion of human fibroblasts, with α5β1 integrin nanoclusters being at the periphery of IAC and αVβ3 integrin nanoclusters randomly distributed. Furthermore, a surprisingly high percentage of inactive integrins within IAC and relatively low spatial integrin colocalisation with adaptor proteins has been shown.

Strengths:

This is a very thoroughly performed STORM-based assessment of the nanodistribution of α5β1 and αVβ3 nanoclusters within IAC (and outside). The image quality is outstanding, and the authors have meticulously executed the experiments and the image analyses.

Weaknesses:

The only weakness is maybe that the manuscript remains descriptive. However, the high quality of the "description" of the nano-organisation of IAC by this scrupulous study is really important to better understand the inner workings of IAC. It provides a very solid foundation to look deeper into the (patho)physiological implications of this organisation, see recommendations (which are rather suggestions in this case).

Reviewer #2 (Public review):

Summary:

In this study, dual-color super-resolution microscopy analysis was performed to study the co-operation between integrins and focal adhesion proteins in human fibroblast cells. The study focused on two integrins which have been previously found to be mainly responsible for focal adhesions, namely α5β1 and αvβ3.

Specifically, the study tried to shed light on the nanoclustering of integrins in focal adhesions.

In the current study, more integrin nanoclusters were observed in focal adhesions compared to other cell-matrix adhesion structures. The study revealed that both α5β1 and αvβ3 form nanoclusters, and those appear segregated from each other. While αvβ3 nanoclusters organize randomly inside focal adhesions regardless of their activation state, α5β1 nanoclusters, and particularly the nanoclusters containing β1-integrin in active conformation, preferentially organized at the edges of focal adhesions. The nanoclusters formed by each integrin were similar in size.

Cytoplasmic adapter proteins appeared less in nanocluster assemblies, suggesting that integrin nanoclusters are also forming without the studied cytoplasmic adapter proteins (talin, vinculin, paxillin). Active integrins were identified with the help of conformation-specific antibodies, and this enabled us to study the colocalization between integrins and their cytoplasmic adapter proteins. This analysis revealed that activated integrins are strongly engaged with adapter proteins

Strengths:

The study stems from the thorough computational modelling of the nanoclusters, which enables quantification of the behavior of the clusters, including their mesoscale distribution.

The study strengthens the view that α5β1 and αvβ3 have specific functions in focal adhesions, α5β1 nanoclusters localizing preferentially on focal adhesion edges. The study also revealed that nanoclusters localized at the edges of focal adhesion were enriched for talin and paxillin but not for vinculin.

Analysis of adaptor protein nanoclusters (paxillin, talin, and vinculin) revealed that all adapter protein nanoclusters studied here close to active β1 nanoclusters are enriched on the focal adhesion edge region, whereas integrin adaptor nanoclusters far from active β1 appear to be more uniformly distributed.

Importantly, the current study suggests that integrin subtype-specific nanoclusters are not only present at an early stage of adhesion formation, but integrin nanoclusters remain segregated from each other also in mature focal adhesions, maintaining their sizes and number of molecules.

Interestingly, the study revealed that selected cytoplasmic adaptors (paxillin, talin, and vinculin), also form nanoclusters of similar size and number of single molecule localizations as the integrins, regardless of whether they locate inside or outside focal adhesions. The adapter nanoclusters are enriched in the focal adhesion "belt", colocalizing with the active α5β1 integrin nanoclusters.

Weaknesses:

The current study is highly dependent on the antibodies. It is possible that antibodies containing two binding sites for antigen influence the nanoscale organization (and also activation) of the receptors. Control experiments to study the possible contribution of antibodies to the measured outcome should be performed to verify the main findings. One possible approach could be to use fluorescently tagged integrins available. Alternatively, integrins (or adapter proteins) could be tagged with a small ligand and detected using a monovalent binder.

Only a limited number of integrin adapter proteins were investigated. Given the high number of identified adapter proteins, this is an understandable choice. However, it would be fascinating to understand if the nanoclusters of inactive integrins are dominantly bound with a certain adapter protein, such as tensin.

Reviewer #3 (Public review):

Summary:

In their study, the authors reveal using dual-color super-resolution STORM microscopy modality and immunolabeling in fixed adherent cells, that β1 and β3 integrins as well as adaptors (paxillin, talin and vinculin) are all organized in nanoclusters of similar size (50nm) and molecular density (20 copy number) inside FAs but also outside. Using activity-specific immunolabeling of β1 and β3 integrins, they revealed that active integrin subpopulations were both clustered but in distinct exclusive nano-aggregates in agreement with Spiess et al. (2018). Once more, the "active" integrin nanoclusters displayed similar properties in terms of size and molecular density, suggesting that molecular organization in nanoclusters is an intrinsic property of integrins in plasma membrane multimerizing independently of their location (inside or outside FAs), their level of activation, or their connection to the cytoskeleton. Then the authors followed up by analyzing at the mesoscale how these "universal" nanoclustered adhesive units are distributed spatially. Inspecting the surface density of nanoclusters revealed that the density of integrin nanoclusters in FAs was 5x larger, compared to integrin nanoclusters outside adhesions. Interestingly, whereas the density of total integrin nanoclusters was 2-4x larger than adaptor nanoclusters, the density of "active" integrin nanoclusters stoichiometrically matches that of talin and vinculin nanoclusters, and was slightly outnumbered by paxillin nanoclusters. These findings suggest that inside FAs, among the total number of integrin nanoclusters, the subset of "active" integrin nanoclusters could be engaged with "adaptor" nanoclusters on a 1:1 ratio. Using analysis of the nearest neighbor distance (NND) between distinct integrin clusters and each of the adaptors, the authors report that they found negligible spatial colocalization of integrins with these adaptor proteins and that spatial segregation is essentially determined by the density of nanoclusters within the FAs. As authors reported that α5β1 and αvβ3 do not intermix at the nanoscale, the authors finally highlighted how α5β1 and αvβ3 distinct nanoclusters are differently organized and segregated inside FAs. Adapting the NND analysis in order to inspect how far the nanoclusters are from the edges of FAs they are located in, authors revealed that α5β1 but not αvβ3 integrin nanoclusters are enriched on FA edges and that similar FA edge-enriched distribution for "active" α5β1 and adaptor protein nanoclusters was found for talin and paxillin but not vinculin. The latter results suggest that FA edges could constitute multiprotein hubs for enhanced colocalization and activation for α5β1 integrin nanoclusters and adaptors such as talin and paxillin. Unfortunately NND analysis could not confirm this enhanced colocalization hypothesis.

General Assessment:

While the study presents some valuable findings, it reads currently as a compilation of intriguing but preliminary observations derived primarily from a single methodology (dual-color STORM and DBSCAN clustering analysis). As the initial findings often lack confirmation through additional data analysis (such as the NND analysis the authors used), there's a critical necessity to bolster the methodological approach. This should involve replicating the main findings using alternative single-molecule super-resolution techniques (such as quantitative DNA-PAINT) or employing different clustering analytical tools (such as voronoi-tessellation). Furthermore, the manuscript feels incomplete, focusing solely on describing molecular organization without offering substantial insights into how these observations correlate with the regulation, activation, and functionality of integrins at the cellular level.

The manuscript presents extensive datasets and utilizes methodologies in which the investigators demonstrate expertise. Nevertheless, there's uncertainty regarding the novelty and broad appeal of the findings. For instance, the observation of integrin nanoclustering has been previously reported in several publications (e.g., Changede et al., Dev Cell 2015; Spiess et al., JCB 2018; Fujiwara et al., JCB 2023). Similarly, the accumulation of specific proteins at the periphery of FAs has been documented elsewhere (e.g., Sun et al., NCB 2016; Stubb et al., NatComm 2019; Nunes-Vicente TCB 2023), as well as the differential dynamic organization of α5β1 and αvβ3 integrins inside FAs (e.g., Rossier et al., NCB 2012). Beyond the universal organization of adhesive proteins, there's a need to identify novel insights that significantly advance the field. One potential avenue could involve pinpointing the molecular determinant controlling the FA edge enrichment of active α5β1 integrins and talin nanoclusters. For instance, could there be an interplay between α5β1 and αvβ3 integrin nanoclusters visible on one's organisation when suppressing the other using deletion (KO) or depletion (SiRNA)? Also, could KANK, which also exhibits enrichment and regulates talin activity (e.g., Sun et al., NCB 2016), play a role in this process? Identifying the molecular players that regulate even partially the mesoscale organization of nanoclusters of proteins would really benefit the breadth of this manuscript.

Echoing the previous concern, the manuscript described a novel and rather surprising finding related to molecular clustering of adhesion proteins. Indeed, the fact that nanoclusters exhibit uniform size and molecular density regardless of the protein type, location, or activation level is indeed surprising and raises many questions about the methodology used to assess molecular clustering. I feel that the description and characterization of integrin nanoclusters appear incomplete and need to be expanded by comparing different analytical strategies for protein clustering. Furthermore, a lack of the manuscript in its actual form concerns the quantification of integrin numbers inside the observed nanoclusters. I agree that the path from optical microscopy to protein stoichiometry quantification is hard and full of drawbacks. But the authors do not fully address these issues that are extremely important when discussing protein nanoclustering. This quantitative aspect should be discussed.

First, it is crucial for the authors to carefully examine and discuss in their manuscript whether there are any potential biases or limitations in the experimental techniques (dual-color STORM) or data analysis methods employed (DBSCAN). Second, the authors did not in the current manuscript, but should provide control samples to demonstrate the sensitivity and dynamic range of their experimental strategy.

In STORM images displayed in Figure S1, the authors highlighted localization clusters detected by DBSCAN as a signature for integrin nanoclusters. But the authors do not discuss the localization spots that were not detected by DBSCAN. Could they be individual integrins? And if so, they should also be considered as useful information? This brings me to another related technical question about how DBSCAN handles the case where fluorescent molecules are blinking. This is important as multiple emissions by a single fluorophore could be detected as a nanocluster of several molecules where it would be an artefact due to the photophysics of the fluorophore. Could the authors comment on these points?

Also, using isolated and stochastically physisorbed fluorophores (Ab coupled with activator /reporter pairs used in this study) on glass helped define the signature in STORM of a single isolated molecule. To obtain the signature of clustered fluorophores, the authors could use anti-donkey antibodies to cross-link those STORM-specifically labeled Ab as a means to artificially obtain clustered fluorophores. Ultimately, to avoid the bias effect of the glass surfaces on the photophysics of fluorophores and be in the same imaging conditions as for the described nanoclusters, the authors should use model systems composed of multimers of GFP vs. single GFP, immunolabeled with a GFP-binding monoclonal antibody. This will permit evaluation of the cluster signature obtained with DBSCAN analysis of STORM data for single vs. multimers of known stoichiometry. This would constitute an undisputable molecular stoichiometry ruler.

Due to the surprising finding of the nanoclusters' "universality", it is imperative for the authors to validate the findings through complementary methodologies and analytical tools. This should involve replication of results using alternative super-resolution techniques (quantitative DNA-PAINT) and exploring different clustering algorithms (Voronoï-Tesselation) to ensure the robustness and reliability of the observations.

Reviewer #1 (Public review):

Summary:

Early and accurate diagnosis is critical to treating N. fowleri infections, which often lead to death within 2 weeks of exposure. Current methods-sampling cerebrospinal fluid are invasive, slow, and sometimes unreliable. Therefore, there is a need for a new diagnostic method. Russell et al. address this need by identifying small RNAs secreted by Naegleria fowleri (Figure 1) that are detectable by RT-qPCR in multiple biological fluids including blood and urine. SmallRNA-1 and smallRNA-2 were detectable in plasma samples of mice experimentally infected with 6 different N. fowleri strains, and were not detected in uninfected mouse or human samples (Figure 4). Further, smallRNA-1 is detectable in the urine of experimentally infected mice as early as 24 hours post-infection (Figure 5). The study culminates with testing human samples (obtained from the CDC) from patients with confirmed N. fowleri infections; smallRNA-1 was detectable in cerebrospinal fluid in 6 out of 6 samples (Figure 6B), and in whole blood from 2 out of 2 samples (Figure 6C). These results suggest that smallRNA-1 could be a valuable diagnostic marker for N. fowleri infection, detectable in cerebrospinal fluid, blood, or potentially urine.

Strengths:

This study investigates an important problem, and comes to a potential solution with a new diagnostic test for N. fowleri infection that is fast, less invasive than current methods, and seems robust to multiple N. fowleri strains. The work in mice is convincing that smallRNA1 is detectable in blood and urine early in infection. Analysis of patient blood samples suggest that whole blood (but not plasma) could be tested for smallRNA-1 to diagnose N. fowleri infections.

Weaknesses:

(1) There are not many N. fowleri cases, so the authors were limited in the human samples available for testing. It is difficult to know how robust this biomarker is in whole blood (only 2 samples were tested, both had detectable smallRNA-1), serum (1 out of 1 sample tested negative), or human urine (presumably there is no material available for testing). This limitation is openly discussed in the last paragraph of the discussion section.

(2) There seems to be some noise in the data for uninfected samples (Figures 4B-C, 5B, and 6C), especially for those with serum (2E). While this is often orders of magnitude lower than the positive results, it does raise questions about false positives, especially early in infection when diagnosis would be the most useful. A few additional uninfected human samples may be helpful.

Reviewer #2 (Public review):

Summary:

The authors sought to develop a rapid and non-invasive diagnostic method for primary amoebic meningoencephalitis (PAM), a highly fatal disease caused by Naegleria fowleri. Due to the challenges of early diagnosis, they investigated extracellular vesicles (EVs) from N. fowleri, identifying small RNA biomarkers. They developed an RT-qPCR assay to detect these biomarkers in various biofluids.

Strengths:

(1) This study has a clear methodological approach, which allows for the reproducibility of the experiments.

(2) Early and Non-Invasive Diagnosis - The identification of a small RNA biomarker that can be detected in urine, plasma, and cerebrospinal fluid (CSF) provides a non-invasive diagnostic approach, which is crucial for improving early detection of PAM.

(3) High Sensitivity and Rapid Detection - The RT-qPCR assay developed in the study is highly sensitive, detecting the biomarker in 100% of CSF samples from human PAM cases and in mouse urine as early as 24 hours post-infection. Additionally, the test can be completed in ~3 hours, making it feasible for clinical use.

(4) Potential for Disease Monitoring - Since the biomarker is detectable throughout the course of infection, it could be used not only for early diagnosis but also for tracking disease progression and monitoring treatment efficacy.

(5) Strong Experimental Validation - The study demonstrates biomarker detection across multiple sample types (CSF, urine, whole blood, plasma) in both animal models and human cases, providing robust evidence for its clinical relevance.

(6) Addresses a Critical Unmet Need - With a >97% case fatality rate, PAM urgently requires improved diagnostics. This study provides one of the first viable liquid biopsy-based diagnostic approaches, potentially transforming how PAM is detected and managed.

Weaknesses:

(1) Limited Human Sample Size - While the biomarker was detected in 100% of CSF samples from human PAM cases, the number of human samples analyzed (n=6 for CSF) is relatively small. A larger cohort is needed to validate its diagnostic reliability across diverse populations.

(2) Lack of Pre-Symptomatic or Early-Stage Human Data - Although the biomarker was detected in mouse urine as early as 24 hours post-infection, there is no data on whether it can be reliably detected before symptoms appear in humans, which is crucial for early diagnosis and treatment initiation.

(3) Plasma Detection Challenges - While the biomarker was detected in whole blood, it was not detected in human plasma, which could limit the ease of clinical implementation since plasma-based diagnostics are more common. Further investigation is needed to understand why it is absent in plasma and whether alternative blood-based approaches (e.g., whole blood assays) could be optimized.

Reviewer #1 (Public review):

Summary:

The study investigates how neuropeptidergic signaling affects sleep regulation in Drosophila larvae. The authors first conduct a screen of CRISPR knock-out lines of genes encoding enzymes or receptors for neuropeptides and monoamines. As a result of this screen, the authors follow up on one hit, the hugin receptor, PK2-R1. They use genetic approaches, including mutants and targeted manipulations of PK2-R1 activity in insulin-producing cells (IPCs) to increase total sleep amounts in 2nd instar larvae. Similarly, dilp3 and dilp5 null mutants and genetic silencing of IPCs show increases in sleep. The authors also show that hugin mutants and thermogenetic/optogenetic activation of hugin-expressing neurons caused reductions in sleep. Furthermore, they show through imaging-based approaches that hugin-expressing neurons activate IPCs. A key finding is that wash-on of hugin peptides, Hug-γ and PK-2, in ex vivo brain preparations activates larval IPCs, as assayed by CRTC::GFP imaging. The authors then examine how the PK2-R1, hugin, and IPC manipulations affect adult sleep. Finally, the authors examine how Ca2+ responses through CRTC::GFP imaging in adult IPCs are influenced by the wash-on of hugin peptides. The conclusions of this paper are somewhat well supported by data, but some aspects of the experimental approach and sleep analysis need to be clarified and extended.

Strengths:

(1) This paper builds on previously published studies that examine Drosophila larval sleep regulation. Through the power of Drosophila genetics, this study yields additional insights into what role neuropeptides play in the regulation of Drosophila larval sleep.

(2) This study utilizes several diverse approaches to examine larval and adult sleep regulation, neural activity, and circuit connections. The impressive array of distinct analyses provides new understanding into how Drosophila sleep-wake circuitry in regulated across the lifespan.

(3) The imaging approaches used to examine IPC activation upon hugin manipulation (either thermogenetic activation or wash-on of peptides) demonstrate a powerful approach for examining how changes in neuropeptidergic signaling affect downstream neurons. These experiments involve precise manipulations as the authors use both in vivo and ex vivo conditions to observe an effect on IPC activity.

Weaknesses:

Although the paper does have some strengths in principle, these strengths are not fully supported by the experimental approaches used by the authors. In particular:

(1) The authors show total sleep amount over an 18-hour period for all the measures of 2nd instar larval sleep throughout the paper. However, published studies have shown that sleep changes over the course of 2nd instar development, so more precise time windows are necessary for the analyses in this study.

(2) Previously published reports of sleep metrics in both Drosophila larvae and adults include the average number of sleep episodes (bout number) and the average length of sleep episodes (bout length). Neither of these metrics is included in the paper for either the larval sleep or adult sleep data. Not including these metrics makes it difficult for readers to compare the findings in this study to previously published papers in the established Drosophila sleep literature.

(3) Because Drosophila adult & larval sleep is based on locomotion, the authors need to show the activity values for the experiments supporting their key conclusions. They do show travel distances in Figure 2 - Figure Supplement 1, however, it is not clear how these distances were calculated or how the distances relate to the overall activity of individual larvae during sleep experiments. It is also concerning that inactivation of the PK2-R1-expressing neurons causes a reduction in locomotion speed. This could partially explain the increase in sleep that they observe.

(4) The authors rely on homozygous mutant larvae and adult flies to support many of their conclusions. They also rely on Gal4 lines with fairly broad expression in the Drosophila brain to support their conclusions. Adding more precise tissue-specific manipulations, including thermogenetic activation and inhibition of smaller populations of neurons in the study would be needed to increase confidence in the presented results. Similarly, demonstrating that larval development and feeding are not affected by the broad manipulations would strengthen the conclusions.

(5) Many of the experiments presented in this study would benefit from genetic and temperature controls. These controls would increase confidence in the presented results.

(6) The authors claim that their findings in larvae uncover the circuit basis for larval sleep regulation. However, there is very little comparison to published studies demonstrating that neuropeptides like Dh44 regulate larval sleep. Because hugin-expressing neurons have been shown to be downstream of Dh44 neurons, the authors need to include this as part of their discussion. The authors also do not explain why other neuropeptides in the initial screen are not pursued in the study. Given the effect that these manipulations have on larval sleep in their initial screen, it seems likely that other neuropeptidergic circuits regulate larval sleep.

Reviewer #2 (Public review):

Summary:

This study examines larval sleep patterns and compares them to sleep regulation in adult flies. The authors demonstrate hallmark sleep characteristics in larvae, including sleep rebound and increased arousal thresholds. Through genetic and behavioral analyses, they identify PK2-R1 as a key receptor involved in sleep modulation, likely via the HuginPC-IPC signaling pathway. Loss of PK2-R1 results in increased sleep, which aligns with previous findings in hugin knockout mutants. While the study presents significant contributions to the field, further investigation is needed to address discrepancies with earlier research and strengthen mechanistic claims.

Strengths:

(1) The study explores a relatively understudied aspect of sleep regulation, focusing on larval development.

(2) The use of an automated behavioral measurement system ensures precise quantification of sleep patterns.

(3) The findings provide strong genetic and behavioral evidence supporting the role of the HuginPC-IPC pathway in sleep regulation.

(4) The study has broader implications for understanding the evolution and functional divergence of sleep circuits.

Weaknesses:

(1) The manuscript does not sufficiently discuss previous studies, particularly concerning hugin mutants and their metabolic effects.

(2) The specificity of IPC secretion mechanisms is unclear, particularly regarding potential indirect effects on Dilp2.

(3) Alternative circuits, such as the HuginPC-DH44 pathway, require further consideration.

(4) Functional connectivity between HuginPC neurons and IPCs is not directly validated.

(5) Developmental differences in sleep regulatory mechanisms are not thoroughly examined.

Reviewer #3 (Public review):

Summary:

Sleep affects cognition and metabolism, evolving throughout development. In mammals, infants have fast sleep-wake cycles that stabilize in adults via circadian regulation. In this study, the author performed a genetic screen for neurotransmitters/peptides regulating sleep and identified the neuropeptide Hugin and its receptor PK2-R1 as essential components for sleep in Drosophila larvae. They showed that IPCs express Pk2-R1 and silencing IPCs resulted in a significant increase in the sleep amount, which was consistent with the effect they observed in PK2-R1 knock-out mutants. They also showed that Hugin peptides, secreted by a subset of Hugin neurons (Hug-PC), activate IPCs through the PK2-R1 receptor. This activation prompts IPCs to release insulin-like peptides (Dilps), which are implicated in the modulation of sleep. They showed that Hugin peptides induce a PK2-R1 dependent calcium (Ca²⁺) increase in IPCs, which they linked to the release of Dilp3, showing a connection between Hugin signaling to IPCs, Dilp3 release, and sleep regulation. Additionally, the activation of Hug-PC neurons reduced sleep amounts, while silencing them had the opposite effect. In contrast to the larval stage, the Hugin/PK2-R1 axis was not critical for sleep regulation in Drosophila adults, suggesting that this neuropeptidergic circuitry has divergent roles in sleep regulation across different stages of development.

Strengths:

This study used an updated system for sleep quantification in Drosophila larvae, and this method allowed precise measurement of larval sleep patterns which is essential for the understanding of sleep regulation.

The authors performed unbiased genetics screening and successfully identified novel regulators for larval sleep, Hugin and its receptor PK2-R1, making a substantial contribution to the understanding of neuropeptidergic control of sleep regulation.

They clearly demonstrated the mechanism by which Hugin-expressing neurons influence sleep through the activation of IPCs via PK2-R1 with Ca2+ responses and can modulate sleep.

Based on the demonstrated activation of PK2-R1 by the human Hugin orthologue Neuromedin U, research on human sleep disorders may benefit from the discoveries from Drosophila since sleep-regulating mechanisms are conserved across species.

Weaknesses:

The study primarily focused on sleep regulation in Drosophila larvae, showing that the Hugin/PK2-R1 axis is critical for larval sleep but not necessary for adult sleep. The effects of the Hugin axis in the adult are, however, incompletely explained and somewhat inconsistent. PK2-R1 knockout adults also display increased sleep, as does HugPC silencing, at least for daytime sleep. The difference lies in Dilp3/5 mutant animals showing decreased sleep and IPCs seemingly responding with reduced Dilp3 release to PK-2 treatment (Figure 6). It seems difficult to reconcile the author's conclusions regarding this point without additional data. It could be argued that PK2-R1 still regulates adult sleep, but not via Hugin and IPCs/Dilps.

Another issue might be that the authors show relative sleep levels for adults using Trikinetics monitoring. From the methods, it is not clear if the authors backcrossed their line to an isogenic wild-type background to normalize for line-specific effects on sleep. Thus, it is likely that each line has differences in total sleep time due to background effects, e.g., their Kir2.1 control line showed reduced sleep relative to the compared genotypes. This might limit the conclusions on the role of Hugin/PK2-R1 on adult sleep.

Reviewer #1 (Public review):

Summary:

Using highly specific antibody reagents for biological research is of prime importance. In the past few years, novel approaches have been proposed to gain easier access to such reagents. This manuscript describes an important step forward toward the rapid and widespread isolation of antibody reagents. Via the refinement and improvement of previous approaches, the Perrimon lab describes a novel phage-displayed synthetic library for nanobody isolation. They used the library to isolate nanobodies targeting Drosophila secreted proteins. They used these nanobodies in immunostainings and immunoblottings, as well as in tissue immunostainings and live cell assays (by tethering the antigens on the cell surface).

Since the library is made freely available, it will contribute to gaining access to better research reagents for non-profit use, an important step towards the democratisation of science.

Strengths:

(1) New design for a phage-displayed library of high content.

(2) Isolation of valuble novel tools.

(3) Detailed description of the methods such that they can be used by many other labs.

Weaknesses:

My comments largely concentrate on the representation of the data in the different Figures.

Reviewer #2 (Public review):

Summary:

In this study, the authors propose an alternative platform for nanobody discovery using a phage-displayed synthetic library. The authors relied on DNA templates originally created by McMahon et al. (2018) to build the yeast-displayed synthetic library. To validate their platform, the authors screened for nanobodies against 8 Drosophila secreted proteins. Nanobody screening has been performed with phage-displayed nanobody libraries followed by an enzyme-linked immunosorbent assay (ELISA) to validate positive hits. Nanobodies with higher affinity have been tested for immunostaining and immunoblotting applications using Drosophila adult guts and hemolymph, respectively.

Strengths:

The authors presented a detailed protocol with various and complementary approaches to select nanobodies and test their application for immunostaining and immunoblotting experiments. Data are convincing and the manuscript is well-written, clear, and easy to read.

Weaknesses:

On the eight Drosophila secreted proteins selected to screen for nanobodies, the authors failed to identify nanobodies for three of them. While the authors mentioned potential improvements of the protocol in the discussion, none of them have been tested in this manuscript.

The same comment applies to the experiments using membrane-tethered forms of the antigens to test the affinity of nanobodies identified by ELISA. Many nanobodies fail to recognize the antigens. While authors suggested a low affinity of these nanobodies for their antigens, this hypothesis has not been tested in the manuscript.

Improving the protocol at each step for nanobody selection would greatly increase the success rate for the discovery of nanobodies with high affinity.

Reviewer #1 (Public review):

Summary:

The authors wanted to better understand how the various septin-associated kinases contribute to septin organization and function in budding yeast. This question has been recently addressed by similar kinds of studies but there are still some open questions, particularly as regards to what extent the kinases may interact with and/or modify components of the contractile ring that drives cytokinesis.

Strengths:

This study uses sensitive imaging with good temporal and spatial resolution to monitor the localization of various proteins in living cells. Particularly informative is the use of a GFP/GFP-binding-protein "tethering" approach to ask if the requirement for one protein can be bypassed by physically tethering another protein to a third protein. Results from a yeast two-hybrid assay for measuring protein-protein interactions in vivo are buttressed by direct in vitro binding assays using purified proteins, which is important given the likelihood of "bridging" interactions between yeast proteins in the two-hybrid approach. The authors' conclusions are quite well supported by the data.

Weaknesses:

A control for non-specific binding is missing from the in vitro binding assay. The figures suffer sometimes from the very small text in the labels, which obscures understanding. Ultimately, while the study provides some interesting and novel insights, we still don't understand which phosphorylation events on which proteins are important for the events occurring at the molecular level, so the advance in knowledge is somewhat incremental.

Reviewer #2 (Public review):

Summary:

In this paper, Bhojappa et al. provide insights into the function of septin-related kinases Elm1, Gin4, Hsl1, and Kcc4 in septin organization and actomyosin ring (AMR) structure and constriction. Their findings are both corroborative of and complementary to previous related studies.

First, the authors provide a comparative analysis of the dynamic localization of these kinases at the bud neck, as well as a comparative analysis of defects in septin localization, splitting dynamics, AMR constriction rates, and cell morphology in kinase-deficient cells. They find that septin localization and splitting kinetics, as well as AMR constriction rates, are significantly perturbed in elm1∆ and gin4∆ mutants but remain largely unaffected in hsl1∆ and kcc4∆. A similar trend is observed in terms of cell morphology and viability.

Next, the authors focus on elm1∆ and gin4∆ cells, demonstrating that the residence time of the F-BAR protein Hof1 is significantly increased and defective in these mutants. Using yeast two-hybrid (Y2H) and in vitro binding assays, they show that the KA1 domain of Gin4 interacts with the F-BAR domain of Hof1, which may explain the cytokinesis-related functions of Elm1 and Gin4. Supporting this, they find that Gin4's role in septin localization, AMR constriction kinetics, and Hof1 bud neck localization is kinase-independent.

The authors then conduct a series of artificial tethering experiments given their bud neck localization is mostly interdependent. They first demonstrate that artificially tethering Gin4 to the bud neck rescues the morphology defects of elm1∆ cells, with the strongest rescue observed when Gin4 was forced to interact with Hsl1-an effect that was also kinase-independent. Additionally, artificial tethering of Hsl1 to the bud neck restores the morphology of elm1∆ cells in a KA1 domain-dependent manner, suggesting that Hsl1 functions downstream of Elm1 to maintain normal cell morphology. Consistently, artificial tethering of Elm1 to the bud neck in gin4∆ cells rescues morphology defects, as well as defects in Myo1 localization and AMR constriction, but only in the presence of full-length Hsl1. The rescue fails in the absence of Hsl1 or when using a version of Hsl1 lacking the KA1 domain, which supports the role of Hsl1 downstream to Elm1 in cytokinesis.

Strengths

Altogether, this study offers valuable insights into the mode of cytokinesis regulation mediated by the septin-related kinases, mainly Elm1, Gin4, and Hsl1, and would be an important contribution to the field of septins and cytokinesis after addressing current weaknesses.

Weaknesses

(1) When assessing rescue of the elm1∆ phenotype, it needs to become clearer whether only morphology or also cytokinesis and septin organization are rescued.

(2) The quantification of the microscopy data does not always match up with the example images, and it's not always clear how the authors quantitatively analyzed their data.

(3) The forced tethering data are key to the paper, but the lack of a summarizing table makes it difficult to grasp the full picture.

(4) Novel results and those confirming earlier results could be better distinguished.

Reviewer #3 (Public review):

Summary:

The study by Bhojappa et al. brings new and interesting elements about the stability of the septin ring and the crosstalk between septin and actomyosin ring assemblies. The study focuses on the four kinases associated with the septin ring, Elm1p, Gin4p, Hsl1p, and Kcc4p. Elm1 and Gin4 show strong knock-out phenotypes, whereas Hsl1p and Kcc4p show weak knock-out phenotypes. The Elm1p/Kccp1p and Gin4p/Hsl1p pairs show similar timing at the bud neck. While these kinases share redundant functions, Gin4 appears to have a unique interaction with the BAR domain protein Hof1, revealing a novel direct interaction between the septin and actomyosin rings. Interestingly, the kinase activity of Gin4 is not required for its role in septin organisation and AMR constriction. The last part of the manuscript shows an original protein tethering protocol used to show that Hsl1 and its membrane binding ability are required for phenotype rescue of gin4null cells.

Strengths:

The combination of genetics, cell imaging, and biochemical characterization of protein-protein interactions is attractive.

Weaknesses:

(1) Imaging and data analysis is the main weakness of this manuscript. The authors must avoid manual counting and selection when easy analysis software can be used to limit bias. Instead of presenting unclear statistics of "percentage phenotypes", they need to define clear metrics to offer meaningful phenotype analysis.

(2) This manuscript examines a very complex mechanism with four kinases of overlapping function using new data and existing literature. A clearer picture/model at the end of the manuscript that synthesizes the current knowledge would be beneficial.

Reviewer #1 (Public review):

Summary:

Using single-unit recording in 4 regions of non-human primate brains, the authors tested whether these regions encode computational variables related to model-based and model-free reinforcement learning strategies. While some of the variables seem to be encoded by all regions, there is clear evidence for stronger encoding of model-based information in the anterior cingulate cortex and caudate.

Strengths:

The analyses are thorough, the writing is clear, and the work is well-motivated by prior theory and empirical studies.

Weaknesses:

My comments here are quite minor.

The correlation between transition and reward coefficients is interesting, but I'm a little worried that this might be an artifact. I suspect that reward probability is higher after common transitions, due to the fact that animals are choosing actions they think will lead to higher reward. This suggests that the coefficients might be inevitably correlated by virtue of the task design and the fact that all regions are sensitive to reward. Can the authors rule out this possibility (e.g., by simulation)?

The explore/exploit section seems somewhat randomly tacked on. Is this really relevant? If yes, then I think it needs to be integrated more coherently.

Reviewer #2 (Public review):

Summary:

The authors investigate single-neuron activity in rhesus macaques during model-based (MB) and model-free (MF) reinforcement learning (RL). Using a well-established two-step choice task, they analyze neural correlates of MB and MF learning across four brain regions: the anterior cingulate cortex (ACC), dorsolateral PFC (DLPFC), caudate, and putamen. The study provides strong evidence that these regions encode distinct RL-related signals, with ACC playing a dominant role in MB learning and caudate updating value representations after rare transitions. The authors apply rigorous statistical analyses to characterize neural encoding at both population and single-neuron levels.

Strengths:

(1) The research fills a gap in the literature, which has been limited in directly dissociating MB vs. MF learning at the single unit level and across brain areas known to be involved in reinforcement learning. This study advances our understanding of how different brain regions are involved in RL computations.

(2) The study used a two-step choice task Miranda et al., (2020), which was previously established for distinguishing MB and MF reinforcement learning strategies.

(3) The use of multiple brain regions (ACC, DLPFC, caudate, and putamen) in the study enabled comparisons across cortical and subcortical structures.

(4) The study used multiple GLMs, population-level encoding analyses, and decoding approaches. With each analysis, they conducted the appropriate controls for multiple comparisons and described their methods clearly.

(5) They implemented control regressors to account for neural drift and temporal autocorrelation.

(6) The authors showed evidence for three main findings:<br /> a) ACC as the strongest encoder of MB variables from the four areas, which emphasizes its role in tracking transition structures and reward-based learning. The ACC also showed sustained representation of feedback that went into the next trial.<br /> b) ACC was the only area to represent both MB and MF value representations.<br /> c) The caudate selectively updates value representations when rare transitions occur, supporting its role in MB updating.

(7) The findings support the idea that MB and MF reinforcement learning operate in parallel rather than strictly competing.

(8) The paper also discusses how MB computations could be an extension of sophisticated MF strategies.

Weaknesses: o

(1) There is limited evidence for a causal relationship between neural activity and behavior. The authors cite previous lesion studies, but causality between neural encoding in ACC, caudate, and putamen and behavioral reliance on MB or MF learning is not established.

(2) There is a heavy emphasis on ACC versus other areas, but it is unclear how much of this signal drives behavior relative to the caudate.

(3) The role of the putamen is somewhat underexplored here.

(4) The authors mention the monkeys were overtrained before recording, which might have led to a bias in the MB versus MF strategy.

(5) The GLM3 model combines MB and MF value estimates but does not clearly mention how hyperparameters were optimized to prevent overfitting. While the hybrid model explains behavior well, it does not clarify whether MB/MF weighting changes dynamically over time.

(6) It was unclear from the task description whether the images used changed periodically or how the transition effect (e.g., in Figure 3) could be disambiguated from a visual response to the pair of cues.

Reviewer #1 (Public review):

In the manuscript entitled "Rtf1 HMD domain facilitates global histone H2B monoubiquitination and regulates morphogenesis and virulence in the meningitis-causing pathogen Cryptococcus neoformans" by Jiang et al., the authors employ a combination of molecular genetics and biochemical approaches, along with phenotypic evaluations and animal models, to identify the conserved subunit of the Paf1 complex (Paf1C), Rtf1, and functionally characterize its critical roles in mediating H2B monoubiquitination (H2Bub1) and the consequent regulation of gene expression, fungal development, and virulence traits in C. deneoformans or C. neoformans. Specially, the authors found that the histone modification domain (HMD) of Rtf1 is sufficient to promote H2B monoubiquitination (H2Bub1) and the expression of genes related to fungal mating and filamentation, and restores the fungal morphogenesis and pathogenicity defects caused by RTF1 deletion. These findings highlight the critical contribution of Rtf1's HMD to epigenetic regulation and cryptococcal virulence. This work will be of interest to fungal biologists and medical mycologists, particularly those studying fungal epigenetic regulation and fungal morphogenesis.

Comments on revisions:

The revised manuscript addresses all my previous concerns satisfactorily.

Reviewer #1 (Public review):

Summary:

The authors show for the first time that deleting GLS from rod photoreceptors results in the rapid death of these cells. The death of photoreceptor cells could result from loss of synaptic activity because of a decrease in glutamate, as has been shown in neurons, changes in redox balance, or nutrient deprivation.

Strengths:

The strength of this manuscript is that the author shows a similar phenotype in the mice when Gls was knocked out early in rod development or the adult rod. They showed that rapid cell death is through apoptosis, and there is an increase in the expression of genes responsive to oxidative stress.

Comments on revisions:

The authors addressed all of my concerns in their responses to reviewers.

Reviewer #2 (Public review):

Summary:

Photoreceptor neurons are crucial for vision, and discovering pathways necessary for photoreceptor health and survival can open new avenues for therapeutics. Studies have shown that metabolic dysfunction can cause photoreceptor degeneration and vision loss, but the metabolic pathways maintaining photoreceptor health are not well understood. This is a fundamental study that shows that glutamine catabolism is critical for photoreceptor cell health using in vivo model systems.

Strengths:

The data are compelling, and the consideration of potential confounding factors (such as glutaminase 2 expression) and additional experiments to examine the synaptic connectivity and inner retina added strength to this work. The authors were also careful not to overstate their claims, but to provide solid conclusions that fit the results and data provided in their study. The findings linking asparagine supplementation and the inhibition of the integrated stress response to glutamine catabolism within the rod photoreceptor cell are intriguing and innovative. Overall, the authors provide convincing data to highlight that photoreceptors utilize various fuel sources to meet their metabolic needs, and that glutamine is critical to these cells for their biomass, redox balance, function and survival.

Reviewer #3 (Public review):

Summary:

The authors explored the role of GLS, a glutaminase, which is an enzyme catalyzes the conversion of glutamine to glutamate, in rod photoreceptor function and survival. The loss of GLS was found to cause rapid autonomous death of rod photoreceptors.

Strengths:

Interesting and novel phenotype. Two types of cre-lines were rigorously used to knockout Gls gene in rods. Both of the conditional knockouts led to a similar phenotype, i.e. rod death. Histology and ERG were carefully done to characterize the loss of rods over specific ages. Necessary metabolomic study was performed and appreciated. Some rescue experiments were performed, and revealed possible mechanism.

Weaknesses:

No major weaknesses. Mechanism of GLS-loss induced rod death could be followed up in the future, and same for GLS's role in cones. Authors have addressed all minor points raised by this reviewer.

Reviewer #2 (Public review):

Summary:

The study investigates the potential influence of the response criterion on neural decoding accuracy in consciousness and unconsciousness, utilizing either simulated data or reanalyzing experimental data with post-hoc sorting data.

Strengths:

When comparing the neural decoding performance of Target versus NonTarget with or without post-hoc sorting based on subject reports, it is evident that response criterion can influence the results. This was observed in simulated data as well as in two experiments that manipulated the subject response criterion to be either more liberal or more conservative. One experiment involved a two-level response (seen vs unseen), while the other included a more detailed four-level response (ranging from 0 for no experience to 3 for a clear experience). The findings consistently indicated that adopting a more conservative response criterion could enhance neural decoding performance, whether in conscious or unconscious states, depending on the sensitivity or overall response threshold.

The uneven distribution of trails for Target (75%) and NonTarget (25%) was identified as a potential weakness in the initial review of this study. Nevertheless, we support the authors' assertion that their analysis methodology validates comparing liberal and conservative approaches. Future investigations could further explore differences between liberal and conservative on different ratios of Target vs NonTarget, particularly when the proportion of Target matches or falls below that of NonTarget.

Reviewer #1 (Public review):

Guo, Hue et al., is focused on understanding the epigenetic activity and functional dependencies for two different fusions found in spindle cell rhabdomyosarcoma, VGLL2::NCOA2 and TEAD1::NCOA2. They use a variety of models and methods; specifically, ectopic expression of the fusions in human 293T cells to perform RNAseq (both fusions), CUT&RUN (VGLL2::NCOA2) and BioID mass spec (both fusions). These data identify that the VGLL2::NCOA2 fusion has peaks that are enriched for TEAD motifs. Further, CPB/p300 CUT&RUN support an enrichment of binding sites and three TEAD targets in VGLL2::NCOA2 and TEAD1::NCOA2 expressing cells. They also functionally evaluate genetic and chemical dependencies (TEAD inhibition), and found this was only effective for the VGLL2::NCOA2 fusion, and not for TEAD1::NCOA2. Using complementary biochemical approaches, they suggest (with other supporting data) the fusions regulate TEAD transcriptional outputs via a YAP/TAZ independent mechanism. Further, they expand into a C2C12 myoblast model and show that TEAD1::NCOA2 is transforming in colony formation assays and in mouse allograft. These strategies for TEAD1-NCOA2 are consistent with previous published strategies using VGLL2::NCOA2. Importantly, they show that a CBP/p300 (a binding partner found in their BioID mass spec) small molecule inhibitor suppresses tumor formation using this mouse allograft model, and that the tumors are less proliferative, and have a reduction in transcriptional of three TEAD target genes. They complement in vivo data with biochemical approaches, and suggest this interface with p300 (for VGLL2::NCOA2) is through the NCOA2 fusion partner, as Co-IP in HEK293T with a mutant fusion that does not contain NCOA2 loses the association with endogenous p300. The data is interesting and suggests new biology for these fusion-oncogenes. However, the choice of 293T may limit the broad applicability of the findings. Strikingly, in 293T there was more transcriptional overlap with the VGLL2-NCOA2 fusion with the YAP5SA mutant than with TEAD1-NCOA2. Further, there is an additional opportunity to directly compare transcriptional profiles in 293T to the human disease and in the mouse allograft system to directly compare and discuss VGLL2-NCOA2 and TEAD1-NCOA2 histological differences or how A485 treatment may change the histology. Overall, the breadth of methods used in this study, and comparison of the two fusion-oncogene's biology is of interest to the fusion-oncogene, pediatric sarcoma, and epigenetic therapeutic targeting fields.

Reviewer #2 (Public review):

In the manuscript entitled "VGLL2 and TEAD1 fusion proteins drive YAP/TAZ-independent transcription and tumorigenesis by engaging p300", Gu et al. investigated two Hippo pathway-related gene fusion events (i.e., VGLL2-NCOA2, TEAD1-NCOA2) in spindle cell rhabdomyosarcoma (scRMS). They demonstrate that these fusion proteins activate Hippo downstream gene transcription independently of YAP/TAZ. Using BioID-based mass spectrometry analysis, the authors identify histone acetyltransferase CBP/p300 as a specific binding protein for VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins. Pharmacologically targeting p300 inhibits the fusion proteins-induced Hippo downstream gene transcription and tumorigenesis.

Overall, this work provides novel mechanistic insights into scRMS-associated gene fusions in tumorigenesis and reveals potential therapeutic targets for cancer treatment. The manuscript is well-written and easy to follow. Below are a few comments based on the revised study.

(1) While the study majorly focuses on Hippo downstream gene transcription, a significant portion of genes regulated by the VGLL2-NCOA2 and TEAD1-NCOA2 fusion proteins are non-Hippo downstream genes (Fig. 3). Further characterization of how both Hippo and non-Hippo downstream genes contribute to fusion proteins-induced oncogenesis would enhance our understanding of scRMS etiology.

(2) A potential limitation of this study is the reliance on overexpression approaches to investigate VGLL2-NCOA2 and TEAD1-NCOA2 fusion genes, which may not fully reflect pathological conditions in scRMS patients. Despite this, the significant study offers valuable mechanistic insights into fusion genes-induced scRMS and provides molecular foundation for developing targeted therapies.

Reviewer #1 (Public review):

Summary:

Giménez-Orenga et al. investigate the origin and pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). Using RNA microarrays, the authors compare the expression profiles and evaluate the biomarker potential of human endogenous retroviruses (HERV) in these two conditions. Altogether, the authors show that HERV expression is distinct between ME/CFS and FM patients, and HERV dysregulation is associated with higher symptom intensity in ME/CFS. HERV expression in ME/CFS patients is associated with impaired immune function and higher estimated levels of plasma cells and resting CD4 memory T cells. This work provides interesting insights into the pathophysiology of ME/CFS and FM, creating opportunities for several follow-up studies.

Strengths:

(1) Overall, the data is convincing and supports the authors' claims. The manuscript is clear and easy to understand, and the methods are generally well-detailed. It was quite enjoyable to read.<br /> (2) The authors combined several unbiased approaches to analyse HERV expression in ME/CFS and FM. The tools, thresholds, and statistical models used all seem appropriate to answer their biological questions.<br /> (3) The authors propose an interesting alternative to diagnosing these two conditions. Transcriptomic analysis of blood samples using an RNA microarray could allow a minimally invasive and reproducible way of diagnosing ME/CFS and FM.

Weakness:<br /> (1) While this work makes several intriguing observations, some results will need to be validated in future studies using experimental approaches.

Reviewer #2 (Public review):

Summary:

Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in expression of HERVs in patients suffering from ME/CFS, FM or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables presents solid support for the findings. However, some statements made by the authors seem incomplete and would benefit by a more thorough literature review. Overall, this work will be of interest to the medical community seeking in better understanding the co-occurrence of these pathologies, hinting at a novel angle by integrating HERVs, which are often overlooked, into their assessment.

Strengths:

- The work is well-presented, allowing the reader to understand the overall workflow and how the specific aims contribute to filling the knowledge gap in the field.

- The analyses carried out to understand the potential impact on gene expression mediated by HERVs are in line with previous works, making it solid and robust in the context of this study.

Weaknesses:

- The authors claim to obtain genome-wide HERV expression profiles. However, the array used was developed using hg19, while the genomic analysis of this work are carried out using a liftover to hg38. It would improve the statement and findings to include a comparation of the differences in HERVs available in hg38, and how this could impact the "genome-wide" findings.

- The authors in some points are not thorough with the cited literature. Two examples are:<br /> (1) Lines 396-397 the authors say "the MLT1, usually found enriched near DE genes (Bogdan et al., 2020)". I checked the work by Bogdan, and they studied bacterial infection. A single work in a specific topic is not sufficient to support the statement that MLT1 is "usually" in close vicinity to differentially expressed genes. More works are needed to support this.<br /> (2) After the previous statement, the authors go on to mention "contributing to the coding of conserved lncRNAs (Ramsay et al., 2017)". First, lnc = long non-coding, so this doesn't make sense. Second, in the work by Ramsay they mention "that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved", which is different to what the authors in this study are trying to convey. Again, additional work and a rephrasing might help to support this idea.

- When presenting the clusters, the authors overlook the fact that cluster 4 is clearly control-specific, and fail to discuss what this means. Could this subset of HERV be used as bona fide markers of healthy individuals in the context of these diseases? Are they associated with DE genes? What could be the impact of such associations?

Appraisals on aims:

The authors set specific questions and presented the results to successfully answer them. The evidence is solid, with some weaknesses discussed above that will methodologically strengthen the work.

Likely impact of work on the field:<br /> This work will be of interest to the medical community looking for novel ways to improve clinical diagnosis. Although future works with a greater population size, and more robust techniques such as RNA-Seq, are needed, this is the first step in presenting a novel way to distinguish these pathologies.

It would be of great benefit to the community to provide a table/spreadsheet indicating the specific genomic locations of the HERVs specific to each condition. This will allow proper provenance for future researchers interesting in expanding on this knowledge, as these genomic coordinates will be independent of the technique used (as was the array used here).

Comments on revisions:

When addressing the comments made in the previous round, there are some answers that lack substance and don't seem to be incorporated in the manuscript. For example, the authors say:

Authors' response: This is an important point. However, the low number of probes (less than 100) that were excluded from our analysis by lack of correspondence with hg38 among the 1,290,800 probesets was interpreted as insignificant for "genome-wide" claims. An aspect that will be explained in the revised version of this manuscript.

I checked the revised manuscript with tracked changes, and there doesn't seem to be an updated explanation to this. In which lines is this explained?

For the other response:

Authors' response: Using control DE HERV as bona fide markers of healthy individuals seems like an interesting possibility worth exploring. Control DE HERV (cluster 4) associate with DE genes involved in apoptosis, T cell activation and cell-cell adhesion (modules 1 and 6). The impact of which deserves further study.

I couldn't find an updated mention of this in the discussion.

Another point that I raised was regarding the decision of using an FDR of 0.1 instead of 0.05. The authors only speculate about the impacts in their answer, while I believe that this could have been rigorously addressed. Since this was done in R, and DE analysis are relatively fast, I don't see a reason as to why this part was not repeated and discussed accordingly.

For other analyses, there doesn't seem to be a problem with using 0.05 as threshold. Examples of this are the "Overrepresentation functional analysis", or the "Statistical analysis" part of the methods they say "we used a Fisher exact test to calculate p-value, considering enriched in the provided list if an adjusted p-value (FDR) was less than 0.05".

Just to make this point clear: I'm not asking the authors to repeat all the work using the 0.05 FDR threshold, but rather that they are aware and conscious about the impact of this, and give an idea to the audience on how it would change the DE numbers. This would put in perspective the findings to any future reader.

I think that most of the other answers to both my previous concerns and the other reviewer's concerns are ok. My last outstanding concern is that the probe coordinates apparently can't be shared, which undermines a lot this study reproducibility, and its use by future researches which won't be able to compare their results to this study.

Reviewer #3 (Public review):

Summary:

The authors find that HERV expression patterns can be used as new criteria for differential diagnosis of FM and ME/CFS and patient subtyping. The data are based on transcriptome analysis by microarray for HERVs using patient blood samples, followed by differential expression of ERVs and bioinformatic analyses. This is a standard and solid data processing pipeline, and the results are well presented and support the authors' claim.

Strengths:

It provides an innovative diagnostic approach using ERV profiles to subtype patients and distinguish FM and ME/CFS.

Comments on revisions:

This is a revised manuscript which addresses the comments well.

Reviewer #2 (Public review):

Summary:

This study uses in vivo multimodal high-resolution imaging to track how microglia and neutrophils respond to light-induced retinal injury from soon after injury to 2 months post-injury. The in vivo imaging finding was subsequently verified by ex vivo study. The results suggest that despite the highly active microglia at the injury site, neutrophils were not recruited in response to acute light-induced retinal injury.

Strengths:

An extremely thorough examination of the cellular-level immune activity at the injury site. In vivo imaging observations being verified using ex vivo techniques is a strong plus.

Weaknesses:

This paper is extremely long, and in the perspective of this reviewer, needs to be better organized. Update: Modifications have been made throughout, which has made the manuscript easier to follow.

Study weakness: though the finding prompts more questions and future studies, the findings discussed in this paper is potentially important for us to understand how the immune cells respond differently to different severity level of injury. The study also demonstrated an imaging technology which may help us better understand cellular activity in living tissue during earlier time points.

Comments on revisions:

I appreciate the thorough clarification and re-organization by the authors, and the messages in the manuscript are now more apparent. I recommend also briefly discussing limitations/future improvements in the discussion or conclusion.

Reviewer #3 (Public review):

Summary

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation to the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network, constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience.

Strengths

Adaptive optics imaging of murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is a benefit for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. This model would potentially allow for controlling the size, depth and severity of the laser injury opening interesting avenues for future study.

The time-course, 2D and 3D spatial activation pattern of microglial activation are striking and provide an unprecedented view of the retinal response to mild injury.

Weaknesses

Generalization of the (lack of) neutrophil response to photoreceptor loss - there is ample evidence in literature that neutrophils are heavily recruited in response to severe retinal damage that includes photoreceptor loss. Why the same was not observed here in this article remains an open question. One could hypothesize that neutrophil recruitment might indeed occur under conditions that are more in line with the more extreme damage models, for example, with a stronger and global ablation (substantially more photoreceptor loss over a larger area). This parameter space is unwieldy and sufficiently large to address the question conclusively in the current article, i.e. how much photoreceptor loss leads to neutrophil recruitment? By the same token, the strong and general conclusion in the title - Photoreceptor loss does not recruit neutrophils - cannot be made until an exhaustive exploration be made of the same parameter space. A scaling back may help here, to reflect the specific, mild form of laser damage explored here, for instance - Mild photoreceptor loss does not recruit neutrophils despite...

EIU model - The EIU model was used as a positive control for neutrophil extravasation. Prior work with flow cytometry has shown a substantial increase in neutrophil counts in the EIU model. Yet, in all, the entire article shows exactly 2 examples in vivo and 3 ex vivo (Figure 7) of extravasated neutrophils from the EIU model (n = 2 mice). The general conclusion made about neutrophil recruitment (or lack thereof) is built partly upon this positive control experiment. But these limited examples, especially in the case where literature reports a preponderance of extravasated neutrophils, raise a question on the paradigm(s) used to evaluate this effect in the mild laser damage model.

Overall, the strengths outweigh the weaknesses, provided the conclusions/interpretations are reconsidered.

Reviewer #1 (Public review):

The manuscript now compares the WNet3D quantitatively against other methods on all four datasets:

Figure 1b shows results on the mouse cortex dataset, comparing StarDist, CellPose, SegResNet, SwinUNetR against self-supervised (or learning-free methods) WNet3D and Otsu thresholding.

Figure 2b shows results on an unnamed dataset (presumably the mouse cortex dataset), comparing StarDist, CellPose, SegResNet, SwinUNetR with different levels of training data against WNet3D.

Figure 3 shows results on three datasets (Platynereis-ISH-Nuclei-CBG, Platynereis-Nuclei-CBG, and Mouse-Skull-Nuclei-CBG), comparing StarDist, CellPose against WNet3D and Otsu thresholding.

It is unclear whether the Otsu thresholding baseline was given the same post-processing as the WNet3D. Figure 1b shows two versions for WNet3D ("WNet3D - No artifacts" and "WNet3D"), but only one for Otsu thresholding. Given that post-processing (or artifact removal) seems to have a substantial impact on accuracy, the authors should clarify whether the Otsu thresholding results were treated in the same way and if Otsu thresholding was not post-processed. Figure 2a would also benefit from including the thresholding results (with and without artifact removal).

Reviewer #2 (Public review):

The authors have now addressed the most important points, and they include more comprehensive evaluation of their method and comparisons to other approaches for multiple datasets.

Some points would benefit from clarification:

- Figure 1B now compares "Otsu thresholding", "WNet 3D - No artifacts" and "WNet 3d". Why don't you also report the score for "Otsu thresholding - No Artifacts"? To my understanding this is a post-processing operation to remove small and very large objects, so it could easily be applied to the Otsu thresholding. Given the good results for Otsu thresholding alone (quite close F1-score to WNet 3d), it seems like DL might not really be necessary at all for this dataset and including "Otsu thresholding - No artifacts" would enable evaluating this point.

- CellPose and StarDist perform poorly in all the experiments performed by the authors. In almost all cases they underperform Otsu thresholding, which is in most cases on par with the WNet results (except for "Mouse Skull Nuclei CBG"). This is surprising and contradicts the collective expertise of the community: good CellPose and StarDist models can be trained for the 3D instance segmentation tasks studied here. Perhaps these methods were not trained in an optimal way. Seems unlikely that it is not possible to get much better CellPose or StarDist models for these tasks (current versions are on par or much worse than Otsu!), as I have applied both of these models successfully in similar settings. Specifically, it seems unlikely that the developers of CellPose or StarDist would obtain similarly poor scores on the same data (note I am not one of the developers).

The current experiments still highlight an interesting aspect: the problem of training / fine-tuning these methods correctly on new data and the technical challenges associated with this. But the reported results should by no means be taken as a fair assessment of the capabilities of StarDist or CellPose.

Please note that I did not have time to test the Napari plugin again, so I did not evaluate whether it improved in usability.

Reviewer #2 (Public review):

Summary:

The goal of the paper was to trace the transitions hippocampal microglia undergo along aging. ScRNA-seq analysis allowed the authors to predict a trajectory and hypothesize about possible molecular checkpoints, which keep the pace of microglial aging. E.g. TGF1b was predicted as a molecule slowing down the microglial aging path and indeed, loss of TGF1 in microglia led to premature microglia aging, which was associated with premature loss of cognitive ability. The authors also used the parabiosis model to show how peripheral, blood-derived signals from the old organism can "push" microglia forward on the aging path.

Strengths:

A major strength and uniqueness of this work is the in-depth single-cell dataset, which may be a useful resource for the community, as well as the data showing what happens to young microglia in heterochronic parabiosis setting and upon loss of TGFb in their environment.

Weaknesses:

All weaknesses were addressed during revision.

Overall:

In general, I think the authors did a good job following the initial observations and devised clever ways to test the emerging hypotheses. The resulting data are an important addition to what we know about microglial aging and can be fruitfully used by other researchers, e.g. those working on microglia in a disease context.

Comments on revisions:

All my comments were addressed.

Reviewer #1 (Public review):

Summary:

This study addresses the question of how task-relevant sensory information affects activity in motor cortex. The authors use various approaches to address this question, looking at single units and population activity. They find that there are three subtypes of modulation by sensory information at the single unit level. Population analyses reveal that sensory information affects the neural activity orthogonally to motor output. The authors then compare both single unit and population activity to computational models to investigate how encoding of sensory information at the single-unit level is coordinated in a network. They find that an RNN that displays similar orbital dynamics and sensory modulation to motor cortex also contains nodes that are modulated similarly to the three subtypes identified by the single unit analysis.

Strengths:

The strengths of this study lie in the population analyses and the approach of comparing single-unit encoding to population dynamics. In particular, the analysis in Figure 3 is very elegant and informative about the effect of sensory information on motor cortical activity. The task is also well designed to suit the questions being asked and well controlled.

It is commendable that the authors compare single-unit to population modulation. The addition of the RNN model and perturbations strengthen the conclusion that the subtypes of individual units all contribute to the population dynamics.

Weaknesses:

The main weaknesses of the study lie in the categorization of the single units into PD shift, gain and addition types. The single units exhibit clear mixed selectivity, as the authors highlight. Therefore, the subsequent analyses looking only at the individual classes in the RNN are a little limited. Another weakness of the paper is that the choice of windows for analyses is not properly justified and the dependence of the results on the time windows chosen for single unit analyses is not assessed. This is particularly pertinent because tuning curves are known to rotate during movements (Sergio et al. 2005 Journal of Neurophysiology).

This study uses insights from single-unit analysis to inform mechanistic models of these population dynamics, which is a powerful approach, but is dependent on the validity of the single-cell analysis, which I have expanded on below.

I have clarified some of the areas that would benefit from further analysis below:

Task:

The task is well designed, although it would have benefited from perhaps one more target speed (for each direction). One monkey appears to have experienced one more target speed than the others (seen in Figure 3C). It would have been nice to have this data for all monkeys, although, of course, unfeasible given that the study has been concluded.

Single unit analyses:

The choice of the three categories (PD shift, gain addition) is not completely justified in a satisfactory way. It would be nice to see whether these three main categories are confirmed by unsupervised methods.

The decoder analyses in Figure 2 provide evidence that target speed modulation may change over the trial. Therefore, it is important to see how the window considered for the firing rate in Figure 1 (currently 100ms pre - 100ms post movement onset) affects the results. Whilst it is of course understandable that a window must be chosen and will always be slightly arbitrary, using different windows and comparing the results of two or three different sizes or timed windows would be more convincing that the results are not dependent on this particular window.

RNN:

Mixed selectivity is not analysed in the RNN, which would help to compare the model to the real data where mixed selectivity is common. The CCA and Procrustes analysis are a good start to validate the claim of similarity between RNN and neural dynamics, rather than allowing comparisons to be dominated by geometric similarities that may be features of the task. However, some of the disparity values for the Procrustes analysis are quite high, albeit below that of the shuffle. Maybe a comment about this in the text should be included. There is also an absence of alternate models to compare the perturbation model results to.

Reviewer #2 (Public review):

Summary:

In this manuscript, Zhang et al. examine neural activity in motor cortex as monkeys make reaches in a novel target interception task. Zhang et al. begin by examining the single neuron tuning properties across different moving target conditions, finding several classes of neurons: those that shift their preferred direction, those that change their modulation gain, and those that shift their baseline firing rates. The authors go on to find an interesting, tilted ring structure of the neural population activity, depending on the target speed, and find that 1) the reach direction has consistent positioning around the ring, and 2) the tilt of the ring is highly predictive of the target movement speed. The authors then model the neural activity with a single neuron representational model and a recurrent neural network model, concluding that this population structure requires a mixture of the three types of single neurons described at the beginning of the manuscript.

Strengths:

I find the task the authors present here to be novel and exciting. It slots nicely into an overall trend to break away from a simple reach-to-static-target tasks to better characterize the breadth of how motor cortex generates movements. I also appreciate the movement from single neuron characterization to population activity exploration, which generally serves to anchor the results and make them concrete. Further, the orbital ring structure of population activity is fascinating, and the modeling work at the end serves as a useful baseline control to see how it might arise.

Weaknesses:

While I find the behavioral task presented here to be excitingly novel, I find the presented analyses and results to be far less interesting than they could be. Key to this, I think, is that the authors are examining this task and related neural activity primarily with a single-neuron representational lens. This would be fine as an initial analysis, since the population activity is of course composed of individual neurons, but the field seems to have largely moved towards a more abstract "computation through dynamics" framework that has, in the last several years, provided much more understanding of motor control than the representational framework has. As the manuscript stands now, I'm not entirely sure what interpretation to take away from the representational conclusions the authors made (i.e. the fact that the orbital population geometry arises from a mixture of different tuning types). As such, by the end of the manuscript, I'm not sure I understand any better how motor cortex or its neural geometry might be contributing to the execution of this novel task.

Main Comments:

My main suggestions to the authors revolve around bringing in the computation through a dynamics framework to strengthen their population results. The authors cite the Vyas et al. review paper on the subject, so I believe they are aware of this framework. I have three suggestions for improving or adding to the population results:

(1) Examination of delay period activity: one of the most interesting aspects of the task was the fact that the monkey had a random-length delay period before he could move to intercept the target. Presumably, the monkey had to prepare to intercept at any time between 400 and 800 ms, which means that there may be some interesting preparatory activity dynamics during this period. For example, after 400ms, does the preparatory activity rotate with the target such that once the go cue happens, the correct interception can be executed? There is some analysis of the delay period population activity in the supplement, but it doesn't quite get at the question of how the interception movement is prepared. This is perhaps the most interesting question that can be asked with this experiment, and it's one that I think may be quite novel for the field--it is a shame that it isn't discussed.

(2) Supervised examination of population structure via potent and null spaces: simply examining the first three principal components revealed an orbital structure, with a seemingly conserved motor output space and a dimension orthogonal to it that relates to the visual input. However, the authors don't push this insight any further. One way to do that would be to find the "potent space" of motor cortical activity by regression to the arm movement and examine how the tilted rings look in that space. Presumably, then, the null space should contain information about the target movement. The ring tilt will likely be evident if the authors look at the highest variance neural dimension orthogonal to the potent space (the "null space")--this is akin to PC3 in the current figures, but it would be nice to see what comes out when you look in the data for it.

The authors attempt this sort of analysis in the supplement, alongside their dPCA results, but the results seem misinterpreted. The authors do identify one kind of output-potent space using the reach direction components of dPCA, and the reach directions are indeed aligned here. However, they then go on to interpret the target-velocity space as the output-null space, orthogonal to the potent space. There are two problems with this. 1) The target-velocity space is not necessarily orthogonal to the reach-direction space. This is a key aspect of dPCA--while the individual components within a particular marginalization space are orthogonal, the marginalization spaces themselves are not necessarily orthogonal unless they are forced to be (which the authors don't mention doing). 2) Even if the target-velocity space were orthogonal to the reach-direction space, it would not comprise the whole output-null space--such a null space would also include dimensions of neural population activity that have target-velocity/reach-direction interaction, which the authors show is a major component of neural population variance. Incidentally, the dPCA analysis the authors present shows what I would expect from their unsupervised results, but as it is written, the dPCA results are interpreted in a strange or potentially misleading way.

(3) RNN perturbations: as it's currently written, the RNN modeling has promise, but the perturbations performed don't provide me with much insight. I think this is because the authors are trying to use the RNN to interpret the single neuron tuning, but it's unclear to me what was learned from perturbing the connectivity between what seems to me almost arbitrary groups of neurons. It seems to me that a better perturbation might be to move the neural state before the movement onset to see how it changes the output. For example, the authors could move the neural state from one tilted ring to another to see if the virtual hand then reaches a completely different (yet predictable) target. Moreover, if the authors can more clearly characterize the preparatory movement, perhaps perturbations in the delay period would provide even more insight into how the interception might be prepared.

Reviewer #3 (Public review):

Summary:

This experimental study investigates the influence of sensory information on neural population activity in M1 during a delayed reaching task. In the experiment, monkeys are trained to perform a delayed interception reach task, in which the goal is to intercept a potentially moving target.

This paradigm allows the authors to investigate how, given a fixed reach end point (which is assumed to correspond to a fixed motor output), the sensory information regarding the target motion is encoded in neural activity.

At the level of single neurons, the authors find that target motion modulates the activity is three main ways: gain modulation (scaling of the neural activity depending on the target direction), shift (shift of the preferred direction of neurons tuned to reach direction), or addition (offset to the neural activity).

At the level of the neural population, target motion information was largely encoded along the 3rd PC of the neural activity, leading to a tilt of the manifold along which reach direction was encoded that was proportional to target speed. The tilt of the neural manifold was found to be largely driven by the variation of activity of the population of gain modulated neurons.

Finally, the authors study the behaviour of an RNN trained to generate the correct hand velocity given the sensory input and reach direction. The RNN units are found to similarly exhibit mixed selectivity to the sensory information, and the geometry of the « neural population » resembles that observed in the monkeys.

Overall, the experiment is well set up to address the question of how sensory information that is directly relevant to the behaviour but does not lead to a direct change in behavioural output modulates motor cortical activity.<br /> The finding that sensory information modulates the neural activity in M1 during motor preparation and execution is non trivial, given that this modulation of the activity must occur in the nullspace of the movement.<br /> The authors provide analyses at both the single neuron and the population level, leading to a relatively complete characterization of the effect of the target motion on neural activity.<br /> Additionally, they start exploring the link between the population geometry and the mixed selectivity of the single neurons in their RNN model. While they could be extended in future work, the analyses of the RNN provide a good starting point to address how exactly the task setup and constraints on the network shape the single neuron selectivity and the population geometry.

Reviewer #2 (Public review):

Summary:

The authors describe a "beads-on-a-string" (BOAS) immunogen, where they link, using a non-flexible glycine linker, up to eight distinct hemagglutinin (HA) head domains from circulating and non-circulating influenzas and assess their immunogenicity. They also display some of their immunogens on ferritin NP and compare the immunogenicity. They conclude that this new platform can be useful to elicit robust immune responses to multiple influenza subtypes using one immunogen and that it can also be used for other viral proteins.

Strengths:

The paper is clearly written. While the use of flexible linkers has been used many times, this particular approach (linking different HA subtypes in the same construct resembling adding beads on a string, as the authors describe their display platform) is novel and could be of interest.

Comments on revisions:

The authors have addressed most comments. Some mistakes/issues remain:

TI should be defined earlier on line 61 not on line 196

No legend for Figure 3E - it looks like this is where the authors did the first immunization with the "mix" to compare to the BOAs but strangely they do not mention this in the response to reviewers letter and only mention fig 6G and 7<br /> Maybe add "mix" to the title of Figure 3?

In Figure 6G they do show the response to the mix but do not mention it in the immunizations for that figure. Also weird because obviously the mix is not a NP while this figure addresses NP format.

Line 796 - pseudo viruses

The authors should add some clarification in the paper as they did in response to reviewers.

Reviewer #3 (Public review):

This work describes the tandem linkage of influenza hemagglutinin (HA) receptor binding domains of diverse subtypes to create 'beads on a string' (BOAS) immunogens. They show that these immunogens elicit ELISA binding titers against full-length HA trimers in mice, as well as varying degrees of vaccine mismatched responses and neutralization titers. They also compare these to BOAS conjugated on ferritin nanoparticles and find that this did not largely improve immune responses. This work offers a new type of vaccine platform for influenza vaccines, and this could be useful for further studies on the effects of conformation and immunodominance on the resulting immune response. 

Overall, the central claims of immunogenicity in a murine model of the BOAS immunogens described here are supported by the data. 

Strengths included the adaptability of the approach to include several, diverse subtypes of HAs. The determination of an optimal composition of strains in the 5-BOAS that overall yielded the best immune responses was an interesting finding and one that could also be adapted to other vaccine platforms. Lastly, as the authors discuss, the ease of translation to an mRNA vaccine is indeed a strength of this platform. 

One interesting and counter-intuitive result is the high levels of neutralization titers seen to vaccine-mismatched, group 2 H7 in the 5-BOAS group that differs from the 4-BOAS with the addition of a group 1 H5 RBD. At the same time, no H5 neutralization titers were observed for any of the BOAS immunogens, yet they were seen for the BOAS-NP. Uncovering where these immune responses are being directed and why these discrepancies are being observed would be informative future work. 

There are a few caveats in the data that should be noted: 

(1) 20 ug is a pretty high dose for a mouse and the majority of the serology presented is after 3 doses at 20 ug. By comparison, 0.5-5 ug is a more typical range (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380945/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9980174/). Also, the authors state that 20 ug per immunogen was used, including for the BOAS-NP group, which would mean that the BOAS-NP group was given a lower gram dose of HA RBD relative to the BOAS groups. 

(2) Serum was pooled from all animals per group for neutralization assays, instead of testing individual animals. This could mean that a single animal with higher immune responses than the rest in the group could dominate the signal and potentially skew the interpretation of this data. 

(3) In Figure S2, it looks like an apparent increase in MW by changing the order of strains here, which may be due to differences in glycosylation. Further analysis would be needed to determine if there are discrepancies in glycosylation amongst the BOAS immunogens and how those differ from native HAs. 

Comments on revisions:

The authors have addressed all concerns upon revision.

Reviewer #1 (Public review):

Summary:

Rossi et al. asked whether gait adaptation is solely a matter of slow perceptual realignment or if it also involves fast/flexible stimulus-response mapping mechanisms. To test this, they conducted a series of split-belt treadmill experiments with ramped perturbations, revealing behavior indicative of a flexible, automatic stimulus-response mapping mechanism.

Strengths:

(1) The study includes a perceptual test of leg speed, which correlates with the perceptual realignment component of motor aftereffects. This indicates that changes in motor performance are not fully accounted for by perceptual realignment.

(2) The study evaluates the possible contributions of explicit strategy using a framework (Tsay et al., 2024) and provides evidence for minimal strategy involvement in split-belt adaptation through subjective reports.

(3) The study incorporates qualitatively distinct, hypothesis-driven models of adaptation and proposes a new framework that integrates these mechanisms. Relatedly, the study considers a range of alternative models, demonstrating that perceptual recalibration and remapping uniquely explain the patterns of behavior and aftereffects, ruling out models that focus solely on a single process (e.g., PReMo, PEA, memory of errors, optimal feedback control) and others that do not incorporate remapping (dual rate state space models).

Reviewer #2 (Public review):

Recent findings in the field of motor learning have pointed to the combined action of multiple mechanisms that potentially contribute to changes in motor output during adaptation. A nearly ubiquitous motor learning process occurs via the trial-by-trial compensation of motor errors, often attributed to cerebellar-dependent updating. This error-based learning process is slow and largely unconscious. Additional learning processes that are rapid (e.g., explicit strategy-based compensation) have been described in discrete movements like goal-directed reaching adaptation. However, the role of rapid motor updating during continuous movements such as walking has been either under explored or inconsistent with those found during adaptation of discrete movements. Indeed, previous results have largely discounted the role of explicit strategy-based mechanisms for locomotor learning. In the current manuscript, Rossi et al. provide convincing evidence for a previously unknown rapid updating mechanism for locomotor adaptation. Unlike the now well-studied explicit strategies employed during reaching movements, the authors demonstrate that this stimulus-response mapping process is largely unconscious. The authors show that in approximately half of subjects, the mapping process appears to be memory based while the remainder of subjects appear to perform structural learning of the task design. The participants that learned using a structural approach had the capability to rapidly generalize to previously unexplored regions of the perturbation space.

One result that will likely be particularly important to the field of motor learning is the authors' quite convincing correlation between the magnitude of proprioceptive recalibration and the magnitude error-based updating. This result beautifully parallels results in other motor learning tasks and appears to provide a robust marker for the magnitude of the mapping process (by means of subtracting off the contribution of error-based motor learning). This is a fascinating result with implications for the motor learning field well beyond the current study.

A major strength of this manuscript is the large sample size across experiments and the extent of replication performed by the authors in multiple control experiments.

Finally, I commend the authors on extending their original observations via Experiment 2. While it seems that participants use a range of mapping mechanisms (or indeed a combination of multiple mapping mechanisms), future experiments may be able to tease apart why some subjects use memory versus structural mapping. A future ability to push subjects to learn structurally-based mapping rules has the potential to inform rehabilitation strategies.

Overall, the manuscript is well written, the results are clear, and the data and analyses are convincing.

Strengths:

(1) Convincing behavioral data supporting the existence of multiple learning processes during split-belt adaptation. Further convincing correlations typing the extent of forward-model based adaptation with proprioceptive recalibration.<br /> (2) The authors test a veritable "zoo" of prior motor learning models to show that these models do not account for their behavioral results.<br /> (3) The authors develop a convincing alternative model (PM-ReMap) that appears to account for their behavioral results by explicitly modeling forward-model based adaptation in parallel with goal remapping.

Reviewer #3 (Public review):

Summary:

In this work, Rossi et al. use a novel split-belt treadmill learning task to reveal distinct sub-components of gait adaptation. The task involved following a standard adaptation phase with a "ramp-down" phase that helped them dissociate implicit recalibration and more deliberate SR map learning. Combined with modeling and re-analysis of previous studies, the authors show multiple lines of evidence that both processes run simultaneously, with implicit learning saturating based on intrinsic learning constraints and SR learning showing sensitivity to a "perceptual" error. These results offer a parallel with work in reaching adaptation showing both explicit and implicit processes contributing to behavior; however, in the case of gait adaptation the deliberate learning component does not appear to be strategic but is instead a more implicit SR learning process.

The authors have done a commendable job responding to my comments and critiques. I have updated the S/W below to reflect that.

Strengths:

- The task design is very clever and the "ramp down" phase offers a novel way to attempt to dissociate competing models of multiple processes in gait adaptation<br /> - The analyses are thorough, as is the re-analysis of multiple previous data sets; the expanded modeling analyses are strong<br /> - The querying of perception of the different relative belt speeds is a very nice addition, allowing the authors to connect different learning components with error perception<br /> - The conceptual framework is compelling, highlighting parallels with work in reaching but also emphasizing differences, especially w/r/t SR learning versus strategic behaviors. Thus the discovery of an SR learning process in gait adaptation would be both novel and also help conjoin different siloed subfields of motor learning research.

Weaknesses:

- The expanded modeling analyses are useful although the SR process still seems somewhat mysterious (is it explicit/implicit? how exactly is it interacting with re-calibration?); however, understanding this system more could be a fruitful topic for future work<br /> - The sample size for the individual difference analysis is somewhat modest

Reviewer #1 (Public Review):

Summary:

Zhang et al. demonstrate that CD4+ single positive (SP) thymocytes, CD4+ recent thymic emigrants (RTE), and CD4+ T naive (Tn) cells from Cd11c-p28-flox mice, which lack IL-27p28 selectively in Cd11c+ cells, exhibit a hyper-Th1 phenotype instead of the expected hyper Th2 phenotype. Using IL-27R-deficient mice, the authors confirm that this hyper-Th1 phenotype is due to IL-27 signaling via IL-27R, rather than the effects of monomeric IL-27p28. They also crossed Cd11c-p28-flox mice with autoimmune-prone Aire-deficient mice and showed that both T cell responses and tissue pathology are enhanced, suggesting that SP, RTE, and Tn cells from Cd11c-p28-flox mice are poised to become Th1 cells in response to self-antigens. Regarding mechanism, the authors demonstrate that SP, RTE, and Tn cells from Cd11c-p28-flox mice have reduced DNA methylation at the IFN-g and Tbx21 loci, indicating 'de-repression', along with enhanced histone tri-methylation at H3K4, indicating a 'permissive' transcriptional state. They also find evidence for enhanced STAT1 activity, which is relevant given the well-established role of STAT1 in promoting Th1 responses, and surprising given IL-27 is a potent STAT1 activator. This latter finding suggests that the Th1-inhibiting property of thymic IL-27 may not be due to direct effects on the T cells themselves.

Strengths:

Overall the data presented are high quality and the manuscript is well-reasoned and composed. The basic finding - that thymic IL-27 production limits the Th1 potential of SP, RTE, and Tn cells - is both unexpected and well described.

Weaknesses from the original round of review:

A credible mechanistic explanation, cellular or molecular, is lacking. The authors convincingly affirm the hyper-Th1 phenotype at epigenetic level but it remains unclear whether the observed changes reflect the capacity of IL-27 to directly elicit epigenetic remodeling in developing thymocytes or knock-on effects from other cell types which, in turn, elicit the epigenetic changes (presumably via cytokines). The authors propose that increased STAT1 activity is a driving force for the epigenetic changes and resultant hyper-Th1 phenotype. That conclusion is logical given the data at hand but the alternative hypothesis - that the hyper-STAT1 response is just a downstream consequence of the hyper-Th1 phenotype - remains equally likely. Thus, while the discovery of a new anti-inflammatory function for IL-27 within the thymus is compelling, further mechanistic studies are needed to advance the finding beyond phenomenology.

Reviewer #2 (Public Review):

Summary:

Naïve CD4 T cells in CD11c-Cre p28-floxed mice express highly elevated levels of proinflammatory IFNg and the transcription factor T-bet. This phenotype turned out to be imposed by thymic dendritic cells (DCs) during CD4SP T cell development in the thymus [PMID: 23175475]. The current study affirms these observations, first, by developmentally mapping the IFNg dysregulation to newly generated thymic CD4SP cells [PMID: 23175475], second, by demonstrating increased STAT1 activation being associated with increased T-bet expression in CD11c-Cre p28-floxed CD4 T cells [PMID: 36109504], and lastly, by confirming IL-27 as the key cytokine in this process [PMID: 27469302]. The authors further demonstrate that such dysregulated cytokine expression is specific to the Th1 cytokine IFNg, without affecting the expression of the Th2 cytokine IL-4, thus proposing a role for thymic DC-derived p28 in shaping the cytokine response of newly generated CD4 helper T cells. Mechanistically, CD4SP cells of CD11c-Cre p28-floxed mice were found to display epigenetic changes in the Ifng and Tbx21 gene loci that were consistent with increased transcriptional activities of IFNg and T-bet mRNA expression. Moreover, in autoimmune Aire-deficiency settings, CD11c-Cre p28-floxed CD4 T cells still expressed significantly increased amounts of IFNg, exacerbating the autoimmune response and disease severity. Based on these results, the investigators propose a model where thymic DC-derived IL-27 is necessary to suppress IFNg expression by CD4SP cells and thus would impose a Th2-skewed predisposition of newly generated CD4 T cells in the thymus, potentially relevant in autoimmunity.

Strengths:

Experiments are well-designed and executed. The conclusions are convincing and supported by the experimental results.

Weaknesses from the original round of review:

The premise of the current study is confusing as it tries to use the CD11c-p28 floxed mouse model to explain the Th2-prone immune profile of newly generated CD4SP thymocytes. Instead, it would be more helpful to (1) give full credit to the original study which already described the proinflammatory IFNg+ phenotype of CD4 T cells in CD11c-p28 floxed mice to be mediated by thymic dendritic cells [PMID: 23175475], and then, (2) build on that to explain that this study is aimed to understand the molecular basis of the original finding.

In its essence, this study mostly rediscovers and reaffirms previously reported findings, but with different tools. While the mapping of epigenetic changes in the IFNg and T-bet gene loci and the STAT1 gene signature in CD4SP cells are interesting, these are expected results, and they only reaffirm what would be assumed from the literature. Thus, there is only incremental gain in new insights and information on the role of DC-derived IL-27 in driving the Th1 phenotype of CD4SP cells in CD11c-p28 floxed mice.

Altogether, the major issues of this study remain unresolved:

(1) It is still unclear why the p28-deficiency in thymic dendritic cells would result in increased STAT1 activation in CD4SP cells. Based on their in vitro experiments with blocking anti-IFNg antibodies, the authors conclude that it is unlikely that the constitutive activation of STAT1 would be a secondary effect due to autocrine IFNg production by CD4SP cells. However, this possibility should be further tested with in vivo models, such as Ifng-deficient CD11c-p28 floxed mice. Alternatively, is this an indirect effect by other IFNg producers in the thymus, such as iNKT cells? It is necessary to explain what drives the STAT1 activation in CD11c-p28 floxed CD4SP cells in the first place.

(2) It is also unclear whether CD4SP cells are the direct targets of IL-27 p28. The cell-intrinsic effects of IL-27 p28 signaling in CD4SP cells should be assessed and demonstrated, ideally by CD4SP-specific deletion of IL-27Ra, or by establishing bone marrow chimeras of IL-27Ra germline KO mice.

[Editors' note: The resubmitted paper was minimally revised, and many of the initial concerns remain unresolved.]

Reviewer #1 (Public review):

Summary:

The authors define the principles that, based on first principles, should be guiding the optimisation of trascription factors with intrinsically disordered regions (IDR). The first part of the study defines the following principles to optimize the binding affinities to the genome in the receiving region that is called the "antenna": (i) reduce the target to IDR-binding distance on the genome, (ii) optimise the distance betwee the DNA binding domain and the binding sites on the IDR to be as close as possible to the distance between their binding sites on the genome; (iii) keep the same number of binding sites and their targets and modulate this number with binding strength, reducing them with increased strenght; (iv) modulate the binding strenght to be above a threshold that depends on the proportion of IDR binding sites in the antenna. The second part defines the scaling of the seach time in function of key parameters such as the volume of the nucleus, and the size of the antenna, derived as a combination of 3D search of the antenna and 1D "octopusing" on the antenna. The third part focuses on validation, where the current results are compared to binding probabilith data from a single experiment, and new experiment are proposed to further validate the model as well as testing designed transcription factors.

Strengths:

The strength of this work is that it provides simple, interpretable and testable theoretical conclusions. This will allow the derived design principles to be understood, evaluated and improved in the future. The theoretical derivations are rigorous. The authors provides a comparison to experiments, and also propose new experiments to be performed in the future, this is a great value in the paper since it will set the stage and inspire new experimental techniques. Further, the field needs inspiration and motivations to develop these techniques, since they are required to benchmark the transcription factors designed with the methods presented in this paper, as well as to develop novel data based or in vivo methods that would greatly benefit the field. As such, this paper is a fundamental contribution to the field.

Weaknesses:

The model assumption that the interaction between the transcription factor and the DNA outside of the antenna region is negligible is probably too strong for many/most transcription factors, particularly in organisms with a longer genome than yeasts. The model presents many first principles to drive the design of transcription factor, but arguably, other principles and mechanisms might also play a role by being beneficial to the search and binding process. Specifically: (i) a role of the IDR in complex formation and cooperativity between multiple trascription factors, (ii) ability of the IDR to do parallel searching based on multiple DNA binding sites spaced by disordered regions, (iii) affinity of the IDR to specific compartmentalisations in the nucleus reducing the search time, etc. The paper would be improved by a discussion over alternative mechanisms.

Reviewer #2 (Public review):

Summary:

This is an interesting theoretical exploration of how a flexible protein domain, which has multiple DNA-binding sites along it, affects the stability of the protein-DNA complex. It proposes a mechanism ("octopusing") for protein doing a random walk while bound to DNA which simultaneously enables exploration of the DNA strand and stability of the bound state.

Strengths:

Stability of the protein-DNA bound state and the ability of the protein to perform 1d diffusion along the DNA are two properties of a transcription factor that are usually seen as being in opposition of each other. The octopusing mechanism is an elegant resolution of the puzzle of how both could be accommodated. This mechanism has interesting biological implications for the functional role of intrinsically disordered domains in transcription factor (TF) proteins. They show theoretically how these domains, if flexible and able to make multiple weak contacts with the DNA, can enhance the ability of the TF to efficiently find their binding site on the DNA from which they exert control over the transcription of their target gene. The paper concludes with a comparison of model predictions with experimental data which gives further support to the proposed model. Overall, this is an interesting and well executed theoretical paper that proposes an interesting idea about the functional role for IDR domains in TFs.

Weaknesses:

IDR domains are assumed flexible which I believe is not always the case. Also, I'm not sure how ubiquitous are the assumed binding sites on the DNA for multiple subdomains along the IDR. These assumptions though seem like interesting points of departure for further experiments.

Reviewer #1 (Public review):

Summary:

This paper uses state-of-the-art techniques to define the cellular composition and its complexity in two rodent species (mice and rats). The study is built on available datasets but extends those in a way that future research will be facilitated. The study will be of high impact for the study of metabolic control.

Strengths:

(1) The study is based on experiments that are combined with two exceptional data sets to provide compelling evidence for the cellular composition of the DVC.

(2) The use of two rodent species is very useful.

Weaknesses:<br /> There is no conceptual weakness, the performance of experiments is state-of-the-art, and the discussion of results is appropriate. One minor point that would further strengthen the data is a more distinct analysis of receptors that are characteristic of the different populations of neuronal and non-neuronal cells; this part could be improved. Currently, it is only briefly mentioned, e.g., line 585ff. See also lines 603ff; it is true that the previous studies lack some information about the neurotransmitter profile of cells, but combining all data sets should result in an analysis of the receptors as well, e.g. in the form of an easy-to-read table.

Reviewer #2 (Public review):

In this manuscript, Hes et al. present a comprehensive multi-species atlas of the dorsal vagal complex (DVC) using single-nucleus RNA sequencing, identifying over 180,000 cells and 123 cell types across five levels of granularity in mice and rats. Intriguingly, the analysis uncovered previously uncharacterized cell populations, including Kcnj3-expressing astrocytes, neurons co-expressing Th and Cck, and a population of leptin receptor-expressing neurons in the rat area postrema, which also express the progenitor marker Pdgfra. These findings suggest species-specific differences in appetite regulation. This study provides a valuable resource for investigating the intricate cellular landscape of the DVC and its role in metabolic control, with potential implications for refining obesity treatments targeting this hindbrain region.

In line with previous work published by the PI, the topic is of clear scientific relevance, and the data presented in this manuscript are both novel and compelling. Additionally, the manuscript is well-structured, and the conclusions are robust and supported by the data. Overall, this study significantly enhances our understanding of the DVC and sheds light on key differences between rats and mice.

I applaud the authors for the depth of their analysis. However, I have a few major concerns, comments, and suggestions that should be addressed.

(1) If I understand the methodology correctly, mice were fasted overnight and then re-fed for 2 hours before being sacrificed (lines 91-92), which occurred 4 hours after the onset of the light phase (line 111). This means that the re-fed animals had access and consequently consumed food when they typically would not. While I completely recognize that every timepoint has its limitations, the strong influence of the circadian rhythm on the DVC gene expression (highlighted by the work published by Lukasz Chrobok), and the fact that timing of food/eating is a potent Zeitgeber, might have an impact on the analysis and should be mentioned as a potential limitation in the discussion (along with citing Dr Chrobok's work). Could this (i.e., eating during a time when the animals are not "primed by their own circadian clock to eat" potentially explain why the meal-related changes in gene expression were relatively small?

(2) In the Materials and Methods section, LiCl is mentioned as one of the treatment conditions; however, very little corresponding data are presented or discussed. Please include these results and elaborate on the rationale for selecting LiCl over other anorectic compounds.

(3) The number of animals used differs significantly between species, which the authors acknowledge as a limitation in the discussion. Since the authors took advantage of previously published mouse data sets (Ludwig and Dowsett data sets), I wonder if the authors could compare/integrate any rat data set currently available in rats as well to partially address the sample size disparity.

(4) Dividing cells in AP vs NTS vs DMX clusters and analyzing potential species differences would significantly enhance the quality of the manuscript, given the partially diverse functions of these regions. This could be done by leveraging existing published datasets that employed spatial transcriptomics or more classical methodologies (e.g., PMID: 39171288, PMID: 39629676, PMID: 38092916). I would be interested to hear the authors' perspective on the feasibility of such an analysis.

(5) Given the manuscript's focus on feeding and metabolism, I believe a more detailed description and comparison of the transcription profile of known receptors, neurotransmitters, and neuropeptides involved in food intake and energy homeostasis between mice and rats would add value. Adding a curated list of key genes related to feeding regulation would be particularly informative.

Reviewer #3 (Public review):

Summary:

This manuscript from Cecilia H et al provides a compelling resource for single nuclei RNA sequencing data with an emphasis on facilitating the integration of future data sets across mouse and rat data sets.

Strengths:

There are also several interesting findings that are highlighted, even though without a functional assay the importance remains unclear. However, the manuscript properly addresses where conclusions are speculative.

As with other snRNA seq datasets the manuscript demonstrates convincingly an increased level of complexity, while other neuronal populations like Cck and Th neurons were reproduced. Several recent findings from other groups are well addressed and put into a new context, e.g., DMV expression of AgRP (and Hcrt) was found to result from non-coding sequences, co-localization of Cck/Th was identified in a small subset. These statements are informative.

The integration of rat data into the mouse data sets is excellent, and the comparison of cellular groups is very detailed, with interesting differences between mouse and rat data.<br /> All data sets are easily accessible and usable on open platforms, this will be an impactful resource for other researchers.

Weaknesses:

The data analysis seems incomplete. The title indicates the integration of mouse and rat data into a unified rodent dataset. But the discrepancy of animal numbers (30 mice vs. 2 rats) does not fit well with that focus.

On the other hand, the mouse group is further separated into different treatments to study genetic changes that are associated with distinct energy states of fed/fasting/refeeding responses. Yet, this aspect is not addressed in depth.

While the authors find transcriptional changes in all neuronal and non-neuronal cell types, which is interesting, the verification of known transcriptional changes (e.g., cFos) is unaddressed. cFos is a common gene upregulated with refeeding that was surprisingly not investigated, even though this should be a strong maker of proper meal-induced neuronal activation in the DMV. This is a missed opportunity either to verify the data set or to highlight important limitations if that had been attempted without success.

Additional considerations:

(1) The focus on transmitter classification is highlighted, but surprisingly, the well-accepted distinction of GABAergic neurons by Slc32a1 was not used, instead, Gad1 and Gad2 were used as GABAergic markers. While this may be proper for the DMV, given numerous findings that Gad1/2 are not proper markers for GABAergic neurons and often co-expressed in glutamatergic populations, this confound should have been addressed to make a case if and why they would be proper markers in the DMV.

(2) Figure S3 for anatomical localization of clusters is excellent, but several of the cluster gene names do not have a good signal in the DMV. Specifically, the mixed neurons that do not seem to have clear marker genes. What top markers (top 10?) were used to identify these anatomical locations? At least some examples should be shown for anatomical areas to support Figure S3.

(3) Page 15, lines 410-411: "...could not find clusters sharing all markers with our neuronal classes...". Are the authors trying to say that the DMV has more diverse neurons than other brain sites? It seems not too unusual that the hypothalamus is different from the brainstem. Maybe this could be stated more clearly, and the importance of this could be clarified.

(4) The finding of GIRK1 astrocytes is interesting, but the emphasis that this means these astrocytes are highly/more excitable is confusing. This was not experimentally addressed and should be put into context that astrocyte activation is very different from neuronal activation. This should be better clarified in the results and discussion.

(5) The Pdgfra IHC as verification is great, but images are not very convincing in distinguishing the 2 (mouse) or 3 (rat) classes of cells. Why not compare Pdgfra and HuC/D co-localization by IHC and snRNAseq data (using the genes for HuC/D) in the mouse and in the rat? That would also clarify how specific HuC/D is for DMV neurons, or if it may also be expressed in non-neuronal populations.

Reviewer #1 (Public review):

This was a clearly written manuscript that did an excellent job summarizing complex data. In this manuscript, Cuevas-Zuviría et al. use protein modeling to generate over 5,000 predicted structures of nitrogenase components, encompassing both extant and ancestral forms across different clades. The study highlights that key insertions define the various Nif groups. The authors also examined the structures of three ancestral nitrogenase variants that had been previously identified and experimentally tested. These ancestral forms were shown in earlier studies to exhibit reduced activity in Azotobacter vinelandii, a model diazotroph.

This work provides a useful resource for studying nitrogenase evolution. However, its impact is somewhat limited due to a lack of evidence linking the observed structural differences to functional changes. For example, in the ancestral nitrogenase structures, only a small set of residues (lines 421-431) were identified as potentially affecting interactions between nitrogenase components. Why didn't the authors test whether reverting these residues to their extant counterparts could improve nitrogenase activity of the ancestral variants?

Additionally, the paper feels somewhat disconnected. The predicted nitrogenase structures discussed in the first half of the manuscript were not well integrated with the findings from the ancestral structures. For instance, do the ancestral nitrogenase structures align with the predicted models? This comparison was never explicitly made and could have strengthened the study's conclusions.

Reviewer #2 (Public review):

This work aims to study the evolution of nitrogenanses, understanding how their structure and function adapted to changes in the environment, including oxygen levels and changes in metal availability.

The study predicts > 5000 structures of nitrogenases, corresponding to extant, ancestral, and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive undertaking that is certain to be a resource for the community. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes.

The challenge with this study is that all (or nearly all) of the quantitative analyses presented are based on RMSD calculations, many of which are under 2 angstroms. For all intents and purposes, two structures with RMSD < 2 angstroms could be considered 'structurally identical'. A lot of insight generated is based on minuscule differences in RMSD, for which it is not clear that they are significantly different. The suggestion would be to find a way to evaluate the RMSD metric and determine whether these values, as obtained for structures being compared, are reliable. Some options are provided in earlier studies: PMID: 11514933, PMID: 17218333, PMID: 11420449, PMID: 8289285 (and others).

It could also be valuable to focus more on site-specific RMSDs rather than Global RMSDs. The high conservation in the nitrogenases likely ensures that the global RMSDs will remain low across the family. Focusing on specific regions might reveal interesting differences between clades that are more informative regarding the evolution of structure in tandem with environment/time.