Reviewer #1 (Public review):

Summary:

This manuscript follows up previous work from this group using a conditional TCF4 mouse where Cre-expression turns "on" expression of TCF4 to investigate whether postnatal re-expression of TCF4 is effective to correct phenotypes related to Pitt-Hopkins Syndrome (PTHS) in humans. Results may inform gene therapy human PTHS gene therapy efforts on effective developmental windows for gene therapy. The authors demonstrate that re-expression of TCF4, induced by retro-orbital (RO) AAV-PHP.eB-Cre, during 2-4th postnatal week, does not rescue brain or body weight, anxiety-like or nest-building behaviors, but rescues an object location memory task, a measure of cognition. These results are novel and interesting in that they reveal distinct developmental roles for TCF4 in distinct behaviors and suggest that TCF4 plays a role in the mature brain in hippocampal and memory-related plasticity. Results may inform gene therapy design in PTHS.

Strengths:

The results are rigorous and high quality. Multiple methods are used to assess AAV-mediated re-expression of Cre, reactivation of TCF4, and the developmental time course of expression. Multiple behavioral phenotypes and molecular rescue are assessed. Most behavioral phenotypes are reproducible and robust, and it is clear whether a rescue was observed.

Weaknesses:

(1) Although the authors demonstrate the time course and spatial extent of Cre and a Cre-reporter (TdTom) in the brain with the AAV-Cre, it is unclear how many cells are transduced. Similarly, the authors do not measure TCF4 levels with immunohistochemistry or western blot. So the level of protein reactivation is unknown. A possible reason the rescue is incomplete is that the TCF4 protein is not induced in a large % of neurons in specific brain regions that mediate specific behaviors, such as the hippocampus vs. the striatum.

(2) The authors perform bulk qPCR to demonstrate a 20% increase in TCF4 RNA with Cre-mediated activation. It is unclear why the full gene reactivation is not observed. An alternative interpretation of the incomplete rescue of the phenotypes is that full TCF4 expression is required at later developmental time points.

Reviewer #2 (Public review):

Summary:

The basic helix-loop-helix transcription factor TCF4 (also known, as ITF2, SEF2, or E2-2) is a protein involved in the development and functioning of many different cell types. TCF4 plays important roles in the nervous system, both in health and disease. Its importance in the nervous system is underlined by its association with common and rare cognitive disorders. Specifically, variants of the TCF4 gene are implicated in increased susceptibility to schizophrenia, and mutations in the TCF4 gene cause Pitt-Hopkins syndrome (PTHS) or mild to moderate non-syndromic intellectual disability.

In this manuscript, the authors have studied whether reinstating TCF4 later in postnatal development in juvenile PTHS model mice could reverse behavioral phenotypes, thereby simulating gene therapy. Previous research by the same group has demonstrated that restoring TCF4 during embryonic or neonatal stages, corresponding to prenatal or neonatal periods in humans, improved phenotypes in a PTHS mouse model. In the current study, a conditional TCF4 reinstatement mouse model, Tcf4-lox-stop-lox (Tcf4-LSL), previously developed and characterized by their lab, where Cre-mediated recombination removes a floxed transcriptional stop cassette downstream of exon 17, leading to reinstatement of all TCF4 isoforms at appropriate levels in neurons, was used. The study showed that this later intervention failed to correct most phenotypes, suggesting that perinatal reinstatement of TCF4 holds the greatest potential to treat behavioral symptoms of PTHS. However, the study also suggests that some cognitive behaviors may still be responsive to TCF4 reinstatement later in life.

Strengths:

This is a very important study aimed at developing gene therapy for PTHS. The study is technically very well performed and written.

Weaknesses:

The only weakness is that a human disease is modelled in a mouse, which is evolutionarily not the closest mammal to humans. Hopefully, in the future, similar studies will also be performed in a nonhuman primate model, for example rhesus macaque.

Reviewer #1 (Public review):

Summary:

The authors aimed to develop a translational framework for predicting vaccine reactogenicity by training a penalized ordinal regression model on mouse muscle transcriptomics and applying it across tissues and species to rank human vaccines by their inflammatory potential.

Strengths:

The study addresses an important gap in preclinical vaccine safety assessment. The identification of IL6/JAK/STAT3 signaling as a key pathway implicated in reactogenicity is biologically plausible, and the observation of coordinated changes between muscle and blood compartments supports the biological relevance of the signature. The model achieves near-perfect classification in mouse muscle tissue and successfully identifies Fluad (MF59-adjuvanted) as the most reactogenic among licensed human vaccines, consistent with clinical safety data.

Weaknesses:

The methodological foundation has several concerns. The reactogenicity class definitions rely on PC1 scores with modest variance explained, yet no sensitivity analyses demonstrate robustness to different normalization strategies, feature selection approaches, or dimensionality reduction methods. I suggest performing sensitivity analyses demonstrating that reactogenicity class definitions are robust to alternative normalization methods, feature selection criteria, and dimensionality reduction approaches.

The combined mouse analysis reveals that tissue effects dominate over vaccine-induced variation, and no explicit batch or compartment correction was reported. The authors can apply batch/compartment correction (e.g., SVA) when analyzing combined mouse muscle and blood data, then recompute PCA and downstream analyses.

The central claim regarding cross-species ranking capability is not fully supported. In human blood, the model largely distinguishes Fluad from other vaccines but shows limited separation among non-Fluad formulations, with many pairwise comparisons yielding non-significant adjusted p-values. This pattern suggests the model may be tuned to detect large inflammatory magnitudes-likely a consequence of training on extreme stimuli such as LPS and whole-cell pertussis-rather than capturing the finer gradations relevant for distinguishing licensed vaccines with moderate reactogenicity profiles. I highly suggest retraining the model, excluding extreme stimuli (LPS, Pentavac), to evaluate whether mid-range separations among licensed vaccines can be recovered.

Impact:

While the conceptual framework is promising, the current evidence does not convincingly demonstrate that the model can rank vaccines beyond identifying highly inflammatory outliers. The utility for preclinical assessment of novel vaccine candidates with moderate reactogenicity profiles remains uncertain.

Reviewer #2 (Public review):

Summary:

The authors derived a time-specific signature of reactogenicity from mouse muscle following exposure to vaccines /TLRs for capturing the reactogenicity patterns. They tested this reactogenicity signature in mouse blood, and then they applied the reactogenicity signature to human blood from subjects having received different vaccines. They identified biomarkers in mouse muscle which are also observed in mouse and human blood and could be used as a reactogenicity signature in mice, instead of CRP.

Strengths:

(1) The authors used transcriptomic response following vaccination and used common genes to human and mice for defining a reactogenic signature.

(2) As the authors used different formulations in mice, the model was trained across a broad reactogenicity spectrum, which has the advantage of being used for evaluating new vaccines/vaccine platforms.

Weaknesses:

(1) The muscle gene signature reflects local reactogenicity. Systemic reactogenicity is not specifically addressed, except where overlapping gene signatures are observed for both local and systemic reactogenicity.

(2) In the same logic, could we find additional genes in the blood which are not captured in the muscle?

(3) The peak of the reactogenicity is usually 24h; it is not certain that additional TPs have helped the findings. If they have, the authors should explain.

Reviewer #1 (Public review):

This manuscript by Toczyski and colleagues explores the role of ubiquitin-dependent degradation in the co-regulation between pro- and anti-apoptotic proteins. The binding of the pro-apoptotic sensor Bim to BCL2 anti-apoptotic proteins sequesters it into inactive complexes, inhibiting BCL2 members but also preventing Bim from activating the apoptotic executors BAX and BAK. The authors now suggest that the E3 ubiquitin ligase Cul5-Wsb2 targets Bim turnover while in complex with BLC2 members. The authors reveal the importance of WSB2 in apoptosis of neuroblastoma cell lines, highlighting the importance of Wsb2 as a cancer biomarker. In sum, this study identifies Bim as a novel Wsb2 target and suggests a novel co-receptor mechanism using BCL-2 members as bridging factors, thus adding a novel mechanistic layer to the apoptosis repressor role of Wsb2. Their experimental approach is sound, and in most cases, the conclusions are justified. However, whether Cul5-Wsb2 targets Bim via BLC2 anti-apoptotic members would require further analysis.

Major comments:

(1) They find that Wsb2 or Cul5 downregulation increases the levels of Puma and Bim isoforms, and that Wsb2 strongly interacts with all Bim isoforms. Moreover, Wsb2 regulates Bim turnover, especially visible for Bim-EL, and controls Bim-L ubiquitylation. Finally, Figure 2E suggests that Wsb2-Bim interaction is bridged by Bcl-xL, and they identify the domain in Bcl-xL/Wsb2 responsible for their binding in Figure 4A-E. However, Figure 4F shows only a mild decrease between Bim-EL and HA-Wsb2EEE, which is inconsistent with their model. This important gap should be backed up by further experimental evidence. For example, by performing (a) coIP studies between Bim and Wsb2 in the presence of Bcl-xlAAA and (b) Bim stability and ubiquitylation analysis in the presence of either Bcl-xlAAA or Wsb2EEE.

(2) The manuscript lacks quantifications and statistical analysis in most figures, which are particularly important for Figure 1D - especially regarding the upregulation of Puma and Bim isoforms upon downregulation of Cul5 and Wsb2, for Fig 3A - also including statistical analyses of Bim1 stability in presence or absence of proteasomal inhibitors, and for Figure 4D, F, especially regarding the interaction of Bim-EL- with WT and mutant Bcl-xL in 4D and with WT and mutant Wsb2 in 4F.

(3) The localization of BCL2 family members at the mitochondrial outer membrane is a crucial step in the implementation of apoptosis, and BCL2 members recruit Bim to the OM. Despite their finding suggesting that Bim insertion into the OM might be dispensable for interaction with Bim, the interaction was abolished by BH3-mimetics that disrupt Bcl-xL interaction with BIM. This suggests that Wsb2 interacts with Bim at the mitochondrial surface. Therefore, it would be interesting to investigate the sub-cellular localization Bim and WSB2 with and without ABT-263.

(4) Wsb2 mildly interacts with Bcl-xL and with Mcl1, but does not interact with Bcl-w or Bcl2. However, they show that Wsb2 recognizes Bcl-xl through a motif conserved between Bcl-xl, Bcl-w and Bcl2. Therefore, it would be helpful to precipitate Bcl-w or Bcl2 and check interaction with Wsb2.

Reviewer #2 (Public review):

Summary

This manuscript proposes an original and conceptually interesting model in which anti-apoptotic BCL-2 family proteins, particularly BCL-XL and MCL-1, not only sequester BIM but also act as adaptor "co-receptors" that recruit BIM to the CUL5-WSB2 ubiquitin ligase complex for degradation. The authors present a mechanistic framework supported by structure-guided mutagenesis, BH3 mimetic perturbations and co-immunoprecipitation assays performed in RPE1 cells. In parallel, the study shows that neuroblastoma cell lines are highly dependent on WSB2 for survival. These observations give the work both conceptual and translational relevance.

Strengths

The principal strength of the study lies in its conceptual novelty. Reframing BCL-XL and MCL-1 not only as sequestration factors but also as adaptors that facilitate substrate engagement by an E3 ligase substantially extends current models of apoptotic regulation. The mechanistic narrative developed in RPE1 cells is clear and internally consistent: the combination of AlphaFold-guided motif identification with complementary mutagenesis provides a persuasive framework for how WSB2 associates with anti-apoptotic BCL-2 family members and promotes BIM turnover. The definition of a BCL-XL/MCL-1 co-receptor mechanism for WSB2-mediated BIM degradation is therefore both intuitive and mechanistically appealing. In parallel, the authors present a distinct experimental series showing that neuroblastoma cells exhibit pronounced sensitivity to WSB2 loss, undergo apoptosis upon its depletion and display reduced competitiveness in mixed-culture assays. Although the mechanistic connection between these observations requires further clarification, the convergence of a well-defined biochemical model with a clear cancer-relevant phenotype enhances the potential biological significance of WSB2 and raises the possibility that its regulation may hold therapeutic relevance.

Weaknesses

There are several limitations that readers should consider when interpreting the study. The most fundamental issue is the disconnect between the mechanistic model established in RPE1 cells and the apoptotic phenotype observed in neuroblastoma. Although the manuscript convincingly demonstrates the WSB2-BCL-XL/MCL-1-BIM axis in RPE1 cells and independently shows that WSB2 loss compromises neuroblastoma viability, it does not examine whether BIM levels are elevated upon WSB2 depletion in neuroblastoma, nor whether apoptosis in these cells requires BIM. Without demonstrating WSB2-BCL-2-BIM complex formation or BIM dependence in the disease-relevant context, it remains unclear whether the co-receptor mechanism characterised in RPE1 cells explains the phenotype. This gap is compounded by the observation that PUMA, another potent pro-apoptotic factor, also increases following WSB2 loss, raising the possibility that multiple death pathways contribute to the outcome. The absence of a genetic rescue experiment, such as re-expression of an shRNA-resistant WSB2 restoring viability and suppressing apoptosis, further limits causal inference regarding WSB2's role in neuroblastoma.

Many central claims rely on single Western blots and pulldown assays without quantification or assessment of reproducibility. This complicates the interpretation of CHX chase experiments (where initial steady-state levels differ between samples) and limits confidence in BH3 mimetic experiments, which use a single concentration and short exposure time. Without dose-response curves, time-course analyses, caspase inhibition, or orthogonal genetic perturbation of BCL-XL or MCL-1, indirect or off-target drug effects cannot be excluded. Reduced co-IP signals in these assays could therefore reflect early apoptotic events or compound instability rather than specific disruption of protein-protein interactions.

A further limitation concerns the inference of a direct WSB2-BCL-XL interaction. The mutagenesis analyses are performed in lysates that contain endogenous or overexpressed BIM, and BH3 mimetics disrupt the WSB2 interaction only when the BCL-XL-BIM heterodimer is dismantled. The study thus cannot distinguish whether the mapped WSB2 motifs mediate direct contact with BCL-XL or whether they influence the architecture or stability of the BCL-XL-BIM complex. Because no purified protein reconstitution or biophysical binding assays are presented, the evidence for direct binding remains suggestive rather than conclusive.

The ubiquitination data also remain incomplete. Although the WSB2 mutation reduces the ubiquitin smear on BIM, the assay does not demonstrate dependence on CUL5, RBX2 or ARIH2, leaving open which ligase components are directly responsible. MLN4924 implicates CRLs more broadly, but the ubiquitination assay itself does not assign activity to the CUL5-WSB2 module.

Finally, several methodological details are insufficiently described, including the generation and validation of the doxycycline-inducible WSB2 and HA-WSB2 lines and the suitability of the WSB2-overexpressing control line used in immunoprecipitations.

Collectively, these issues do not undermine the conceptual interest of the proposed co-receptor model, but they do limit the strength of the mechanistic claims and weaken the connection between the defined mechanism and the neuroblastoma phenotype.

Reviewer #1 (Public review):

Kotzadimitriou et al. investigate how synaptotagmin-7 (syt7) contributes to short-term plasticity at cortical glutamatergic synapses. Using quantal-level iGluSnFR imaging and failure-based analyses at single boutons, the authors distinguish between synchronous and asynchronous glutamate release across boutons with differing baseline efficacy. They show that knocking out syt7 abolishes facilitation of synchronous release while leaving asynchronous facilitation largely intact, although reduced in magnitude. Furthermore, they argue that synchronous and asynchronous events arise from functionally distinct vesicle pools. The manuscript concludes that syt7 is essential for the facilitation of synchronous release, while other calcium sensors govern asynchronous release.

Strengths:

(1) The use of iGluSnFR provides a robust readout of single-synapse activity. Unlike traditional ephys methods that average the activity of thousands of synapses (which may mask the facilitation of low Pr synapses), the authors employ quantal imaging to analyze thousands of individual boutons and stratify them by efficacy. The representative images and traces in Figure 1 are of high quality, and the quantal analysis demonstrating multiple quantal peaks aligns well with previously published work (Mendonca et al., 2022; Wang et al., 2022).

(2) The failure-based analysis is thoughtfully implemented. By isolating trials in which no release occurred, the authors effectively separate facilitation from depletion, strengthening their central argument that syt7 is required for facilitation independent of vesicle depletion.

(3) The proposed model (depicted in Figure 7) is interesting and may reconcile the contradictory roles attributed to syt7, as described by others in the field. Specifically, the authors provide data to address syt7's potential function in facilitation, asynchronous release, and replenishment. However, to further support their model, which argues that "multiple Ca2+ sensors have both unique and overlapping roles in regulating synaptic plasticity," additional experiments are needed (see point 2 below).

Weaknesses:

(1) While the authors use cultures from syt7 knockout mice (and wild-type controls), there are no acute rescue experiments (e.g., syt7 viral transduction in KO cultures) or checks for compensatory changes in other proteins. Previous studies (Bacaj et al., 2013; Jackman et al., 2016) have utilized viral rescues to confirm specificity. Without such experiments, it remains theoretically possible that the chronic loss of syt7 leads to downregulation of another protein essential for facilitation. At a minimum, the authors should perform rescue experiments for at least some of their findings. Additionally, western blots for syt1 and syt7 should be conducted to confirm that their knockout is specific to syt7.

(2) The manuscript acknowledges the possible roles of Doc2a and syt3 but fails to address them experimentally. Recent work (Wu et al., 2024; Weingarten et al., 2024) has identified Doc2a as the primary sensor for asynchronous release. Even if its expression in cortical cultures remains unconfirmed (as claimed by the authors), they should, at the very least, perform Western blots for Doc2a and syt3 in both wild-type (to determine basal expression levels) and syt7 knockout cultures. Without analyzing the levels of these proteins, the mechanism/model behind the "remaining" asynchronous release remains speculative. Is it possible that these other calcium sensors are upregulated in their syt7 KO cultures and could instead explain their results?

Reviewer #2 (Public review):

Summary:

In this elegant study, the authors employ live iGluSnFR-based imaging of glutamate release from cortical boutons to dissect the distinct roles of the Ca²⁺ sensor synaptotagmin-7 (Syt7) in synaptic transmission. Although multiple functions have been attributed to Syt7 over the years, the field remains conflicted. The authors argue that one major obstacle for resolving some of these discrepancies lies in a fundamental limitation of electrophysiological recordings, which aggregate signals across all synapses to yield averaged readouts, dominated by strong, high-release-probability synapses. By using a live glutamate imaging approach combined with sensitive detection of action potential-evoked activity across different stimulation regimes, and a dedicated analysis pipeline, the authors confirm a role for Syt7 in facilitating synchronous release and in regulating the magnitude of asynchronous release. In contrast, they find no evidence that Syt7 contributes to the facilitation of asynchronous release, do not find evidence for a role for Syt7 in synaptic vesicle replenishment during AP trains, and provide evidence suggesting that the maintenance of facilitation by Syt7 may occur independently of vesicle depletion.

Strengths:

This study offers a fresh perspective on a debated issue, using a new experimental approach that the authors previously explored in the context of Synaptotagmin 1 (Mendonca et al. 2022). The authors record the response to a series of pair-pulse stimulations, followed by an AP train. By carefully quantifying individual events and by sorting events based on their efficacy, the authors extract quantitative information that they assign to different properties of synaptic function. They also devised an interesting approach for monitoring aspects of facilitation, in which they isolate PPR events where the first response did not elicit detectable release (thus regarding the release in response to the second AP as facilitating), and compare them with successful events. Together, the authors provide semi-quantitative descriptions of synchronous and asynchronous release during single, paired, and AP trains, yielding a weighted estimate of Syt7's contribution to distinct features of synaptic vesicle release that are independent of postsynaptic readouts. A major strength of the study is the confirmation of two principal proposed functions of Syt7: facilitation of synchronous release and regulation of the magnitude of asynchronous release.

Weaknesses:

The experimental approach presented here is elegant and well-executed. However, a principal limitation lies in translating electrophysiological terminology to imaging-based measurements. For instance, interpreting signals persisting beyond 10 ms as a proxy for asynchronous release relies on assumptions that would be good to experimentally justify. Could such signals arise from iGluSnFR saturation, or be affected by desensitization?. Moreover, the quantification of asynchronous release is based on very small signals that represent only a fraction of the already small synchronous release component, raising concerns about signal-to-noise limitations. A key issue is that failures to evoke glutamate release may arise from AP failures, such that the second response in a PPR does not necessarily represent facilitation. Given that many of the findings largely confirm existing literature, the study might have benefited from a different framing, for example, as an additional validation of the correspondence between electrophysiological measures and the authors' imaging-based readouts. Another point concerns the analysis of synaptic vesicle replenishment following depletion, which would ideally be addressed using alternative stimulation protocols, such as quantifying the response/success rate to single APs at varying time points after a train. Although the authors are appropriately cautious in their conclusions (e.g., with respect to Figure 5b), this limitation remains. Finally, the use of heterogeneous cortical neuronal cultures is likely to introduce substantial variability, as the authors themselves acknowledge, which may arise from the co-expression of multiple Ca²⁺ sensors across diverse cell types.

In summary, the authors were able to confirm previously-described changes in neurotransmission properties upon the loss of Syt7 using live imaging of glutamate release at the level of single boutons. They also present preliminary evidence for the interdependence of Syt7 function, synaptic vesicle replenishment, and the facilitation of asynchronous release, although these results will need to be substantiated in future studies using alternative stimulation protocols and complementary methodologies. Taken together with the group's prior work on synaptotagmin-1, this study illustrates that live imaging of glutamate release offers an alternative approach that recapitulates some elements detectable via electrophysiological analysis, while possibly revealing new insights into the function of synaptic proteins. As a whole, taking a live imaging approach may be a broadly accessible way forward to analyze synaptic function. The potential of studying synaptic proteins in diverse cell types that are difficult to access with patch-clamp electrophysiology is particularly compelling.

Reviewer #3 (Public review):

In this manuscript, the authors examine the role of Syt7 in the plasticity of synchronous and asynchronous release in cultured neurons. The experimental approach is the imaging of SF-iGluSnFR.A184V expressed in cultured neurons while delivering stimulation through whole-cell patch clamping of single neurons in the culture. In this manner, they could examine the optical signature of glutamate release in single presynaptic terminals, while separating the release events into synchronous (<10ms) and asynchronous (>10ms) while delivering both paired pulses or trains of stimuli (at 20 Hz, 50 ms between stimuli).

This manuscript employs techniques previously reported by the research group in their Mendoca et al., Nat Comms 2022 paper. This paper uses this approach to specifically examine the role of Syt7. The use of iGluSnFR in this manner provides significant rigor to the paper. The most significant weakness is that some of the events the authors discuss in this manuscript are rare, and the strength of the conclusions regarding those is somewhat unclear.

The main novel contribution of this manuscript is that single-bouton high-frequency imaging allowed them to examine paired-pulse plasticity in boutons that had not released neurotransmitter during the first pulse (failure-based analysis), thus separating between the effects of vesicle depletion and facilitation of the release machinery. This approach also allowed them to segregate their observations according to bouton-specific release efficacy. Both examinations are unavailable when performing cell-level analysis of neurotransmitter release, as is reported by most electrophysiological approaches.

The authors conclude that Syt7 contributes specifically to facilitation of synchronous release, not asynchronous release, while reducing the magnitude of the asynchronous component. Finally, the authors suggest segregation of synchronous and asynchronous release, either by differential use of calcium sensors or spatial segregation of the vesicles contributing to both modes of release.

This report contributes significantly to the discussion of the control of synaptic plasticity by different molecular players. It is not the first to examine Syt7, but its contribution to the examination of this protein is significant.

I find this report to be well executed and reasoned. In my opinion, the authors could improve the manuscript by clarifying the description of several methodological and experimental sections. Furthermore, in my opinion, some of the conclusions are overstated.

The authors state: "Because boutons along a single axon originate from the same presynaptic neuron, they are expected to share broadly similar molecular components of the vesicular release machinery and experience comparable presynaptic action potential waveforms." The authors should address the idea that presynaptic terminals from the same neuron on different postsynaptic targets can differ in the molecular components, as well as in the presynaptic side. There is ample evidence for differences between synapses onto glutamatergic and GABAergic neurons of the same neuron.

The authors used 4ms-long frames, but the stimuli are delivered at 20Hz (50ms apart). Therefore, in paired pulse stimulation, isn't there going to be a difference between the first and second stimuli regarding the timing of the frames relative to the stimulus? Isn't the deconvolution sensitive to such an offset?

A 10ms threshold for defining synchronous vs. asynchronous release full in-between frames. Doesn't this increase the chance of assigning borderline events to the wrong category?

On page 11 of the conclusion, the authors state that "Our data indicate that in our conditions during paired-pulse protocol Syt7 primarily enhances release probability rather than increasing the RRP size." While I understand the reasoning behind this statement, it should be toned down. The authors did not directly address the RRP size.

In failure-based analysis, the number of failure events in high-efficiency boutons is expected to be low. How does this affect the conclusions of the authors concerning the effects of Syt7 deletion on facilitation in high-efficiency boutons?<br /> SourceData.xlsx was not available to me, as far as I could tell.

How can the conclusions of the authors on the differential molecular composition of vesicles contributing to synchronous and asynchronous release be related to the reported effect of strontium on the nature of release? (see 10.1523/JNEUROSCI.20-12-04414.2000)

Is this the first use of failure-based analysis? If not, the authors should cite precedents. In 10.1016/s0896-6273(00)80338-4, failure of release during the 1st AP was presented, with facilitation during the 2nd, although no formal analysis was performed.

Reviewer #1 (Public review):

Summary:

This preprint investigates the molecular mechanism by which warm temperature induces female-to-male sex reversal in the ricefield eel (Monopterus albus), a protogynous hermaphroditic fish of significant aquacultural value in China. The study identifies Trpv4 - a temperature-sensitive Ca²⁺ channel - as a putative thermosensor linking environmental temperature to sex determination. The authors propose that Trpv4 causes Ca²⁺ influx, leading to activation of Stat3 (pStat3). pStat3 then transcriptionally upregulates the histone demethylase Kdm6b (aka Jmjd3), leading to increased dmrt1 gene expression and ovo-testes development. This work aims to bridge ecological cues with molecular and epigenetic regulators of sex change and has potential implications for sex control in aquaculture.

Strengths:

(1) This study proposes the first mechanistic pathway linking thermal cues to natural sex reversal in adult ricefield eel, extending the temperature-dependent sex determination paradigm beyond embryonic reptiles and saltwater fish

(2) The findings could have applications for aquaculture, where skewed sex ratios apparently limit breeding efficiency

Weaknesses:

Although the revised manuscript represents an improvement over the original version, substantial weaknesses remain.

Scientific Concerns

(1) Western blot normalization and exposure: The loading controls (GAPDH) in Fig. S3C appear overexposed, as do several Foxl2 blots. Because these signals are likely outside the linear range, I am not convinced that normalization is reliable. This raises concerns about the validity of the quantified results.

(2) Antibody validation and referencing (Line 776): The authors need to refer explicitly to figures demonstrating antibody validation. At present, these data are provided only as a supplementary file that is not cited in the manuscript. In addition, the Sox9a antibody appears to yield indistinguishable signals in control and RNAi conditions, suggesting that it may not recognize eel Sox9a. This issue is not addressed by the authors. Furthermore, antibody validation Western blots should be quantified.

(3) Unclear sample sizes (N values): Sample sizes remain unclear for several figures:

(a) Fig. 3F - No N value is provided. Each graph shows three data points; does this indicate that only three samples were quantified? If ten samples were collected, why were all not quantified?

(b) Fig. 4 - No N values are reported.

(c) Fig. 5A - Again, only three data points are shown per group, despite the apparent availability of twelve samples. The rationale for this discrepancy is not explained.

(4) qRT-PCR normalization: The manuscript does not specify the reference gene(s) used for qRT-PCR normalization. Although expression levels are reported as "relative," neither the identity of the reference gene(s) nor the justification for their selection is provided.

(5) Specificity of key antibodies: While the authors have made some effort to validate anti-Amh, anti-Sox9, and anti-Dmrt antibodies, the results remain incomplete. The Amh and Dmrt antibodies detect reduced protein levels following knockdown of their respective targets, which is encouraging. However, the Sox9a antibody shows no difference between control and RNAi conditions, suggesting it does not recognize eel Sox9. This is not acknowledged in the manuscript. In addition, no validation data are presented for Foxl2. Antibody validation data must be clearly referenced in the main text and presented in an interpretable and quantitative manner.

(6) Immunofluorescence data quality: The immunofluorescence images remain difficult to interpret. I strongly encourage the authors to enlarge the image panels and to present monochrome images (white signal on black background). The current presentation severely limits interpretability.

(7) Unreferenced supplementary figure: Fig. S4 is included in the submission but is not referenced anywhere in the manuscript text.

(8) Fig. 5B image resolution: The micrographs in Fig. 5B are too small to allow meaningful evaluation of the data.

(9) Unexplained data inclusion (Fig. 5E): Fig. 5E includes a pERK blot that is not mentioned in the Results section. The rationale for including these data is unclear.

(10) Poor blot quality (Fig. S3C): The blots in Fig. S3C exhibit high background and overexposure. I am concerned about the reliability of the quantification shown in panel D.

(11) Poor blot quality (Fig. S5G): The Stat3 blots in Fig. S5G contain numerous white artifacts, raising concerns about their suitability for normalization in panel H.

(12) Missing controls (Fig. 6E): Fig. 6E lacks controls for HO-3867 and Colivelin treatments alone. Without these controls, it is not possible to determine whether the reported effects are meaningful.

(13) Graphical presentation: The use of a light blue-to-pink gradient in bar graphs throughout the manuscript does not aid interpretation. I recommend using more distinct colors (e.g., red, orange, green, blue, purple, gray, black) to improve clarity. In summary, the interpretation of the study remains limited by persistent issues related to data presentation, image quality, and reagent specificity.

Reviewer #1 (Public review):

Summary:

Abdelmageed et al. investigate age-related changes in the subcellular localization of DNA polymerase kappa (POLK) in the brains of mice. POLK has been actively investigated for its role in translesion DNA synthesis and involvement in other DNA repair pathways in proliferating cells, very little is known about POLK in a tissue-specific context or let alone in post-mitotic cells. The authors investigated POLK subcellular distribution in the brains of young, middle-aged, and old mice via immunoblotting of fractioned tissue extracts and immunofluorescence (IF). Immunoblotting revealed a progressive decrease in the abundance of nuclear POLK, while cytoplasmic POLK levels concomitantly increased. Similar findings were present when IF was performed on brain sections. Further IF studies of cingulate cortex (Cg1), motor cortex (M1, M2), and somatosensory (S1) cortical regions all showed an age-related decline in nuclear POLK. Nuclear speckles of POLK decrease in each region, meanwhile the number of cytoplasmic POLK granules decreases in all four regions, but granule size is increasing. The authors report similar findings for REV1, another Y-family DNA polymerase.

The authors then investigate the colocalization of POLK with other DNA damage response (DDR) proteins in either pyramidal neurons or inhibitory interneurons. At 18 months of age, DNA damage marker gH2AX demonstrated colocalization with nuclear POLK, while strong colocalization of POLK and 8-oxo-dG was present in geriatric mice. The authors find that cytoplasmic POLK granules colocalize with stress granule marker G3BP1, suggesting that the accumulated POLK ends up in the lysosome.

Brain regions were further stained to identify POLK patterns in NeuN+ neurons, GABAergic neurons, and other non-neuronal cell types present in the cortex. Microglia associated with pyramidal neurons or inhibitory interneurons were found to have higher abundance of cytoplasmic POLK. The authors also report that POLK localization can be regulated by neuronal activity induced by Kainic acid treatment. Lastly, the authors suggest that POLK could serve as an aging clock for brain tissue, but POLK deserves further characterization and correlation to functional changes before being considered for a biomarker.

Strengths:

Investigation of TLS polymerases in specific tissues and in post-mitotic cells is largely understudied. The potential changes in sub cellular localization of POLK and potentially other TLS polymerases opens up many questions about DNA repair and damage tolerance in the brain and how it can change with age.

Weaknesses:

The work is quite novel and interesting, and the authors do suggest some potentially interesting roles for POLK in the brain, but these are in of themselves a bit speculative. The majority of the findings of this paper draw upon findings from POLK antibody and its presumed specificity for POLK. However, this antibody has not been fully validated and would benefit from further validation of the different band sizes. More mechanistic investigation is needed before POLK could be considered as a brain aging clock but does not preclude the potential for using POLK as a biological "dating" system for the brain.

Comments on revisions:

The revised manuscript is suitably improved and addresses reviewer comments.

Reviewer #2 (Public review):

Summary:

Abdelmageed et al., demonstrate POLK expression in nervous tissue and focus mainly on neurons. Here, they describe an exciting age-dependent change in POLK subcellular localization, from the nucleus in young tissue to the cytoplasm in old tissue. They argue that the cytosolic POLK associates with stress granules. They also investigate cell-type specific expression of POLK, and quantitate expression changes induced by cell autonomous (activity) and cell nonautonomous (microglia) factors.

Comments on revisions:

Do the authors have any explanation or reason for why they weren't able to achieve a higher knockdown of POLK using siRNA in Figure 1A2? It does not seem statistically different by eye, as all values in the KD overlap with the control. This does not seem like strong evidence that their antibody works.

Reviewer #1 (Public review):

Summary:

GID/CTLH-type RING ligases are huge multi-protein complexes that play an important role in protein ubiquitylation. The subunits of its core complex are distinct and form a defined structural arrangement, but there can be variations in subunit composition, such as exchange of RanBP9 and RanBP10. In this study, van gen Hassend and Schindelin provide new crystal structures of (parts of) key subunits and use those structures to elucidate the molecular details of the pairwise binding between those subunits. They identify key residues that mediate binding partner specificity. Using in vitro binding assays with purified protein, they show that altering those residues can switch specificity to a different binding partner.

Strengths:

This is a technically demanding study that sheds light on an interesting structural biology problem in residue-level detail. The combination of crystallization, structural modeling, and binding assays with purified mutant proteins is elegant and, in my eyes, convincing.

Weaknesses:

I mainly have some suggestions for further clarification, especially for a broad audience beyond the structural biology community.

(1) The authors establish what they call an 'engineering toolkit' for the controlled assembly of alternative compositions of the GID complex. The mutagenesis results are great for the specific questions asked in this manuscript. It would be great if they could elaborate on the more general significance of this 'toolkit' - is there anything from a technical point of view that can be generalized? Is there a biological interest in altering the ring composition for functional studies?

(2) Along the same lines, the mutagenesis required to rewire Twa1 binding was very complex (8 mutations). While this is impressive work, the 'big picture conclusion' from this part is not as clear as for the simpler RanBP9/10. It would be great if the authors could provide more context as to what this is useful for (e.g., potential for in vivo or in vitro functional studies, maybe even with clinical significance?)

(3) For many new crystal structures, the authors used truncated, fused, or otherwise modified versions of the proteins for technical reasons. It would be helpful if the authors could provide reasoning why those modifications are unlikely to change the conclusions of those experiments compared to the full-length proteins (which are challenging to work with for technical reasons). For instance, could the authors use folding prediction (AlphaFold) that incorporates information of their resolved structures and predicts the impact of the omitted parts of the proteins? The authors used AlphaFold for some aspects of the study, which could be expanded.

Reviewer #2 (Public review):

Summary:

This is a very interesting study focusing on a remarkable oligomerization domain, the LisH-CTLH-CRA module. The module is found in a diverse set of proteins across evolution. The present manuscript focuses on the extraordinary elaboration of this domain in GID/CTLH RING E3 ubiquitin ligases, which assemble into a gigantic, highly ordered, oval-shaped megadalton complex with strict subunit specificity. The arrangement of LisH-CTLH-CRA modules from several distinct subunits is required to form the oval on the outside of the assembly, allowing functional entities to recruit and modify substrates in the center. Although previous structures had shown that data revealed that CTLH-CRA dimerization interfaces share a conserved helical architecture, the molecular rules that govern subunit pairing have not been explored. This was a daunting task in protein biochemistry that was achieved in the present study, which defines this "assembly specificity code" at the structural and residue-specific level.

The authors used X-ray crystallography to solve high-resolution structures of mammalian CTLH-CRA domains, including RANBP9, RANBP10, TWA1, MAEA, and the heterodimeric complex between RANBP9 and MKLN. They further examined and characterized assemblies by quantitative methods (ITC and SEC-MALS) and qualitatively using nondenaturing gels. Some of their ITC measurements were particularly clever and involved competitive titrations and titrations of varying partners depending on protein behavior. The experiments allowed the authors to discover that affinities for interactions between partners is exceptionally tight, in the pM-nM range, and to distill the basis for specificity while also inferring that additional interactions beyond the LisH-CTLH-CRA modules likely also contribute to stability. Beyond discovering how the native pairings are achieved, the authors were able to use this new structural knowledge to reengineer interfaces to achieve different preferred partnerings.

Strengths:

Nearly everything about this work is exceptionally strong.

(1) The question is interesting for the native complexes, and even beyond that, has potential implications for the design of novel molecular machines.

(2) The experimental data and analyses are quantitative, rigorous, and thorough.

(3) The paper is a great read - scholarly and really interesting.

(4) The figures are exceptional in every possible way. They present very complex and intricate interactions with exquisite clarity. The authors are to be commended for outstanding use of color and color-coding throughout the study, including in cartoons to help track what was studied in what experiments. And the figures are also outstanding aesthetically.

Weaknesses:

There are no major weaknesses of note, but I can make a few recommendations for editing the text.

Reviewer #3 (Public review):

Summary:

Protein complexes, like the GID/CTLH-type E3 ligase, adopt a complex three-dimensional structure, which is of functional importance. Several domains are known to be involved in shaping the complexes. Structural information based on cryo-EM is available, but its resolution does not always provide detailed information on protein-protein interactions. The work by van gen Hassend and Schindelin provides additional structural data based on crystal structures.

Strengths:

The work is solid and very carefully performed. It provides high-resolution insights into the domain architecture, which helps to understand the protein-protein interactions on a detailed molecular level. They also include mutant data and can thereby draw conclusions on the specificity of the domain interactions. These data are probably very helpful for others who work on a functional level with protein complexes containing these domains.

Weaknesses:

The manuscript contains a lot of useful, very detailed information. This information is likely very helpful to investigate functional and regulatory aspects of the protein complexes, whose assembly relies on the LisH-CTLH-CRA modules. However, this goes beyond the scope of this manuscript.

Reviewer #1 (Public review):

Summary:

This study investigates the molecular mechanisms allowing the KSM mite to infest tea plants, a host that is toxic to the closely related TSSM mite due to high concentrations of phenolic catechins. The authors utilize a comparative approach involving tea-adapted KSM, non-adapted KSM, and TSSM to assess behavioral avoidance and physiological tolerance to catechins. The main finding is that tea-adapted KSM possesses a specific detoxification mechanism mediated by an enzyme, TkDOG15, which was acquired via horizontal gene transfer. The study demonstrates that adaptation is a two-step process: (1) structural refinement of the TkDOG15 enzyme through amino acid substitutions that enhance enzymatic efficiency against catechins, and (2) significant transcriptional upregulation of this gene in response to tea feeding. This enzymatic adaptation allows the mites to cleave and detoxify tea catechins, enabling survival on a toxic host plant.

Strengths:

A multiomics approach (transcriptomics and proteomics) provided a compelling cross-validation of its findings. Functional bioassays, such as RNAi and recombinant enzyme assays, demonstrated that the adapted mite has higher activity against catechins via TkDOG15. Other methodologies, like feeding assay using a parafilm-covered leaf disc, were effective in avoiding contact chemosensation.

Weaknesses:

Although TkDOG15 is assumed to "detoxify" catechins by ring cleavage, the study doesn't identify or characterize the breakdown metabolic products. If the metabolites are indeed non-toxic compared to the parent catechins, that would strengthen the detoxification hypothesis. Also, the transcriptomic and proteomic analyses identified other potential detoxification enzymes, such as CCEs, UGTs, and ABC (Supplementary Tables 3-1 & 3-2), which were also upregulated. The manuscript focuses almost exclusively on TkDOG15, potentially overlooking a multigenic adaptation mechanism, where these other enzymes might play synergistic roles, although it was mentioned in the discussion section.

Reviewer #2 (Public review):

Summary:

The fascinating topic of the host range of arthropods, including insects, and the detoxification of host secondary metabolites has been elucidated through studies of the host specificity of two closely related species. The discovery that key genes were acquired from fungi through horizontal gene transfer (HGT) is particularly significant.

Strengths:

(1) The discovery that the TkDOG15 enzyme, acquired through HGT from fungi, plays a key role in the detoxification of green tea catechins in the Kanzawa mite, revealing a new mechanism of plant-herbivore interactions, is highly encouraging.

(2) The verification of this finding through various experiments, including behavioral, toxicological, transcriptomic, and proteomic analyses, RNAi-based gene function analysis, and recombinant enzyme activity assays, is also highly commendable.

(3) By proposing a two-step model in which amino acid substitutions and expression regulation of a specific enzyme gene (TkDOG15) enable host adaptive evolution, this study contributes significantly to our understanding of the evolutionary mechanisms of speciation and plant defense overcoming.

Weaknesses:

While transcriptome/proteome analyses reported changes in the expression of other detoxification-related enzymes, including CCEs, UGTs, ABC transporters, DOG1, DOG4, and DOG7, it is regrettable that the contribution of each enzyme, including its interaction with TkDOG15 and the functional analysis of each enzyme within the overall catechin detoxification system, was not investigated.

Reviewer #1 (Public review):

Summary:

This manuscript presents high-resolution cryoEM structures of VPS34-complex II bound to Rab5A at 3.2A resolution. The Williams group previously reported the structure of VPS34 complex II bound to Rab5A on liposomes using tomography, and therefore, the previous structure, although very informative, was at lower resolution.

The first new structure they present is of the 'REIE>AAAA' mutant complex bound to RAB5A. The structure resembles the previously determined one, except that an additional molecule of RAB5A was observed bound to the complex in a new position, interacting with the solenoid of VPS15.

Although this second binding site exhibited reduced occupancy of RAB5A in the structure, the authors determined an additional structure in which the primary binding site was mutated to prevent RAB5A binding ('REIE>ERIR'). In this structure, there is no RAB5A bound to the primary binding site on VPS34, but the RAB5A bound to VPS15 now has strong density. The authors note that the way in which RAB5A interacts with each site is distinct, though both interfaces involve the switch regions. The authors confirm the location of this additional binding site using HDX-MS.

The authors then determine multiple structures of the wild-type complex bound to RAB5A from a single sample, as they use 3D classifications to separate out versions of the complex bound to 0, 1, or 2 copies of RAB5A. Overall, the structure of VPS34-Complex II does not change between the different states, and the data indicate that both RAB5A binding sites can be occupied at the same time.

The authors then design a new mutant form of the complex (SHMIT>DDMIE) that is expected to disrupt the interaction at the secondary site between VPS15 and RAB5A. This mutation had a minor impact on the Kd for RAB5A binding, but when combined with the REIE>ERIR mutation of the primary binding site, RAB5A binding to the complex was abolished.

Comparison of sequences across species indicated that the RAB5A binding site on VPS15 was conserved in yeast, while the RAB5A binding site on VPS34 is not.

The authors tested the impact of a corresponding yeast Vps15 mutation (SHLITY>DDLIEY) predicted to disrupt interaction with yeast Rab5/Vps21, and found that this mutant Vps15 protein was mislocalized and caused defective CPY processing.

The authors then compare these structures of the RAB5A-class II complex to recently published structures from the Hurley group of the RAB1A-class I complex, and find that in both complexes the Rab protein is bound to the VPS34 binding site in a somewhat similar manner. However, a key difference is that the position of VPS34 is slightly different in the two complexes because of the unique ATL14L and UVRAG subunits in the class I and class II complexes, respectively. This difference creates a different RAB binding pocket that explains the difference in RAB specificity between the two complexes.

Finally, the higher resolution structures enable the authors to now model portions of BECLIN1 and UVRAG that were not previously modeled in the cryoET structure.

Strengths:

Overall, I found this to be an interesting and comprehensive study of the structural basis for the interaction of RAB5A with VPS34-complex II. The authors have performed experiments to validate their structural interpretations, and they present a clear and thorough comparative analysis of the Rab binding sites in the two different VPS34 complexes. The result is a much better understanding of how two different Rab GTPases specifically recruit two different, but highly similar complexes to the membrane surface.

Weaknesses:

No significant weaknesses were noted.

Reviewer #2 (Public review):

The work by Spokaite et al describes the discovery of a novel Rab5 binding site present in complex II of class III PI3K using a combination of HDX and Cryo EM. Extensive mutational and sequence analysis define this as the primordial Rab5 interface. The data presented are convincing that this is indeed a biologically relevant interface, and is important in defining mechanistically how VPS34 complexes are regulated.

This paper is a very nice expansion of their previous cryo-ET work from 2021, and is an excellent companion piece on high-resolution cryo-EM of the complex I class III complex bound to Rab1 from the Hurley lab in 2025. Overall, this work is of excellent technical quality and answers important unexplained observations on some unexpected mutational analysis from the previous work.

They used their increased affinity VPS34 mutant to determine the 3.2 ang structure of Rab5 bound to VPS34-CII. Clear density was seen for the original Rab5 interface, but an additional site was observed. Based on this structure, they mutated out the VPS34 interface, allowing for a high-resolution structure of the Rab5 bound at the VPS15 interface.

They extensively validated the VPS15 interface in the yeast variant of VPS34, showing that the Vp215-Rab5 (VPS21) interface identified is critical in controlling complex II VPS34 recruitment.

The major strengths of this paper are that the experiments appear to be done carefully and rigorously, and I have very few experimental suggestions.

Here is what I recommend based on some very minor weaknesses I observed

(1) My main concern has to do a little bit with presentation. My main issue is how the authors use mutant description. They clearly indicate the mutant sequence in the human isoform (for example, see Figure 2A, VPS15 described as 579-SHMIT-583>DDMIE); however, when they shift to the yeast version, they shift to saying VPS15 mutant, but don't define the mutant, Figure 2G). I would recommend they just include the same sequence numbering and WT to mutant replacement every time a new mutant (or species) is described. It is always easier to interpret what is being shown when the authors are jumping between species, when the exact mutant is included. This is particularly important in this paper, where we are jumping between different subunits and different species, so a clear description in the figure/figure legends makes it much easier to read for non-specialists.

(2) The HDX data very clearly shows that Rab5 is likely able to bind at both sites, which back ups the cryo EM data nicely. I am slightly confused by some of the HDX statements described in the methods.

(3) The authors state, "Only statistically significant peptides showing a difference greater than 0.25 Da and greater than 5% for at least two timepoints were kept." This seems to be confusing as to why they required multiple timepoints, and before they also describe that they required a p-value of less than 0.05. It might be clearer to state that significant differences required a 0.25 Da, 5%, and p-value of <0.05 (n=3). Also, what do they mean by kept? Does this mean that they only fully processed the peptides with differences?

(4) They show peptide traces for a selection in the supplement, but it would be ideal to include the full set of HDX data as an Excel file, including peptides with no differences, as there is a lot of additional information (deuteration levels for everything) that would be useful to share, as recommended from the Masson et al 2019 recommendations paper. This may be attached, but this reviewer could not see an example of it in the shared data dropbox folder.

Reviewer #3 (Public review):

Summary:

The manuscript of Spokaite et al. focuses on the Vps34 complex involved in PI3P production. This complex exists in two variants, one (class I) specific for autophagy, and a second one (class II) specific for the endocytic system. Both differ only in one subunit. The authors previously showed that the Vps34 complexes interact with Rab GTPases, Rab1 or Rab5 (for class II), and the identified site was found at Vps34. Now, the authors identify a conserved and overlooked Rab5 binding site in Vps15, which is required for the function of the Class II complex. In support of this, they show cryo-EM data with a second Rab5 bound to Vps15, identify the corresponding residues, and show by mutant analysis that impaired Rab5 binding also results in defects using yeast as a model system.

Overall, this is a most complete study with little to criticize. The paper shows convincingly that the two Rab5 binding sites are required for Vps34 complex II function, with the Vps15 binding site being critical for endosomal localization. The structural data is very much complete. What I am missing are a few controls that show that the mutations in Vps15 do not affect autophagy. I also found the last paragraph of the results section a bit out of place, even though this is a nice observation that the N-terminal part of BECLIN has these domains. However, what does it add to the story?

Joint Public review:

Summary

This interesting work by Shuhao Li and colleagues suggests that developmental sleep and feeding behavior in larval flies is genetically programmed to prepare the animal for adult contingencies, such as in the case of flies living in harsh ecological environments, such as deserts. Thus, the work proposes that desert-dwelling flies such as Drosophila mojavensis sleep less and feed more than D. melanogaster as larvae, which allows them to feed less and sleep more as adults in the harsh desert conditions where they live. The authors argue that this is evidence for developmental sleep reallocation, which helps the adult flies survive in the desert. In general, their results support this compelling hypothesis, so this work provides a new perspective on how sleep might be differentially programmed across developmental stages according to the requirements of an ecological niche. This work is particularly innovative for several reasons. First, it extends the Drosophila sleep field beyond D. melanogaster and directly addresses questions about the evolution of sleep that remain largely unexplored. Second, it investigates the possibility that changes in sleep across development may be adaptive, rather than sleep being a static trait. Overall, this work opens new avenues of research, effectively bridges the fields of sleep biology and evolutionary ecology, and should be of broad interest to a general readership. The manuscript is scientifically sound and clearly written for a generalist audience.

There are, however, two important weaknesses that should be addressed. The first is the implicit assumption that all observed behavioral differences are adaptive; this would benefit from a more cautious framing. Second, the manuscript would be strengthened by a more detailed discussion, and potentially additional data, regarding the ecological differences experienced by D. mojavensis and D. melanogaster at distinct life-cycle stages.

Strengths:

(1) The study astutely uses desert Drosophila species as models to understand how sleep is optimized in a challenging environment. The manuscript is rigorous, experiments are well controlled, the work is very clearly presented, and the results support the main conclusions, which are quite exciting.

(2) The manuscript examines previously unexplored sleep differences in a non-melanogaster species.

(3) The study provides evidence that selective pressure can be restricted to specific developmental stages.

(4) This work offers evolutionary insights into the trade-offs between sleep and feeding across development.

Weaknesses

(1) The authors should soften interpretations so that it is not assumed that any observed difference between mojavensis and melanogaster is necessarily adaptive, or evolved due to food availability or temperature stress.

(2) The study relies on comparisons and correlations. While it seems likely that the observed differences in sleep explain the increased food consumption and energy storage in the larvae of desert flies, demonstrating this through sleep manipulation would strengthen the authors' conclusions.

(3) The question arises regarding whether transiently quiescent larvae are always really sleeping, and whether it is appropriate to treat sleep as a stochastic population-level phenomenon rather than as an individual trait.

(4) The manuscript would benefit from comparative analysis beyond mojavensis and melanogaster.

(5) A deeper discussion of the ecological differences between the 2 Drosophila species would place the results in a broader context.

(6) The feeding parameters used in adults and larvae measure different aspects of feeding, confounding comparisons.

Reviewer #1 (Public review):

Summary:

Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains.

Strengths:

This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.

The authors have adequately addressed all of my concerns. I have no further questions or concerns.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised manuscript, reworked figures make the data easier to interpret.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring vasopressin function in the mammalian brain and for developing hypotheses about cell- and circuit-level function.

Future considerations:

The work contained in the manuscript is substantial and informative. Some questions remain and would be addressed in the current manuscript. How many cells are impacted? Are transcripts spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield?

Reviewer #1 (Public review):

Summary:

The manuscript titled, "Sleep-Wake Transitions Are Impaired in the AppNL-G-F Mouse Model of Early Onset Alzheimer's Disease", is about a study of sleep/wake phenomena in a knockin mouse strain carrying "three mutations in the human App gene associated with elevated risk for early onset AD". Traditional, in-depth characterization of sleep/wake states, EEG parameters, and response to sleep loss are employed to provide evidence, "supporting the use of this strain as a model to investigate interventions that mitigate AD burden during early disease stages". The sleep/wake findings of earlier studies (especially Maezono et al., 2020, as noted by the authors) were extended by several important, genotype-related observations, including age-related hyperactivity onset that is typically associated with increased arousal, a normal response to loss of sleep and to multiple sleep latency testing, and a stronger AD-like phenotype in females. The authors conclude that the AppNL-G-F mice demonstrate many of the human AD prodromal symptoms and suggest that this strain may serve as a model for prodromal AD in humans, confirming the earlier results and conclusions of Maezono et al. Finally, based on state bout frequency and duration analyses, it is suggested that the AppNL-G-F mice may develop disruptions in mechanism(s) involved in state transition.

Strengths:

The study appears to have been, technically, rigorously conducted with high quality, in-depth traditional assessment of both state and EEG characteristics, with the concordant addition of activity and temperature. The major strengths of this study derive from observations that the AppNL-G-F mice: (1) are more hyperactive in association with decreased transitions between states; (2) maintain a normal response to sleep deprivation and have normal MSLT results; and (3) display a sex specific, "stronger" insomnia-like effect of the knockin in females.

Weaknesses:

The weaknesses stem from the study's impact being limited due to its being largely confirmatory of the Maezono et al. study, with advances of importance to a potentially more focused field. Further, the authors conclude that AppNL-G-F mice have disrupted mechanism(s) responsible for state transition; however, these were not directly examined. The rationale for this conclusion is stated by the authors as based on the observations that bouts of both W and NREM tend to be longer in duration and decreased in frequency in AppNL-G-F mice. Although altered mechanism(s) of state transition (it is not clear what mechanisms are referenced here) cannot be ruled out, other explanations might be considered. For example, increased arousal in association with hyperactivity would be expected to result in increased duration of W bouts during the active phase. This would also predictably result in greater sleep pressure that is typically associated with more consolidated NREM bouts, consistent with the observations of bout duration and frequency.

Reviewer #2 (Public review):

Summary:

The authors have used a knock-in mouse model to explore late-in-life amyloid effects on sleep. This is an excellent model as the mutated genes are regulated by the endogenous promoter system. The sleep study techniques and statistical analyses are also first-rate.

The group finds an age-dependent increase in motor activity in advanced age in the NLGF homozygous knock-in mice (NLGF), with a parallel age-dependent increase in body temperature, both effects predominate in the dark period. Interestingly, the sleep patterns do not quite follow the sleep changes. Wake time is increased in NLGF mice, and there is no progression in increased wake over time. NREMS and REM sleep are both reduced, and there is no progression. Sleep-wake effects, however, show a robust light:dark effect with larger effects in the dark period. These findings support distinct effects of this mutation on activity and temperature and on sleep. This is the first description of the temporal pattern of these effects. NLGF mice show wake stability (longer bout durations in the dark period (their active period) and fewer brief arousals from sleep. Sleep homeostasis across the lights-on period is normal. Wake power spectral density is unaffected in NLGF mice at either age. Only REM power spectra are affected, with NLGF mice showing less theta and more delta. There are interesting sex differences, with females showing no gene difference in wake bout number, while males show a gene effect. Similarly, gene effects on NREM bout number seem larger in males than in females. Although there was no difference in homeostatic response, there was normalization of sleep-wake activity after sleep deprivation.

Strengths:

Approach (model extent of sleep phenotyping), analysis.

Weaknesses:

The weaknesses are summarized below and are viewed as "addressable".

(1) The term insomnia. Insomnia is defined as a subjective dissatisfaction with sleep, which cannot be ascertained in a mouse model. The findings across baseline sleep in NLGF mice support increased wake consolidation in the active period. The predominant sleep period (lights on) is largely unaffected, and the active period (lights off) shows increased activity and increased wake with longer bouts. There is a fantastic clue where NLGF effects are consistent with increased hypocretinergic (orexinergic) neuron activity in the dark period, and/or increased drive to hypocretin neurons from PVH.

(2) Sleep-wake transitions are impaired: This should not be termed an impairment. It could actually be beneficial to have greater state stability, especially wake stability in the dark or active period. There is reduced sleep in the model that can be normalized by short-term sleep loss. It is fascinating that recovery sleep normalized sleep in the NLGF in the immediate lights-on and light-off period. This is a key finding.

Reviewer #3 (Public review):

Summary:

In this study, Tisdale et al. studied the sleep/wake patterns in the biological mouse model of Alzheimer's disease. The results in this study, together with the established literature on the relationship of sleep and Alzheimer's disease progression, guided the authors to propose this mouse model for the mechanistic understanding of sleep states that translates to Alzheimer's disease patients. However, the manuscript currently suffers from a disconnect between the physiological data and the mechanistic interpretations. Specifically, the claim of "impaired transitions" is logically at odds with the observed increase in wake-state stability or possible hyperactivity. Additionally, the description of the methods, the quantification, and the figure presentation could be substantially improved. I detail some of my concerns below.

Strengths:

The selection of the knock-in model is a notable strength as it avoids the artifacts associated with APP overexpression and more closely mimics human pathology. The study utilizes continuous 14-day EEG recordings, providing a unique dataset for assessing chronic changes in arousal states. The assessment of sex as a biological variable identifies a more severe "insomniac-like" phenotype in females, which aligns with the higher prevalence and severity of Alzheimer's disease in women.

Weaknesses:

The study seems to lack a clear hypothesis-driven approach and relies mostly on explorative investigations. Moreover, lack of quantitative analytical methods as well as shaky logical conclusions, possibly not supported by data in its current form, leaves room for major improvement.

Since this paper studied sleep states, the "Methods" section is quite unclear on what specific criteria were used to classify sleep states. There is no quantitative description of classifying sleep based on clear, reproducible procedures. There are many reasonably well-characterized sleep scoring systems used in rat electrophysiological literature, which could be useful here. The authors are generally expected to describe movement speed and/or EMG and/or EEG (theta/delta/gamma) criteria used to classify these epochs. The subjective (manual) nature of this procedure provides no verifiable validation of the accuracy and interpretability of the results.

One of the bigger claims is that "state transition mechanism(s)" are impaired. However, Figure 7 shows that model mice exhibit significantly more long wake bouts (>260s) and fewer short wake bouts (<60s). Logically, an "impaired switch" (the flip-flop model, Saper et al., 2010) results in state fragmentation. The data here show the opposite: the wake state has become too stable. This suggests the primary defect is not in the transition mechanism itself, but possibly in a pathological increase in arousal drive (hyper-arousal), likely linked to the dark-phase hyperactivity shown in Figures 4 and 5. Also, a point to note is that this finding is not new.

Figure 3 heatmaps lack color bars and units. Spectral power must be quantitatively defined and methods well-explained in the Methods section. Without these, the reader cannot discern if the "reduced power" in females is a global suppression of signal or a frequency-specific shift. Additionally, the representative example used to claim shorter sleep bouts lacks the statistical weight required for a major physiological conclusion. How does a cooler color (not clear what range and what the interpretation is) mean shorter sleep bout in female mice? The authors should clearly mark the frequency ranges that support their claims. In this figure, there is a question mark following the theta/delta range. The authors should avoid speculation and state their claims based on facts. They should also add the theta and delta ranges in the plot, such that readers can draw their own conclusions.

Figure 8 and the MSLT results show that model mice are "no sleepier than WT mice" and have a functional homeostatic rebound. This presents a logical flaw in the "insomnia" narrative. True insomnia in AD patients typically involves a failure of the homeostatic process or a debilitating accumulation of sleep debt. If these mice do not show increased sleepiness (shorter latency) despite ~19% less sleep, the authors might be describing a "reduced need" for sleep or a "hyper-aroused" state, possibly not a clinical insomnia phenotype.

In Figure 9, LFP power shown and compared in percentages is problematic, as LFP power distribution is known to be skewed (follows power law). This is particularly problematic here because all the frequencies above ~20 Hz seem to be totally flattened or nonexistent, which makes this comparison of power severely limited and biased towards the relative frequency in the highly skewed portion of the LFP power spectrum, i.e., very low frequency ranges like delta, theta, and possibly beta. This ignores low, mid, and high gamma as well as ripple band frequencies. NREM sleep is known to have relatively greater ripple band (100-250 Hz) power bursts in hippocampal regions, and REM sleep is known to have synchronous theta-gamma relationships.

Reviewer #1 (Public review):

While the revised manuscript includes additional methodological details and a supplementary comparison with conventional NMF, it would be great if the authors could add the point below as limitations in the manuscript or change the title and abstract accordingly, since core issues remain:

(1) The study claims to evaluate rehabilitation outcomes without demonstrating that patients actually improved functionally

(2) The comparison with existing methods lacks the quantitative rigor needed to establish superiority

(3) The added value of this complex framework over much simpler alternatives has not been demonstrated

The strength of evidence supporting the main claims remains incomplete. I would encourage the authors to consider discussing these points

(1) including or adding a limitation section about functional outcome measures that go beyond clinical scale scores, (2) providing/discussing quantitative benchmarks showing their method outperforms alternatives on specific, predefined metrics, and (3) clarifying the clinical pathway by which these biomarkers would inform treatment decisions.

There are specific, relatively minor points, that require attention

The authors write: "we did not focus on such complementary evidence in this study." This is a weakness for a paper claiming to provide "biomarkers of therapeutic responsiveness." The FMA-UE threshold defines responders, but there's no independent validation that patients actually functioned better in daily life. Can you please clarify?

Maybe I missed the exact point about this, but with the added NMF plot, the authors list 'lower dimensionality' among their framework's advantages, but the basis for this claim is not clear because given that 12 network components were extracted compared to 11 "conventional" synergies. Can you please clarify, as it is not clear. You claim 'lower dimensionality' as an advantage of the proposed framework (in the Supplementary Materials), yet you extracted 12 components (5 redundant + 7 synergistic networks) compared to 11 synergies from the conventional NMF approach, which does not support a clinical / outcome advantage of this method. Please clarify.

Reviewer #2 (Public review):

This study presents an important analysis of how interactions between muscles can serve as biomarkers to quantify therapeutic responses in post-stroke patients. To do so, the authors employ an information-theoretical metric (co-information) to define muscle networks and perform cluster analysis.

I thank the authors for improving the clarity of the Methods section; the newly added Figure 5 is very helpful.

One minor suggestion is that the authors should avoid overloading the notation "m" for both the EEG measurement and the matrix of II values (Eq. 1.1), which I now realise was the source of some of my initial confusion. I suggest that the authors use separate notation for these two quantities.

Reviewer #1 (Public review):

Summary:

The study by Wu et al. uses endogenous bruchpilot expression in a cell-type-specific manner to assess synaptic heterogeneity in adult Drosophila melanogaster mushroom body output neurons. The authors performed genomic on locus tagging of the presynaptic scaffold protein bruchpilot (brp) with one part of splitGFP (GFP11) using the CRISPR/Cas9 methodology and co-expressed the other part of splitGFP (GFP1-10) using the GAL4/UAS system. Upon expression of both parts of splitGFP, fluorescent GFP is assembled at the C-terminus of brp, exactly where brp is endogenously expressed in active zones. For manageable analysis, a high-throughput pipeline was developed. This analysis evaluated parameters like location of brp clusters, volume of clusters, and cluster intensity as a direct measure of the relative amount of brp expression levels on site using publicly available 3D analysis tools that are integrated in Fiji. Analysis was conducted for different mushroom body cell types in different mushroom body lobes using various specific GAL4 drivers. Further validation was provided by extending analysis to R8 photoreceptors that reside in the fly medulla. To test this new method of synapse assessment, Wu et al. performed an associative learning experiment in which an odor was paired with an aversive stimulus and found that in a specific time frame after conditioning, the new analysis solidly revealed changes in brp levels at specific synapses that are associated with aversive learning. Additionally, brp levels were assessed in R8 photoreceptor terminals upon extended exposure to light.

Strengths:

Expression of splitGFP bound to brp enables intensity analysis of brp expression levels as exactly one GFP molecule is expressed per brp. This is a great tool for synapse assessment. This tool can be widely used for any synapse as long as driver lines are available to co-express the other part of splitGFP in a cell-type-specific manner. As neuropils and thus brp label can be extremely dense, the analysis pipeline developed here is very useful and important. The authors have chosen an exceptionally dense neuropil - the mushroom bodies - for their analysis and compellingly show that brp assessment can be achieved even with such densely packed active zones. The result that brp levels change upon associative learning in an experiment with odor presentation paired with punishment is likewise compelling and strongly suggests that the tool and pipeline developed here can be used in an in vivo context. Thus, the tool and its uses have the potential to fundamentally advance protein analysis not only at the synapse but especially there.

Weaknesses:

The weaknesses I perceived originally were satisfactorily explained and refuted.

Reviewer #2 (Public review):

Summary:

The authors developed a cell-type-specific fluorescence-tagging approach using a CRISPR/Cas9 induced spilt-GFP reconstitution system to visualize endogenous Bruchpilot (BRP) clusters at presynaptic active zones (AZ) in specific cell types of the mushroom body (MB) in the adult Drosophila brain. This AZ profiling approach was implemented in a high-throughput quantification process allowing to compare synapse profiles within single cells, cell-types, MB compartments and between different individuals. Aim is to in more detail analyze neuronal connectivity and circuits in this center of associative learning, notoriously difficult to investigate due to the density of cells and structures within the cells. The authors detect and characterize cell-type specific differences in BRP-dependent profiling of presynapses in different compartments of the MB, while intracellular AZ distribution was found to be stereotyped. Next to the descriptive part characterizing various AZ profiles in the MB, the authors apply an associative learning assay and Rab3 knock-down and detected consequent AZ reorganization.

Strengths:

The strength of this study lies in the outstanding resolution of synapse profiling in the extremely dense compartments of the MB. This detailed analysis will serve as an entry point for many future studies of synapse diversity in connection with functional specificity to uncover the molecular mechanisms underlying learning and memory formation and neuronal network logic. Therefore, this approach is of high importance to the scientific community and represents a valuable tool to investigate and correlate AZ architecture and synapse function in the CNS.

Weaknesses:

The results and conclusions presented in this study are conclusively and well supported by the data presented and appropriate controls. As a comment that could possibly aid and strengthen the manuscript (but not required for acceptance of the manuscript): The experiments in the study are based on spilt-GFP lines (BRP:GFP11 and UAS-GFP1-10). The authors clearly validate the new on-locus construct with a genomic GFP insertion (qPCR, confocal and STED imaging of the brain with anti-BRP (Nc82), MB morphology and memory formation). It would be important to comment on the significant overall intensity decrease of anti-BRP (Nc82) in Fig. S1B (R57C10>BRP::rGFP) and possibly a Western Blot with a correlative antibody staining against BRP might help to show that BRP protein level are not affected. Additionally, it would be important to state, at least in the Materials and Methods section, that the flies are not homozygous viable (and to offer an explanation) and to state that all experiments were performed with heterozygous flies.

Reviewer #3 (Public review):

Summary:

The authors develop a tool for marking presynaptic active zones in Drosophila brains, dependent on the GAL4 construct used to express a fragment of GFP, which will incorporate with a genome-engineered partial GFP attached to the active zone protein bruchpilot - signal will be specific to the GAL4 expressing neuronal compartment. They then use various GAL4s to examine innervation onto the mushroom bodies to dissect compartment specific differences in the size and intensity of active zones. After a description of these differences, they induce learning in flies with classic odour/electric shock pairing and observe changes after conditioning that are specific to the paired conditioning/learning paradigm.

Strengths:

The imaging and analysis appears strong. The tool is novel and exciting.

Weaknesses:

I feel that the tool could do with a little more characterisation. It is assumed that the puncta observed are AZs with no further definition or characterisation. It is not resolved if the AZs visualised here simply tagged, or are the constructs incorporated to be an active functional part of the AZ.

Comments on revisions:

Apologies, I should have thought of this in the first round of review. An experiment I would suggest (and it is not a difficult one) to address the functionality of the marker: It is mentioned that the genetically tagged half of the construct is homozygous lethal. Can this be placed in trans to a brp null, with a neuronal UAS-expression of the other half of Brp-GFP - Are the animals then 1) alive, and 2) able to fly (brp mutants can't fly, hence the name 'crashpilot') - a rescue would suggest (and that is all that would be needed here) that the reconstituted brp-GFP has function.

On another note, the paper keeps switching between different DAN-GAL4 lines. In 1H, 2Band 4A, there are informative cartoons showing the extension of the neurons for PPL1, APL and DPM neurons - could these be incorporated into figures 5, 6 and 7, and the supplementary figures to help orient the reader. Ideally they would refer to a figure (in Fig 1?) -to refer to the groups of DANs in the adult brain that are known to innervate the MBs (e.g. Fig1 in Mao and Davis, Front in Neural Circuits 2009). I suggest this because I feel that this tool will be widely used, and if non-MB aficionados can follow what's being done here I feel it will be more widely accepted.

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

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

Weaknesses:

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

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

I have two main suggestions:

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

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

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

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

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

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

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

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

Other issues:

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

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

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

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

Outliers and t-values:

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

Random slopes:

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

Reviewer #2 (Public review):

Summary:

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

Strength:

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

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

Weakness:

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

Reviewer #1 (Public review):

Summary:

Using high-precision eyetracking, the authors measure foveolar sensitivity modulations before, during, and after instructed microsaccades to a centrally cued orientation stimulus.

Strengths:

The article is clearly written, and the stimulus presentation method is sophisticated and well-established. The data provide interesting insights that will be useful for comparisons between trans-saccadic and trans-microsaccadic sensitivity modulations.

Weaknesses:

Nonetheless, I have major concerns regarding the interpretation of the measured time courses (in particular, inconsistencies in distinguishing enhancement from suppression), the attempt to disentangle these effects from endogenous attention shifts, and the overstatement of the findings' novelty.

(1) Overstatement of novelty

The authors motivate their study by stating that "the temporal dynamics of these pre-microsaccadic modulations remain unknown" (l. 55-56). However, Shelchkova & Poletti (2020) already report a microsaccade-aligned sensitivity time course. I understand that the present study uses shorter target durations and thus provides a more resolved estimate. Nonetheless, a fairer characterization of the study's novelty would be that observers' discrimination performance is continuously measured across the pre-, intra-, and post-movement interval, within the same observers and experimental design. Relatedly, the authors state that it is unclear whether pre-microsaccadic sensitivity modulations reflect "suppression at the non-foveated location, enhancement at the microsaccade target, or both" (l. 70). Guzhang et al. (2024) examined the spatial spread of pre-microsaccadic sensitivity modulations by measuring performance at the PRL, the movement target, and several other equidistant locations. They report that "whereas fine spatial vision is enhanced at the microsaccade goal location, it drops at the very center of gaze". The current authors' reasoning seems to be that performances at locations that are neither the target nor the PRL may behave differently. Why would that be the case? If my understanding is correct, I would recommend incorporating these clarifications into the motivation paragraph, so that readers less familiar with the literature do not overestimate the novelty of the findings. Moreover, and related to point 3, I am unsure if the current analyses provide decisive evidence to distinguish enhancement from suppression, as claimed by the authors.

(2) Distinction from endogenous attention

To "rule out the possible influence of covert attention" (l. 232), the authors compute a cue-aligned in addition to the movement-aligned performance time course. A difference in alignment cannot rule out the influence of a certain mechanism; it can only dilute it. Just like endogenous attention may contribute to the movement-aligned time course, movement preparation will necessarily contribute to the cue-aligned time course, since these timelines are intrinsically correlated: as the trial progresses, observers will be in later and later stages of saccade preparation. For this and several additional reasons, an effect in the cue-aligned time course is in fact expected-and, in my view, clearly present (see below). As the authors themselves note, endogenous attention has been shown to operate within the foveola and should therefore be engaged in the present experiment in addition to movement-related attentional shifts (unless the authors believe that specific design features, e.g., stimulus timing, preclude its involvement?). Regardless of the theoretical considerations, the empirical data show a pronounced, near-linear increase in performance at the target location, with d′ doubling from approximately 1 to 2. Although the interaction between condition and time does not reach significance (p = 0.09), this result should not be taken as conclusive evidence against a plausible and perhaps expected contribution of endogenous attention. I suggest an additional analysis that could more directly address these issues. In previous work (Rolfs & Carrasco, 2012; Kroell & Rolfs, 2025; see Figure 3), the relative contributions of cue-alinged influences and pre-saccadic attention were disentangled by reweighting each data point according to its position on both the cue-locked and saccade-locked timelines. Applied to the present study, the authors could compute, for each cue-to-target offset bin, its proportional contribution to each pre-movement time bin. Microsaccade-locked sensitivities could then be reweighted based on these proportions. As a result, each movement-locked time bin would contain equal contributions from all cue-locked time bins, effectively isolating the effect of microsaccade preparation.

(3) Interpretation and analysis of the time course

(3.1) Discrimination before microsaccade onset<br /> In lines 151-153, the author state "While the enhancement at the target location did not reach significance relative to baseline, the impairment at the non-target location did", suggesting that pre-movement sensitivity advantages for information presented at the target location are due to a decrease in performance at the non-target location and not an enhancement at the target location per se. After analyzing the difference between the two locations, the authors state, "These results show that approximately 100 milliseconds before microsaccade onset, discrimination rapidly improved at the intended target location while decreasing at the non-target location." (l. 159-161). How is the statement that discrimination performance rapidly improved (which is repeated throughout the manuscript) justified by the results?

More generally, the authors may benefit from applying bootstrapping or permutation-based analyses to their data. Such approaches would, for example, allow direct comparisons between congruent and incongruent conditions at every individual time point in Figure 3B and may be more sensitive to temporally confined sensitivity variations while requiring fewer assumptions than analyses based on manually segregated temporal bins and aggregate measures. If enhancement at the target location does not reach significance even in these analyses, all corresponding statements should be removed throughout the manuscript. The term "enhancement" should then be rephrased as "detection advantage" or "relative performance benefit" to emphasize the contrast to enhancement effects classically associated with pre-saccadic attention shifts.

Relatedly, the authors state that pre-microsaccadic enhancement peaks around 70 ms before microsaccade onset, which is earlier than sensitivity enhancements preceding large-scale saccades that often increase monotonically up until movement onset. The authors suggest potential reasons for this in the Discussion, yet an additional one seems conceivable based on Figure 3B. Performances at both the cue-congruent and incongruent location decrease leading up to the movement, reaching values even below their early baselines around 100 ms and 25 ms before movement onset for the incongruent and congruent location, respectively. A spatially non-specific decline that drives sensitivities toward a common absolute minimum may thus dictate the time course of detection advantages. In other words, a spatially widespread decrease in foveolar sensitivity likely contributes to both "suppression" at the non-target location and the decrease in "enhancement" at the target location. If this general decrease is due to saccadic suppression, as the authors suggest, it appears to exert a much more pronounced influence on sensitivity modulations than it does before large-scale saccades (which is interesting). Are there other findings suggesting an increased magnitude of micro-saccadic (as compared to saccadic) suppression? Another potentially related phenomenon is the decrease in pre-saccadic foveal detection performances reported twice before (Hanning & Deubel, 2022; Kroell & Rolfs, 2022). It is possible that whatever mechanism triggers this decrease is engaged by the preparation of microsaccadic and saccadic motor programs alike. In any case, I would ask the authors to acknowledge this general decrease early on to clarify that any currently significant advantage for the target location relies on varied degrees of suppression, and not on true enhancement similar to pre-saccadic attention shifts.

Moreover, in Figure 3C, the final 25 ms before microsaccade onset are excluded from the aggregate measure, presumably since including this interval substantially reduces the effect size. Since the last 25 ms before movement onset is the interval most commonly associated with saccadic suppression, I think that this choice can be justified. Nonetheless, it should be mentioned explicitly in the main text. On a minor note, the authors state that "Performance (evaluated as percent of correct responses) was averaged within a 50 millisecond sliding window, advancing in 1 ms steps (with 24 ms overlap)". Why is the overlap not 49 ms?

(3.2) Discrimination during the microsaccade:<br /> The authors state that "in the "during" trials the target must be presented during the peak speed of the microsaccade." Since the target was presented for 50 ms and the average microsaccade duration was around 60 ms, this implies that the intra-microsaccadic condition includes many trials in which the target overlapped with the pre- or post-movement fixation interval. Were there not enough trials to isolate purely intra-microsaccadic presentations? Are the results descriptively comparable?

(4) Additional analyses

Several additional analyses could strengthen the authors' conclusions. If there are enough trials in which observers erroneously saccaded to the uncued (i.e., wrong) location, these trials could experimentally isolate the influence of pre-microsaccadic attention, assuming that endogenous attention went to the cued location. In addition, the authors speculate whether differences in saccadic and microsaccadic movement latencies may underlie the differences in perceptual time courses. The latency distributions provided in the manuscript look sufficiently broad, such that intra-individual variation could be harnessed to explore this question. Do sensitivity time courses differ before microsaccades with shorter vs. longer latencies?

(5) Clarifications regarding the design

At 50 ms, the duration of the to-be discriminated stimulus, although shorter than in previous investigations, is still rather long. What is the reason for this? I would encourage the authors to state in the main text that the duration of the analyzed/plotted time bins is often shorter than the stimulus duration (i.e., there is some overlap between bins that likely introduces smoothing). In Figure 3A, it would be helpful to plot raw data points computed from non-overlapping bins on top of the moving-window estimates, to allow readers to assess the degree of smoothing and potential temporal delays introduced by this analysis. Moreover, I wonder whether the abrupt onset of the target unmasked by flickering noise masks might induce saccadic inhibition, which would manifest as a transient dip in saccade execution probability. The distributions shown in Figure 2B appear too smoothed or fitted to clearly reveal such a dip. How exactly are all distributions in the manuscript computed (e.g., binning, smoothing, fitting procedures)? Finally, on a minor note, explicitly stating on line 105 that two different orientations can be presented at the cued and non-cued location would help avoid potential confusion.

Reviewer #2 (Public review):

Summary and overall evaluation:

The authors assessed how visual discrimination of stimuli in the foveola changes before, during, and after small instructed eye movements (in the "micro" range). Consistent with (and advancing) related prior work, their main finding regards a pre-saccadic modulation of visual performance at the saccade target vs. the opposite location. This pre-saccadic modulation in foveal vision peaks ~70 ms prior to the instructed small saccade.

Strengths:

The study uses an impressive, technically advanced set-up and zooms in on peri-saccadic modulations in visual acuity at the micro scale. The findings build on related prior findings from the literature on smaller and larger eye movements and add temporal granularity over prior work from the same lab. The writing is easy to follow, and the figures are clear.

Weaknesses:

At the same time, the findings remain relatively empirical in nature and do not profoundly advance theoretical understanding beyond adding valuable granularity to existing knowledge. Relevant prior literature could be better introduced and acknowledged. In addition, there remain concerns regarding potential cue-driven attentional influences that may confound the reported effects (leaving the possibility that the reported effects may be related to cue-driven attention, rather than saccade planning/execution per se). There are also some issues regarding specific statistical inferences. I detail these points below.

Major Points:

(1) Novelty framing and introduction of relevant prior literature

At times, this study is introduced as if no prior study explored the time course of changes in visual perception surrounding small (micro) saccades. Yet, it appears that a prior study from the same lab, using a very similar task, already showed a time course (Figure 5 in Shelchkova & Poletti, 2020). While this study is discussed in the introduction, it is not mentioned that at least some pre-saccade time course was already reported there, albeit a more crude one than the one in the current article. Moreover, the 2013 study by Hafed also specifically looked at "peri-microsaccade modulation in visual perception" and also already showed a temporal modulation that peaked ~50 ms before microsaccade onset. I appreciate how the current study differs in a number of ways (focusing on visual acuity in the foveola), but I was nevertheless surprised to see the first reference to this relevant prior finding in the discussion (and without any elaboration). Though more recent, the same could be argued for the 2025 study by Bouhnik et al. on pre-microsaccade modulations in visual processing in V1, which, like the Hafed study, is first mentioned only in the discussion. Perhaps these studies could be introduced in the paragraph starting at line 48, or in the next paragraph, to do better justice to the existing literature on this topic when motivating the study. This would likely also help to better point out the major advances provided by the current study.

Relatedly, in Shelchkova & Poletti (PNAS, 2020), an apparently similar congruency effect on performance was reported >200 ms milliseconds before saccade onset, as evident from Fig 5 in that article. How should readers rhyme this with the current findings? Ideally, the authors would not only acknowledge that such a time course was already reported previously, but also discuss the discrepancies between these findings further: why may the performance effects appear much earlier in this prior study compared to in the current study, where the congruency effect emerges only ~100 ms prior to the instructed small saccade?

(2) Saccade- or cue-driven? (assumption that attention is unaltered in failed saccade trials)

Because the authors used a cue to instruct saccade direction, it remains a possibility that the reported modulations in visual performance may be driven directly by the spatial cue (cue-related attentional allocation), rather than the instructed small saccade per se. While the authors are clearly aware of this potential confound, questions remain regarding the convincingness of the presented control analyses. In my view, a more compelling control would require an additional experiment.

The central argument against a cue-locked (purely attentional) modulation is the absence of a performance modulation in so-called "failed" saccade trials. However, a key assumption here is that putative cue-driven attention was unaltered in these trials. This is never verified and, in my opinion, highly unlikely. Rather, trials with failed microsaccades could very well be the result of failing to process the cue in the first place (indeed, if the task is to make a saccade to the cue, failure to make a saccade equates failure to perform the task). In such trials, any putative cue-driven influences over spatial attention would also be expected to be substantially reduced. Accordingly, just because failed saccade trials show little performance modulation does not rule out cue-driven attention effects, because attention may also have "failed" in these failed saccade trials. The control for potential cue-driven attention effects would be more convincing if the authors included a condition with the same cues, where participants are simply not instructed to make any saccades to the cues. Unfortunately, such an experimental condition appears not to have been included here. The author may still consider adding such a control experiment.

Another argument against a cue-driven effect is that the authors found no interaction with time in the cue-locked data, whereas they did find such an interaction in the saccade-locked data. However, the lack of significance in the cue-locked data but significance in the saccade-locked data is not strong evidence against a cue-driven influence. Statistically, there is no direct comparison here, and more importantly, with longer delays, the cue-locked data may also start to show a dip (this could potentially be tested by the authors if they have enough trials available to extend their cue-locked analysis further in time). Indeed, exogenous attention, that may have been automatically evoked by the spatial cue, is known to be transient and to eventually even reverse after a brief initial facilitation (see e.g., Klein TiCS, 2000).

Finally, the authors consistently refer to "endogenous" attention (starting at line 221) when addressing potential cue-driven attention confounds. However, because the cue is not predictive, but is a spatial cue that differs in a bottom-up manner between left and right cues, "exogenous" attention is a more likely confound here in my view. Specifically, the spatial cue may automatically trigger attention in the direction of the target location it points to (and such exogenous effects would be expected even for unpredictive cues).

(3) Benefit and cost, or just cost?

Line 151 states that no statistically significant benefit for the saccade target was found compared to the neutral baseline. Yet, the claim throughout the article is distinct, such as in line 159: "These results show that approximately 100 milliseconds before microsaccade onset, discrimination rapidly improved at the intended target location". I do not question the robustness of the congruency effect, but the authors should be more careful when inferring "improved" perception at the target location because, as far as I could tell (as well as in the authors' own writing in line 151), this is not substantiated statistically when compared to the neutral baseline.

Related to this point, in Figure 3B, it would be informative to also see the average performance in the neutral cue condition (for example, as a straight line as in some other figures). This would help to better appreciate the relative benefits and/or costs compared to the neutral condition, also in the time-resolved data.

(4) Statistical inference for the comparison between failed and non-failed trials

Currently, the lack of modulation in the failed saccade trials hinges on a null effect. It would be stronger to support the claims with a significant difference in the congruency effect between failed and non-failed trials. Indeed, lack of significance in failed saccade trials does by itself not constitute valid evidence that the congruency effect is larger in saccade compared to failed saccade trials. For this, a significant interaction between saccade-trial-type (failed/non-failed) and congruency (congruent/incongruent) should be established (see e.g., Nieuwenhuis et al., Nat Neurosci, 2011).

(5) Time window justification

While the authors nicely depict their data across the full time axis, all statistics are currently performed on data extracted from specific time windows. How exactly were these time windows determined and justified? Likewise, how were the specific times picked for visualizing and statistically quantifying the data in e.g., Figures 3D and E? It would be reassuring to add justification for these specific time windows and/or to verify (using follow-up analyses) that the presented results are robust when different time windows are chosen.

(6) Microsaccade definition

Microsaccades are explicitly defined as being below half a degree. This appears rather arbitrary and rigid. Does the size of saccades not ultimately depend on the task and stimulus (e.g., Otero-Millan et al., PNAS, 2013) rather than being a fixed biological property? Perhaps this could be stated less rigidly, such as by stating how microsaccades are often observed below 0.5 degrees.

(Relatedly, one may wonder whether the type of instructed saccades that the authors studied here involves the same type of eye movements as the type of fixational microsaccades that have been the focus of ample prior studies. However, I recognize that this specific reflection may open a debate that is beyond the scope of this article.

Reviewer #1 (Public review):

Review of the revised submission:

I thank the authors for their detailed consideration of my comments and for the additional data, analyses, and clarifications they have incorporated. The new behavioral experiments, quantification of targeted manipulations, and expanded methodological details strengthen the manuscript and address many of my initial concerns. While some questions remain for future work, the authors' careful responses and the additional evidence provided help resolve the main issues I raised, and I am generally satisfied with the revisions.

Review of original submission:

Summary

In this article, Kawanabe-Kobayashi et al., aim to examine the mechanisms by which stress can modulate pain in mice. They focus on the contribution of noradrenergic neurons (NA) of the locus coeruleus (LC). The authors use acute restraint stress as a stress paradigm and found that following one hour of restraint stress mice display mechanical hypersensitivity. They show that restraint stress causes the activation of LC NA neurons and the release of NA in the spinal cord dorsal horn (SDH). They then examine the spinal mechanisms by which LC→SDH NA produces mechanical hypersensitivity. The authors provide evidence that NA can act on alphaA1Rs expressed by a class of astrocytes defined by the expression of Hes (Hes+). Furthermore, they found that NA, presumably through astrocytic release of ATP following NA action on alphaA1Rs Hes+ astrocytes, can cause an adenosine-mediated inhibition of SDH inhibitory interneurons. They propose that this disinhibition mechanism could explain how restraint stress can cause the mechanical hypersensitivity they measured in their behavioral experiments.

Strengths:

(1) Significance. Stress profoundly influences pain perception; resolving the mechanisms by which stress alters nociception in rodents may explain the well-known phenomenon of stress-induced analgesia and/or facilitate the development of therapies to mitigate the negative consequences of chronic stress on chronic pain.

(2) Novelty. The authors' findings reveal a crucial contribution of Hes+ spinal astrocytes in the modulation of pain thresholds during stress.

(3) Techniques. This study combines multiple approaches to dissect circuit, cellular, and molecular mechanisms including optical recordings of neural and astrocytic Ca2+ activity in behaving mice, intersectional genetic strategies, cell ablation, optogenetics, chemogenetics, CRISPR-based gene knockdown, slice electrophysiology, and behavior.

Weaknesses:

(1) Mouse model of stress. Although chronic stress can increase sensitivity to somatosensory stimuli and contribute to hyperalgesia and anhedonia, particularly in the context of chronic pain states, acute stress is well known to produce analgesia in humans and rodents. The experimental design used by the authors consists of a single one-hour session of restraint stress followed by 30 min to one hour of habituation and measurement of cutaneous mechanical sensitivity with von Frey filaments. This acute stress behavioral paradigm corresponds to the conditions in which the clinical phenomenon of stress-induced analgesia is observed in humans, as well as in animal models. Surprisingly, however, the authors measured that this acute stressor produced hypersensitivity rather than antinociception. This discrepancy is significant and requires further investigation.

(2) Specifically, is the hypersensitivity to mechanical stimulation also observed in response to heat or cold on a hotplate or coldplate?

(3) Using other stress models, such as a forced swim, do the authors also observe acute stress-induced hypersensitivity instead of stress-induced antinociception?

(4) Measurement of stress hormones in blood would provide an objective measure of the stress of the animals.

(5) Results:

(a) Optical recordings of Ca2+ activity in behaving rodents are particularly useful to investigate the relationship between Ca2+ dynamics and the behaviors displayed by rodents.

(b) The authors report an increase in Ca2+ events in LC NA neurons during restraint stress: Did mice display specific behaviors at the time these Ca2+ events were observed such as movements to escape or orofacial behaviors including head movements or whisking?

(c) Additionally, are similar increases in Ca2+ events in LC NA neurons observed during other stressful behavioral paradigms versus non-stressful paradigms?

(d) Neuronal ablation to reveal the function of a cell population.

(e) The proportion of LC NA neurons and LC→SDH NA neurons expressing DTR-GFP and ablated should be quantified (Figures 1G and J) to validate the methods and permit interpretation of the behavioral data (Figures 1H and K). Importantly, the nocifensive responses and behavior of these mice in other pain assays in the absence of stress (e.g., hotplate) and a few standard assays (open field, rotarod, elevated plus maze) would help determine the consequences of cell ablation on processing of nociceptive information and general behavior.

(f) Confirmation of LC NA neuron function with other methods that alter neuronal excitability or neurotransmission instead of destroying the circuit investigated, such as chemogenetics or chemogenetics, would greatly strengthen the findings. Optogenetics is used in Figure 1M, N but excitation of LC→SDH NA neuron terminals is tested instead of inhibition (to mimic ablation), and in naïve mice instead of stressed mice.

(g) Alpha1Ars. The authors noted that "Adra1a mRNA is also expressed in INs in the SDH".

(h) The authors should comprehensively indicate what other cell types present in the spinal cord and neurons projecting to the spinal cord express alpha1Ars and what is the relative expression level of alpha1Ars in these different cell types.

(i) The conditional KO of alpha1Ars specifically in Hes5+ astrocytes and not in other cell types expressing alpha1Ars should be quantified and validated (Figure 2H).

(j) Depolarization of SDH inhibitory interneurons by NA (Figure 3). The authors' bath applied NA, which presumably activates all NA receptors present in the preparation.

k) The authors' model (Figure 4H) implies that NA released by LC→SDH NA neurons leads to the inhibition of SDH inhibitory interneurons by NA. In other experiments (Figure 1L, Figure 2A), the authors used optogenetics to promote the release of endogenous NA in SDH by LC→SDH NA neurons. This approach would investigate the function of NA endogenously released by LC NA neurons at presynaptic terminals in the SDH and at physiological concentrations and would test the model more convincingly compared to the bath application of NA.

(l) As for other experiments, the proportion of Hes+ astrocytes that express hM3Dq, and the absence of expression in other cells, should be quantified and validated to interpret behavioral data.

(m) Showing that the effect of CNO is dose-dependent would strengthen the authors' findings.

(n) The proportion of SG neurons for which CNO bath application resulted in a reduction in recorded sIPSCs is not clear.

(o) A1Rs. The specific expression of Cas9 and guide RNAs, and the specific KD of A1Rs, in inhibitory interneurons but not in other cell types expressing A1Rs should be quantified and validated.

(6) Methods:

It is unclear how fiber photometry is performed using "optic cannula" during restraint stress while mice are in a 50ml falcon tube (as shown in Figure 1A).

Reviewer #2 (Public review):

Summary:

This study investigates the role of spinal astrocytes in mediating stress-induced pain hypersensitivity, focusing on the LC (locus coeruleus)-to-SDH (spinal dorsal horn) circuit and its mechanisms. The authors aimed to delineate how LC activity contributes to spinal astrocytic activation under stress conditions, explore the role of noradrenaline (NA) signaling in this process, and identify the downstream astrocytic mechanisms that influence pain hypersensitivity.

The authors provide strong evidence that 1-hour restraint stress-induced pain hypersensitivity involves the LC-to-SDH circuit, where NA triggers astrocytic calcium activity via alpha1a adrenoceptors (alpha1aRs). Blockade of alpha1aRs on astrocytes-but not on Vgat-positive SDH neurons-reduced stress-induced pain hypersensitivity. These findings are rigorously supported by well-established behavioral models and advanced genetic techniques, uncovering the critical role of spinal astrocytes in modulating stress-induced pain.

However, the study's third aim-to establish a pathway from astrocyte alpha1aRs to adenosine-mediated inhibition of SDH-Vgat neurons-is less compelling. While pharmacological and behavioral evidence is intriguing, the ex vivo findings are indirect and lack a clear connection to the stress-induced pain model. Despite these limitations, the study advances our understanding of astrocyte-neuron interactions in stress-pain contexts and provides a strong foundation for future research into glial mechanisms in pain hypersensitivity.

Strengths:

The study is built on a robust experimental design using a validated 1-hour restraint stress model, providing a reliable framework to investigate stress-induced pain hypersensitivity. The authors utilized advanced genetic tools, including retrograde AAVs, optogenetics, chemogenetics, and subpopulation-specific knockouts, allowing precise manipulation and interrogation of the LC-SDH circuit and astrocytic roles in pain modulation. Clear evidence demonstrates that NA triggers astrocytic calcium activity via alpha1aRs, and blocking these receptors effectively reduces stress-induced pain hypersensitivity.

Weaknesses:

The study offers mainly indirect evidence for astrocyte-released adenosine acting on SDH-VGAT neurons. The potential contributions of astrocyte-derived D-serine and adenosine to different spinal neuron subtypes, as well as the transient "dip" in astrocytic calcium following LC optostimulation, merit further clarification in future work once appropriate tools become available.

Comments on revisions:

The authors have thoroughly addressed my previous comments, resolving most of the points I raised except those noted in the "Weaknesses" section above. I understand that some of these aspects will require future tool development.

Reviewer #3 (Public review):

Summary

This is an exciting and timely study addressing the role of descending noradrenergic systems in nocifensive responses. While it is well-established that spinally released noradrenaline (aka norepinephrine) generally acts as an inhibitory factor in spinal sensory processing, this system is highly complex. Descending projections from the A6 (locus coeruleus, LC) and the A5 regions typically modulate spinal sensory processing and reduce pain behaviours, but certain subpopulations of LC neurons have been shown to mediate pronociceptive effects, such as those projecting to the prefrontal cortex (Hirshberg et al., PMID: 29027903).

The study proposes that descending cerulean noradrenergic neurons potentiate touch sensation via alpha-1 adrenoceptors on Hes5+ spinal astrocytes, contributing to mechanical hyperalgesia. This finding is consistent with prior work from the same group (dd et al., PMID:). However, caution is needed when generalising about LC projections, as the locus coeruleus is functionally diverse, with differences in targets, neurotransmitter co-release, and behavioural effects. Specifying the subpopulations of LC neurons involved would significantly enhance the impact and interpretability of the findings.

Strengths

The study employs state-of-the-art molecular, genetic, and neurophysiological methods, including precise CRISPR and optogenetic targeting, to investigate the role of Hes5+ astrocytes. This approach is elegant and highlights the often-overlooked contribution of astrocytes in spinal sensory gating. The data convincingly support the role of Hes5+ astrocytes as regulators of touch sensation, coordinated by brain-derived noradrenaline in the spinal dorsal horn, opening new avenues for research into pain and touch modulation.

Furthermore, the data support a model in which superficial dorsal horn (SDH) Hes5+ astrocytes act as non-neuronal gating cells for brain-derived noradrenergic (NA) signalling through their interaction with substantia gelatinosa inhibitory interneurons. Locally released adenosine from NA-stimulated Hes5+ astrocytes, following acute restraint stress, may suppress the function of SDH-Vgat+ inhibitory interneurons, resulting in mechanical pain hypersensitivity. However, the spatially restricted neuron-astrocyte communication underlying this mechanism requires further investigation in future studies.

Comments on revisions:

One important point remains insufficiently resolved. In Figure S4C, two of the three visible neurons in the A5 example appear to show a white "halo" at the cell border, suggesting a merge of eGFP (green) and TH (magenta) and therefore possible transgene positivity. To draw a confident conclusion about the specificity of the approach for the A6 (LC) population, the authors are kindly asked to provide high-resolution images of several representative A5 sections, presented both as merged and as separate colour channels. Ideally, quantification across multiple rostrocaudal sections of A5, A6 and A7 should be provided. This is essential for determining whether any transgene expression occurs within the A5 nucleus, particularly given its several-millimetre rostrocaudal extent. As the behavioural phenotype arises from manipulation of only a small subset of A6 neurons, ruling out any contribution from A5 (or A7) is critical for validating pathway specificity, especially in light of prior reports showing that similar approaches can label A5 fibres.

Reviewer #1 (Public review):

Summary:

In this study, Lamberti et al. investigate how translation initiation and elongation are coordinated at the single-mRNA level in mammalian cells. The authors aim to uncover whether and how cells dynamically adjust initiation rates in response to elongation dynamics, with the overarching goal of understanding how translational homeostasis is maintained. To this end, the study combines single-molecule live-cell imaging using the SunTag system with a kinetic modeling framework grounded in the Totally Asymmetric Simple Exclusion Process (TASEP). By applying this approach to custom reporter constructs with different coding sequences, and under perturbations of the initiation/elongation factor eIF5A, the authors infer initiation and elongation rates from individual mRNAs and examine how these rates covary.

The central finding is that initiation and elongation rates are strongly correlated across a range of coding sequences, resulting in consistently low ribosome density ({less than or equal to}12% of the coding sequence occupied). This coupling is preserved under partial pharmacological inhibition of eIF5A, which slows elongation but is matched by a proportional decrease in initiation, thereby maintaining ribosome density. However, a complete genetic knockout of eIF5A disrupts this coordination, leading to reduced ribosome density, potentially due to changes in ribosome stalling resolution or degradation.

Strengths:

A key strength of this work is its methodological innovation. The authors develop and validate a TASEP-based Hidden Markov Model (HMM) to infer translation kinetics at single-mRNA resolution. This approach provides a substantial advance over previous population-level or averaged models and enables dynamic reconstruction of ribosome behavior from experimental traces. The model is carefully benchmarked against simulated data and appropriately applied. The experimental design is also strong. The authors construct matched SunTag reporters differing only in codon composition in a defined region of the coding sequence, allowing them to isolate the effects of elongation-related features while controlling for other regulatory elements. The use of both pharmacological and genetic perturbations of eIF5A adds robustness and depth to the biological conclusions. The results are compelling: across all constructs and conditions, ribosome density remains low, and initiation and elongation appear tightly coordinated, suggesting an intrinsic feedback mechanism in translational regulation. These findings challenge the classical view of translation initiation as the sole rate-limiting step and provide new insights into how cells may dynamically maintain translation efficiency and avoid ribosome collisions.

Assessment of Goals and Conclusions:

The authors successfully achieve their stated aims: they quantify translation initiation and elongation at the single-mRNA level and show that these processes are dynamically coupled to maintain low ribosome density. The modeling framework is well suited to this task, and the conclusions are supported by multiple lines of evidence, including inferred kinetic parameters, independent ribosome counts, and consistent behavior under perturbation.

Impact and Utility:

This work makes a significant conceptual and technical contribution to the field of translation biology. The modeling framework developed here opens the door to more detailed and quantitative studies of ribosome dynamics on single mRNAs and could be adapted to other imaging systems or perturbations. The discovery of initiation-elongation coupling as a general feature of translation in mammalian cells will likely influence how researchers think about translational regulation under homeostatic and stress conditions.

The data, models, and tools developed in this study will be of broad utility to the community, particularly for researchers studying translation dynamics, ribosome behavior, or the effects of codon usage and mRNA structure on protein synthesis.

Context and Interpretation:

This study contributes to a growing body of evidence that translation is not merely controlled at initiation but involves feedback between elongation and initiation. It supports the emerging view that ribosome collisions, stalling, and quality control pathways play active roles in regulating initiation rates in cis. The findings are consistent with recent studies in yeast and metazoans showing translation initiation repression following stalling events. However, the mechanistic details of this feedback remain incompletely understood and merit further investigation, particularly in physiological or stress contexts.

In summary, this is a thoughtfully executed and timely study that provides valuable insights into the dynamic regulation of translation and introduces a modeling framework with broad applicability. It will be of interest to a wide audience in molecular biology, systems biology, and quantitative imaging.

Reviewer #2 (Public review):

Summary:

This manuscript uses single-molecule run-off experiments and TASEP/HMM models to estimate biophysical parameters, i.e., ribosomal initiation and elongation rates. Combining inferred initiation and elongation rates, the authors quantify ribosomal density. TASEP modeling was used to simulate the mechanistic dynamics of ribosomal translation, and the HMM is used to link ribosomal dynamics to microscope intensity measurements. The authors' main conclusions and findings are:

- Ribosomal elongation rates and initiation rates are strongly coordinated.

- Elongation rates were estimated between 1 and 4.5 aa/sec. Initiation rates were estimated between 1 and 2 ribosomes/min. These values agree with previously reported ones.

- Ribosomal density was determined to be below 12% for all constructs and conditions.

- eIF5A-perturbations (GC7 inhibition) resulted in non-significant changes in translational bursting and ribosome density.

- eIF5A perturbations affected both elongation and initiation rates.

Strengths:

This manuscript presents an interesting scientific hypothesis to study ribosome initiation and elongation concurrently. This topic is relevant for the field. The manuscript presents a novel quantitative methodology to estimate ribosomal initiation rates from Harringtonine run-off assays. This is relevant because run-off assays have been used to estimate, exclusively, elongation rates.

Comments on revisions:

The authors have addressed my concerns. Specifically, they have expanded the discussion on unexpected eIF5A perturbation results, calculated CAI values for all constructs, and made code and data publicly available via GitHub and Zenodo. The mathematical notation is now consistent, and all variables are properly defined.

Reviewer #3 (Public review):

Disclaimer:

My expertise is in live single-molecule imaging of RNA and transcription, as well as associated data analysis and modeling. While this aligns well with the technical aspects of the manuscript, my background in translation is more limited, and I am not best positioned to assess the novelty of the biological conclusions.

Summary:

This study combines live-cell imaging of nascent proteins on single mRNAs with time-series analysis to investigate the kinetics of mRNA translation.<br /> The authors (i) used a calibration method for estimating absolute ribosome counts, and (ii) developed a new Bayesian approach to infer ribosome counts over time from run-off experiments, enabling estimation of elongation rates and ribosome density across conditions.

They report (i) translational bursting at the single-mRNA level, (ii) low ribosome density (~10% occupancy {plus minus} a few percents), (iii) that ribosome density is minimally affected by perturbations of elongation (using a drug and/or different coding sequences in the reporter), suggesting a homeostatic mechanism potentially involving a feedback of elongation onto initiation, although (iv) this coupling breaks down upon knockout of elongation factor eIF5A.

Strengths:

(1) The manuscript is well written and the conclusions are in general appropriately cautious (besides the few improvements I suggest below).

(2) The time-series inference method is interesting and promising for broader application.

(3) Simulations provide convincing support for the modeling (though some improvements are possible).

(4) The reported homeostatic effect on ribosome density is surprising and carefully validated with multiple perturbations.

(5) Imaging quality and corrections (e.g., flat-fielding, laser power measurements) are robust.

(6) Mathematical modeling is clearly described and precise; a few clarifications could improve it further.

Weaknesses:

(1) The absolute quantification of ribosome numbers (via the measurement of $i_{MP}$​) should be improved. This only affects the finding that ribosome density is low, not that it appears to be under homeostatic control. However, if $i_{MP}$​ turns out to be substantially overestimated (hence ribosome density underestimated), then "ribosomes queuing up to the initiation site and physically blocking initiation" could become a relevant hypothesis. In my first review of this work, I made recommendations, which the authors did not follow. In my view, the robustness of this particular aspect of this study remains moderate.

(2) The proposed initiation-elongation coupling is plausible, but alternative explanations such as changes in abortive elongation frequency should be considered. In their response to my previous comments, the authors indicate that this is "beyond the scope of the present work".

(3) More an opportunity for improvement than a weakness: It is unclear what the single-mRNA nature of the inference method is bringing since it is only used here to report _average_ ribosome elongation rate and density (averaged across mRNAs and across time during the run-off experiments -although the method, in principle, has the power to resolve these two aspects). In response to my previous comment, the authors note that such analyses could be incorporated in future work.

Reviewer #1 (Public review):

Summary:

Drosophila larval type II neuroblasts generate diverse types of neurons by sequentially expressing different temporal identity genes during development. Previous studies have shown that transition from early temporal identity genes (such as Chinmo and Imp) to late temporal identity genes (such as Syp and Broad) depends on the activation of the expression of EcR by Seven-up (Svp) and progression through the G1/S transition of the cell cycle. In this study, Chaya and Syed examined if the expression of Syp and EcR is regulated by cell cycle and cytokinesis by knocking down CDK1 or Pav, respectively, throughout development or at specific developmental stages. They find that knocking down CDK1 or Pav either in all type II neuroblasts throughout the development or in single type neuroblast clones after larval hatching consistently leads to failure to activate late temporal identity genes Syp and EcR. To determine whether the failure of the activation of Syp and EcR is due to impaired Svp expression, they also examined Svp expression using a Svp-lacZ reporter line. They find that Svp is expressed normally in CDK1 RNAi neuroblasts. Further, knocking down CDK1 or Pav after Svp activation still leads to loss of Syp and EcR expression. Finally, they also extended their analysis to type I neuroblasts. They find that knocking down CDK1 or Pav, either at 0 hours or at 42 hours after larval hatching, also results in loss of Syp and EcR expression in type I neuroblasts. Based on these findings, the authors conclude that cycle and cytokinesis are required for the transition from early to late late temporal identity genes in both types of neuroblasts. These findings add mechanistic details to our understanding of the temporal patterning of Drosophila larval neuroblasts.

Strengths:

The data presented in the paper are solid and largely support their conclusion. Images are of high quality. The manuscript is well-written and clear.

Weaknesses:

The authors have addressed all the weaknesses in this revision.

Reviewer #2 (Public review):

Summary:

Neural stem cells produce a wide variety of neurons during development. The regulatory mechanisms of neural diversity are based on the spatial and temporal patterning of neural stem cells. Although the molecular basis of spatial patterning is well-understood, the temporal patterning mechanism remains unclear. In this manuscript, the authors focused on the roles of cell cycle progression and cytokinesis in temporal patterning and found that both are involved in this process.

Strengths:

They conducted RNAi-mediated disruption on cell cycle progression and cytokinesis. As they expected, both disruptions affected temporal patterning in NSCs.

Weaknesses:

Although the authors showed clear results, they needed to provide additional data to support their conclusion sufficiently.

For example, they can examine the effects of cell cycle acceleration on the temporal patterning.

Reviewer #3 (Public review):

Summary:

The manuscript by Chaya and Syed focuses on understanding the link between cell cycle and temporal patterning in central brain type II neural stem cells (NSCs). To investigate this, the authors perturb the progression of the cell cycle by delaying the entry into M phase and preventing cytokinesis. Their results convincingly show that temporal factor expression requires progression of the cell cycle in both Type 1 and Type 2 NSCs in the Drosophila central brain. Overall, this study establishes an important link between the two timing mechanisms of neurogenesis.

Strengths:

The authors provide solid experimental evidence for the coupling of cell cycle and temporal factor progression in Type 2 NSCs. The quantified phenotype shows an all-or-none effect of cell cycle block on the emergence of subsequent temporal factors in the NSCs, strongly suggesting that both nuclear division and cytokinesis are required for temporal progression. The authors also extend this phenotype to Type 1 NSCs in the central brain, providing a generalizable characterization of the relationship between cell cycle and temporal patterning.

Weaknesses:

One major weakness of the study is that the authors do not explore the mechanistic relationship between cell cycle and temporal factor expression. Although their results are quite convincing, they do not provide an explanation as to why Cdk1 depletion affects Syp and EcR expression but not the onset of svp. This result suggests that at least a part of the temporal cascade in NSCs is cell-cycle independent which isn't addressed or sufficiently discussed.

Reviewer #1 (Public review):

Summary:

Here, the authors aim to investigate the potential improvements of ANNs when used to explain brain data using top-down feedback connections found in the neocortex. To do so, they use a retinotopic and tonotopic organization to model each subregion of the ventral visual (V1, V2, V4, and IT) and ventral auditory (A1, Belt, A4) regions using Convolutional Gated Recurrent Units. The top-down feedback connections are inspired by the apical tree of pyramidal neurons, modeled either with a multiplicative effect (change of gain of the activation function) or a composite effect (change of gain and threshold of the activation function).

To assess the functional impact of the top-down connections, the authors compare three architectures: a brain-like architecture derived directly from brain data analysis, a reversed architecture where all feedforward connections become feedback connections and vice versa, and a random connectivity architecture. More specifically, in the brain-like model the visual regions provide feedforward input to all auditory areas, whereas auditory areas provide feedback to visual regions.

First, the authors found that top-down feedback influences audiovisual processing and that the brain-like model exhibits a visual bias in multimodal visual and auditory tasks. Second, they discovered that in the brain-like model, the composite integration of top-down feedback, similar to that found in the neocortex, leads to an inductive bias toward visual stimuli, which is not observed in the feedforward-only model. Furthermore, the authors found that the brain-like model learns to utilize relevant stimuli more quickly while ignoring distractors. Finally, by analyzing the activations of all hidden layers (brain regions), they found that the feedforward and feedback connectivity of a region could determine its functional specializations during the given tasks.

Strengths:

The study introduces a novel methodology for designing connectivity between regions in deep learning models. The authors also employ several tasks based on audiovisual stimuli to support their conclusions. Additionally, the model utilizes backpropagation of error as a learning algorithm, making it applicable across a range of tasks, from various supervised learning scenarios to reinforcement learning agents. Conversely, the presented framework offers a valuable tool for studying top-down feedback connections in cortical models. Thus, it is a very nice study that can also give inspiration to other fields (machine learning) to start exploring new architectures.

Reviewer #2 (Public review):

Summary:

This work addresses the question whether artificial deep neural network models of the brain could be improved by incorporating top-down feedback, inspired by the architecture of neocortex.

In line with known biological features of cortical top-down feedback, the authors model such feedback connections with both, a typical driving effect and a purely modulatory effect on the activation of units in the network.

To asses the functional impact of these top-down connections, they compare different architectures of feedforward and feedback connections in a model that mimics the ventral visual and auditory pathways in cortex on an audiovisual integration task.

Notably, one architecture is inspired by human anatomical data, where higher visual and auditory layers possess modulatory top-down connections to all lower-level layers of the same modality, and visual areas provide feedforward input to auditory layers, whereas auditory areas provide modulatory feedback to visual areas.

First, the authors find that this brain-like architecture imparts the models with a light visual bias similar to what is seen in human data, which is the opposite in a reversed architecture, where auditory areas provide feedforward drive to the visual areas.

Second, they find that, in their model, modulatory feedback should be complemented by a driving component to enable effective audiovisual integration, similar to what is observed in neural data.

Overall, the study shows some possible functional implications when adding feedback connections in a deep artificial neural network that mimic some functional aspects of visual perception in humans.

Strengths:

The study contains innovative ideas, such as incorporating an anatomically inspired architecture into a deep ANN, and comparing its impact on a relevant task to alternative architectures.

Moreover, the simplicity of the model allows it to draw conclusions on how features of the architecture and functional aspects of the top-down feedback affects performance of the network.

This could be a helpful resource for future studies of the impact of top-down connections in deep artificial neural network models of neocortex.

Weaknesses:

Some claims not yet supported.

The problem is that results are phrased quite generally in the abstract and discussion, while the actual results shown in the paper are very specific to certain implementations of top-down feedback and architectures. This could lead to misunderstanding and requires some revisions of the claims in the abstract and discussion (see below).

"Altogether our findings demonstrate that modulatory top-down feedback is a computationally relevant feature of biological brain..."

This claim is not supported, since no performance increase is demonstrated for modulatory feedback. So far, only the second half of the sentence is supported: "...and that incorporating it into ANNs affects their behavior and constrains the solutions it's likely to discover."

"This bias does not impair performance on the audiovisual tasks."

This is only true for the composite top-down feedback that combines driving and modulatory effects, whereas modulatory feedback alone can impair the performance (e.g., in the visual tasks VS1 and VS2). The fact that modulatory feedback alone is insufficient in ANNs to enable effective cross-modal integration and requires some driving component is actually very interesting, but it is not stressed enough in the abstract. This is hinted at in the following sentence, but should be made more explicitly:

"The results further suggest that different configurations of top-down feedback make otherwise identically connected models functionally distinct from each other, and from traditional feedforward and laterally recurrent models."

"Here we develop a deep neural network model that captures the core functional properties of top-down feedback in the neocortex" -> this is too strong, take out "the", because very likely there are other important properties that are not yet incorporated.

"Altogether, our results demonstrate that the distinction between feedforward and feedback inputs has clear computational implications, and that ANN models of the brain should therefore consider top-down feedback as an important biological feature."

This claim is still not substantiated by evidence provided in the paper. First, the wording is a bit imprecise, because mechanistically, it is not really the feedforward versus feedback (a purely feedforward model is not considered at all in the paper), but modulatory versus driving. Moreover, the second part of the sentence is problematic: The results imply that, computationally/functionally, driving connections are doing the job, while modulatory feedback does not really seem to improve performance (best case, it does not do any harm). It is true that it is a feature that is inspired by biology, but I don't see why the results imply that (modulatory) top-down feedback should be considered in ANN models of the brain. This would require to show that such models either improve performance, or do improve the ability to fit neural data, both which are beyond the scope of the paper.

The same argument holds for the following sentence, which is not supported by the results of the paper:

"More broadly, our work supports the conclusion that both the cellular neurophysiology and structure of feed-back inputs have critical functional implications that need to be considered by computational models of brain function."

Additional supplementary material required

Although the second version checked the influence of processing time, this was not done for the most important figure of the paper, Figure 4. A central claim in the abstract "This bias does not impair performance on the audiovisual tasks" relies on this figure, because only with composite feedback the performance is comparable between the the "drive-only" and "brain-like" models. Thus, the supplementary Figure 3 should also include the composite networks and drive only network to check the robustness of the claim with respect to process time. This robustness analysis should then also be mentioned in the text. For example, it should be mentioned whether results in these networks are robust or not with respect to process time, whether there are differences between network architectures or types of feedback in general etc.

Moreover, the current analysis for networks with modulatory feedback is a bit confusing. Why is the performance so low for the reverse model for a process time of 3 and 10? This is a very strong effect that warrants explanation. More details should be added in the caption as well. For example, are the models separately trained for the output after 3 and 10 processing steps for the comparison, or just evaluated at these times? Not training these networks separately might explain the low performance for some networks, so ideally networks are trained for each choice of processing steps.

Reviewer #3 (Public review):

Summary:

This study investigates the computational role of top-down feedback in artificial neural networks (ANNs), a feature that is prevalent in the brain but largely absent in standard ANN architectures. The authors construct hierarchical recurrent ANN models that incorporate key properties of top-down feedback in the neocortex. Using these models in an audiovisual integration task, they find that hierarchical structures introduce a mild visual bias, akin to that observed in human perception, not always compromising task performance.

Strengths:

The study investigates a relevant and current topic of considering top-down feedback in deep neural networks. In designing their brain-like model, they use neurophysiological data, such as externopyramidisation and hierarchical connectivity. Their brain-like model exhibits a visual bias that qualitatively matches human perception.

Weaknesses:

While the model is brain-inspired, it has limited bioplausibility. The model assumes a simplified and fixed hierarchy. The authors acknowledge this limitation in the discussion.

While the brain-like model showed an advantage in ignoring distracting auditory inputs, it struggled when visual information had to be ignored. This suggests that its rigid bias toward visual processing could make it less adaptive in tasks requiring flexible multimodal integration. It hence does not necessarily constitute an improvement over existing ANNs. The study does not evaluate whether the top-down feedback architecture scales well to more complex problems or larger datasets. A valuable future contribution would be to evaluate how the network's behaviour fits to human data.

Reviewer #1 (Public review):

Summary:

Jeay-Bizot and colleagues investigate the neural correlates of the preparation of, and commitment to, a self-initiated motor action. In their introduction, they differentiate between theoretical proposals relating to the timing of such neural correlates relative to the time of a recorded motor action (e.g., a keypress). These are categorised into 'early' and 'late' timing accounts. The authors advocate for 'late' accounts based on several arguments that align well with contemporary models of decision-making in other domains (for example, evidence accumulation models applied to perceptual decisions). They also clearly describe prevalent methodological issues related to the measurement of event-related potentials (ERPs) and time-frequency power to gauge the timing of the commitment to making a motor action. These methodological insights are communicated clearly and denote potentially important limitations on the inferences that can be drawn from a large body of existing work.

To attempt to account for such methodological concerns, the authors devise an innovative experiment that includes an experimental condition whereby participants make a motor action (a right-hand keypress) to make an image disappear. They also include a condition whereby the stimulus presentation program automatically proceeds at a set time that is matched to the response timing in a previous trial. In this latter condition, no motor action is required by the participant. The authors then attempt to determine the times at which they can differentiate between these two conditions (motor action vs no motor action) based on EEG and MEG data, using event-related potential analyses, time-frequency analyses, and multivariate classifiers. They also apply analysis techniques based on comparing M/EEG amplitudes at different time windows (as used in previous work) to compare these results to those of their key analyses.

When using multivariate classifiers to discriminate between conditions, they observed very high classification performance at around -100ms from the time of the motor response or computer-initiated image transition, but lower classification performance and a lack of statistically significant effects across analyses for earlier time points. Based on this, they make the key claim that measured M/EEG responses at the earlier time points (i.e., earlier than around -100ms from the motor action) do not reliably correlate with the execution of a motor action (as opposed to no such action being prepared or made). This is argued to favour 'late' accounts of motor action commitment, aligning with the well-made theoretical arguments in favour of these accounts in the introduction. Although the exact time window related to 'late' accounts is not concretely specified, an effect that occurs around -100ms from response onset is assumed here to fall within that window.

Importantly, this claim relies on accepting the null hypothesis of zero effect for the time points preceding around -100ms based on a somewhat small sample of n=15 and some additional analyses of individual participant datasets. Although the authors argue that their classifiers are sensitive to detecting relevant effects, and the study appears well-powered to detect the (likely to be large magnitude) M/EEG signal differences occurring around the time of the response or computer-initiated image transition, there is no guarantee that the study is adequately sensitive to detect earlier differences in M/EEG signals. These earlier effects are likely to be more subtle and exhibit lower signal-to-noise ratios, but would still be relevant to the 'early' vs 'late' debate framed in the manuscript. This, along with some observed patterns in the data, may substantially reduce the confidence one may have in the key claim about the onset timing of M/EEG signal differences.

Notably, there is some indication of above-chance (above 0.5 AUC) classification performance at time points earlier than -100ms from the response, as visible in Figure 3A for the task-based EEG analyses (EEG OC dataset, blue line). While this was not statistically significantly above chance for their n=15 sample, these results do not appear to be clear evidence in favour of a zero-effect null-hypothesis. In Figures 2A-B, there are also visible differences in the ERPs across conditions, from around the time that motor action-related components have been previously observed (around -500ms from the response). The plotted standard errors in the data are large enough to indicate that the study may not have been adequately powered to differentiate between the conditions.

Although the authors acknowledge this limitation in the discussion section of their manuscript, their counter-argument is that the classifiers could reliably differentiate between conditions at time points very close to the motor response, and in the time-based analyses where substantive confounds are likely to be present, as demonstrated in a set of analyses. Based on this data, the authors imply that the study is sufficiently powered to detect effects across the range of time points used in the analyses. While it's commendable that these extra analyses were run, they do not provide convincing evidence that the study is necessarily sensitive to detecting more subtle effects that may occur at earlier time points. In other words, the ability of classifiers (or other analysis methods) to detect what are likely to be very prominent, large effects around the time of the motor response does not guarantee that such analyses will detect smaller magnitude effects at other time points.

In summary, the authors develop some very important lines of argument for why existing work may have misestimated the timing of neural signals that precede motor actions. This in itself is an important contribution to the field. However, their attempt to better estimate the timing of such signals is limited by a reliance on accepting the null hypothesis based on non-statistically significant results, and arguably a limited degree of sensitivity to detect subtle but meaningful effects.

Strengths:

This manuscript provides compelling reasons why existing studies may have misestimated the timing of the neural correlates of motor action preparation and execution. They provide additional analyses as evidence of the relevant confounds and provide simulations to back up their claims. This will be important to consider for many in the field. They also endeavoured to collect large numbers of trials per participant to also examine effects in individuals, which is commendable and arguably better aligned with contemporary theory (which pertains to how individuals make decisions to act, rather than groups of people).

The innovative control condition in their experiment may also be very useful for providing complementary evidence that can better characterise the neural correlates of motor action preparation and commitment. The method for matching image durations across active and passive conditions is particularly well thought-out and provides a nice control for a range of potential confounding factors.

Weaknesses:

There is a mismatch between the stated theoretical phenomenon of interest (commitment to making a motor action) and what is actually tested in the study (differences in neural responses when an action is prepared and made compared to when no action is required). The assumed link between these concepts could be made more explicit for readers, particularly because it is argued in the manuscript that neural correlates of motor action preparation are not necessarily correlates of motor action commitment.

As mentioned in the summary, the main issue is the strong reliance on accepting the null hypothesis of no differences between motor action and computer initiation conditions based on a lack of statistically significant results from the modest (n=15) sample. Although a larger sample will increase measurement precision at the group level, there are some EEG data processing changes that could increase the signal-to-noise ratio of the analysed data and produce more precise estimates of effects, which may improve the ability to detect more subtle effects, or at least provide more confidence in the claims of null effects.

First, it is stated in the EEG acquisition and preprocessing section that the 64-channel Biosemi EEG data were recorded with a common average reference applied. Unless some non-standard acquisition software was used (of which we are not aware exists), Biosemi systems do not actually apply this reference at recording (it is for display purposes only, but often mistaken to be the actual reference applied). As stated in the Biosemi online documentation, a reference should be subsequently applied offline; otherwise, there is a substantial decrease in the signal-to-noise ratio of the EEG data, and a large portion of ambient alternating current noise is retained in the recordings. This can be easily fixed by applying a referencing scheme (e.g., the common average reference) offline as one of the first steps of data processing. If this was, in fact, done offline, it should be clearly communicated in the manuscript.

In addition, the data is downsampled using a non-integer divisor of the original sampling rate (a 2,048 Hz dataset is downsampled to 500 Hz rather than 512 Hz). Downsampling using a non-integer divisor is not recommended and can lead to substantial artefacts in raw data as a result, as personally observed by this Reviewer in Biosemi data. Finally, although a 30 Hz low-pass filter is applied for visualisation purposes of ERPs, no such filter is applied prior to analyses, and no method is used to account for alternating current noise that is likely to be in the data. As noted above, much of the alternating current noise will be retained when an offline reference is not applied, and this is likely to further degrade the quality of the data and reduce one's ability to identify subtle patterns in EEG signals. Changes in data processing to address these issues would likely lead to more precise estimates of EEG signals (and by extension differences across conditions).

With regard to possible effects extending hundreds of milliseconds before the response, it would be helpful for the authors to more precisely clarify the time windows associated with 'early' and 'late' theories in this case. The EEG data that would be required to support 'early' theories is also not made sufficiently clear. For example, even quite early neural correlates of motor actions in this task (e.g., around -500ms from the response, or earlier) could still be taken as evidence for the 'late' theories if these correlates simply reflect the accumulation of evidence toward making a decision and associated motor action, as implied by the Leaky Stochastic Accumulator model described by the authors. In other words, even observations of neural correlates of motor action preparation that occur much earlier than the response would not constitute clear evidence against the 'late' account if this neural activity represents an antecedent to a decision and action (rather than commitment to the action), as the authors point out in the introduction.

In addition, there is some discrepancy regarding the data that is used by the classifiers to differentiate between the conditions in the EEG data and the claims about the timing of neural responses that differentiate between conditions. Unless we reviewers are mistaken, the Sliding Window section of the methods states that the AUC scores in Figure 3 are based on windows of EEG data that extend from the plotted time point until 0.5 seconds into the past. In other words, an AUC value at -100ms from the response is based on classifiers applied to data ranging from -600 to -100 milliseconds relative to the response. In this case, the range of data used by the classifiers extends much earlier than the time points indicated by Figure 3, and it is difficult to know whether the data at these earlier time points may have contributed (even in subtle ways) to the success of the classifiers. This may undermine the claim that neural responses only become differentiable from around -100ms from response onset. The spans of these windows used for classification could be made more explicit in Figure 3, and classification windows that are narrower could be included in a subset of analyses to ensure that classifiers only using data in a narrow window around the response show the high degree of classification performance in the dataset. If we are mistaken, then perhaps these details could be clarified in the method and results sections.

Reviewer #2 (Public review):

Summary:

The authors set out to investigate how well the onset of a self-initiated movement could be predicted at different times prior to action onset. To do so, they collected EEG and MEG data across 15 human participants who watched natural landscape images on a screen. These participants performed active self-initiated movements or observed passive actions to have a new image appear. By comparing the neural activity prior to active and time-matched passive actions, the authors found that even though a build-up of neural activity is visible close to 1s prior to action, action onset could only be reliably predicted around 100ms prior to action. These results confirm what was already suggested in previous literature: the commitment to action is only clear from the late stages in the visible neural ramp-up to action onset.

Strengths:

(1) The paper presents a well-thought-out methodology to assess the predictive value of neural activity prior to a self-initiated movement and passively observed action, while keeping all other experimental factors identical. This methodology can be applied outside the specific scope of this paper as well, in efforts to assess the correspondence of a neural signature with an observed behavior.

(2) The results are a strong confirmation of what was suggested less clearly in previous research (Trevena & Miller, 2010, Consciousness & Cognition; Schmidt et al., 2016, Neuroscience & Biobehavioral Reviews; Travers et al., 2020, NeuroImage).

Weaknesses:

(1) Although the authors conducted a solid confirmatory study, the importance of this confirmation is less clear to me. How do the current results change our interpretation of the relation between conscious intention and neural preparation for action? Do these results affect our interpretation of free will? Why does it matter at all whether we see neural preparatory activity prior to the report of a conscious intention to act, or prior to action observation? This study does not clarify the relationship between the observed neural phenomenon, the action or the experienced intention. It does not explain whether this relation is causal, correlational or something else.

(2) Whereas Derchi et al. (2023, Scientific Reports) were able to keep the entire experimental context similar across intended and unintended conditions, Jeay-Bizot et al. have one big difference between their passive and active conditions: the presence of a movement. Therefore, the present results explain the presence or absence of a movement rather than the presence or absence of an intention to act.

Reviewer #1 (Public review):

Summary:

This study reports the effects of psilocin on iPSC-derived human cortical neurons.

Strengths:

The characterization was comprehensive, involving immunohistochemistry of various markers, 5-HT2A receptors, BDNF, and TrkB, transcriptomics analyses, morphological determination, electrophysiology, and finally synaptic protein measurements. The results are in close agreement with prior work (PMID 29898390) on rat cultured cortical neurons. Nevertheless, there is value in confirming those earlier findings and furthermore to demonstrate the effects in human neurons, which are important for translation. The genetic, proteomics, and cell structure analyses used in this paper are its major strength. The study supports the value of using iPSC-derived human cortical neurons for drug development involving psychedelics-related compounds.

Weaknesses:

(1) Line 140: 5-HT2A receptor expression was found via immunocytochemistry to reside in the somatodendritic and axonal compartments. However, prior work from ex vivo tissue using electron microscopy has found predominantly 5-HT2A receptor expression in the somatodendritic compartment (PMID: 12535944). Was this antibody validated to be 5-HT2A receptor-specific? Can the authors reason why the discrepancy may arise, and if the axonal expression is specific to the cultured neurons?

(2) Line 143: It would be helpful to specify the dose of psilocin tested, and describe how this dose was chosen.

(3) Figure 1: The interpretation is that the differential internalization in the axonal and somatodendritic compartments is time-dependent. However, given that only one dose is tested, it is also possible that this reflects dose dependence, with the longer time exposure leading to higher dose exposure, so these variables are related. That is, if a higher dose is given, internalization may also be observed after 10 minutes in the dendritic compartment.

(4) Figure 3 & 4: What is the 'control' here? A more appropriate control for the 24 hours after psilocin application would be 24 hours after vehicle application. Here the authors are looking at before and after, but the factor of time elapsed and perturbation via application is not controlled for.

(5) The sample size was not clearly described. In the figure legend, N = the number of neurites is provided, but it is unclear how many cells have been analyzed, and then how many of those cells belong to the same culture. These are important sample size information that should be provided. Relatedly, statistical analyses should consider that the neurites from the same cells are not independent. If the neurites indeed come from the same cells, then the sample size is much smaller and a statistical analysis considering the nested nature of the data should be used.

Comments on revisions:

The authors performed substantial experiments to check validity of the HTR2A antibody for the revision. Briefly, they found that western blot shows a single band, abolished by a blocking peptide, in neural progenitors and iPSC-derived neurons, suggesting positive results. However, they also detected immunofluorescence signals in HEK293 and HeLa cells, which do not express 5-HT2A receptors as scRNAseq analysis of these cells show complete absence of the transcript. Therefore the antibody has epitope-selective binding but also has some non-specific binding, precluding its use. The authors rightfully removed the data related to the antibody in the revised manuscript. The account is repeated here to highlight to anyone who may find the information helpful. Overall, the additional results added rigor to the study.

Reviewer #2 (Public review):

In this article, Schmidt et al use iPSC-derived human cortical neurons to test the effects the psychedelic psilocin in different models of neuroplasticity.

Using human iPSC-derived cortical neurons, the authors test the expression of 5-HT2A and subcellular distribution, as well as the effect of different times of exposure to psilocin on 5-HT2A expression. The authors evaluated the effect of the 5-HT2 antagonist ketanserin, as well as the inhibition of dynamin-dependent endocytic pathways with dynasore. Gene expression and plasticity (structural and functional) was also evaluated after different times of exposure to psilocin.

In general, results are interesting since they use the iPSC to evaluate the potentially translationally relevant effects of psilocin (the active metabolite of the psychedelic psilocybin).

Comments on revisions:

The authors have addressed all of my previous concerns. A particular strength of the rebuttal is that the authors corroborated the lack of selectivity/specificity of the anti-5-HT2A antibody used in earlier versions of the manuscript.

Reviewer #1 (Public review):

Summary:

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

Strengths:

This is excellent interdisciplinary work.

I have commented on the response of the authors, in-line, below. This is to maintain the conversation thread with the authors.

Comment 1:

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

Response:

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

Comment to Response: I get the point. I believe that the outcome is highly dependent on how selection pressure is administered. So, generalizing this over all stresses (as done in the abstract) may not be accurate.

Comment 2:

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

Response:

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

Comment to Response: You indeed saw a case of phenotypic convergence. Converging towards trehalose-rich, mechanically reinforced, quiescent like - are phenotypes that have converged. This is what prevented lysis. The same locus need not be mutated over and over again, if the trehalose pathway is controlled by many processes (it is, and many are still unknown as I point in the next comment), many different mutations on different loci can result in the same regulation! I do not see the decoupling between phenotypic convergence and decoupling of genetic mutations as surprising or novel; molecular and cellular biology is replete with such examples where deletion(mutation) of hundreds of different genes can have the same phenotypic outcome (yeast deletion library screening, indirect effects etc). If this was a specific question unsolved in evolutionary biology, then the matter is different.

A minor point: Here I would also like to point out that the three phenotypes you measure may be linked to each other, so their independent evolution may just be a cause-effect relationship. For example Trehalose accumulation may drive the other two. This has not been deconvoluted in this manuscript.

Comment 3:

Adaptive evolution would work on phenotype, as all of selective evolution is supposed to. So, given that one of the phenotypes well-known in literature to allow free-tolerance is trehalose accumulation, I think it is not surprising that this trait is selected. For me, this is not a case of "non-genetic" adaptation as the authors point out: it is likely because perturbation of many genes can individually result in the same outcome - up-regulation of trehalose accumulation. Thereby, although the adaptation is genetic, it is not homogeneous across the evolving lines - the end result is. Do the authors check that the trait is actually a non-genetic adaptation, i.e., if they regrow the cells for a few generations without the stress, the cells fall back to being similarly only partially fit to freeze-thaw cycles? Additionally, the inability to identify a network that is conserved in the sequencing does not mean that there is no regulatory pathway. A large number of cryptic pathways may exist to alter cellular metabolic states.<br /> This is a point in continuation of point #2, and I would like to understand what I have missed.

Response:

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

Comment to Response: While the last term does explain what is going on, isn't it an outcome that is routine in cell biology (as pointed out in my previous comment to your response)? I apologize for not understanding the punchline that is provided in the last few sentences of the abstract.

Comment 4:

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

Response:

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

Comment to Response: Previous large scale studies have done hundreds of sequencing to oversample the pathway and figure out reproducible loci. With pooled sequencing (as mentioned below) and only 4 sample evolution, I am not sure that you would have the power in your study to conclude in the loci are sampled or not! If there were 10 gene LOFs that control Trehalose levels (which you can find from the published deletion screening experiment), then four of the experiments are likely to go through one of these routes; what is the likely event that you would identify the same route in two pools? It is unlikely, and therefore, sequencing of 4 pools cannot tell you if the mutation path is repeatedly sampled or not.

Comment 5:

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

Response:

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

Comment to Response: Please provide the average sequencing depth of each sequencing run. It is essential to understand the power of this study in identifying mutations. What coverage was used in Xgenome size?

Reviewer #2 (Public review):

Summary:

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

Strengths:

Experimental design, data presentation and interpretation, writing

Weaknesses:

None

Comments on revisions:

The revised manuscript is improved and addresses the reviews concerns adequately.

Reviewer #1 (Public review):

In their manuscript, Papadopoli et al explore the role of ETFDH in transformation. They note that ETFDH protein levels are decreased in cancer, and that deletion of ETFDH in cancer cell lines results in increased tumorigenesis, elevated OXPHOS and glycolysis, and a reduction in lipid and amino acid oxidation. The authors attribute these effects to increased amino acid levels stimulating mTORC1 signaling and driving alterations in BCL6 and EIF4EBP1. They conclude that ETFDH1 is epigenetically silenced in a proportion of neoplasms, suggesting a tumor-suppressive function. Overall, the authors logically present clear data and perform appropriate experiments to support their hypotheses.

Reviewer #2 (Public review):

Summary:

The altered metabolism of tumors enables their growth and survival. Classically, tumor metabolism often involves increased activity of a given pathway in intermediary metabolism to provide energy or substrates needed for growth. Papadopoli et al. investigate the converse - the role of mitochondrial electron transfer flavoprotein dehydrogenase (ETFDH) in cancer metabolism and growth. The authors present compelling evidence that ETFDH insufficiency, which is detrimental in non-malignant tissues, paradoxically enhances bioenergetic capacity and accelerates neoplastic growth in cancer cells in spite of the decreased metabolic fuel flexibility that this affords tumor cells. This is achieved through the retrograde activation of the mTORC1/BCL-6/4E-BP1 axis, leading to metabolic and signaling reprogramming that favors tumor progression.

Strengths:

This review focuses primarily on the cancer metabolism aspects of the manuscript.

The study provides robust evidence linking ETFDH insufficiency to enhanced cancer cell bioenergetics and tumor growth.

The use of multiple cancer cell lines and in vivo models strengthens the generalizability of the findings.

The mechanistic insights into the mTORC1/BCL-6/4E-BP1 axis and its role in metabolic reprogramming are of general interest within and outside the immediate field of tumor metabolism.

Conclusion:

This manuscript provides significant insights into the role of ETFDH insufficiency in cancer metabolism and growth. The findings highlight the potential of targeting the mTORC1/BCL-6/4E-BP1 axis in ETFDH-deficient cancers. The compelling data support the conclusions presented in the manuscript, which will be valuable to the cancer metabolism community.

[Editors' note: The authors have addressed each of the two weaknesses previously listed in the public review, providing new experimental data on nucleotides and showing that the catalytic activity is required via the suggested addback experiment.]

Reviewer #1 (Public review):

Schafer et al. tested whether the hippocampus tracks social interactions as sequences of neural states within an abstract social space defined by the dimensions of affiliation and power, using a narrative-based task in which participants engaged in dynamic social interactions. The study showed that individual social relationships were represented as distinct trajectories of hippocampal activity patterns. These neural trajectories systematically reflected trial-by-trial changes in affiliation and power between the participant and each character, suggesting that the hippocampus encodes sequences of socially relevant events and their relational structure, extending its well-established role beyond spatial representations.

A major strength of this study is the use of a richly structured, narrative-based task that allows social relationships to evolve dynamically over time. The use of representational similarity analysis provides a principled framework for linking behavioral trajectories in social space to neural pattern dynamics.

One potential limitation concerns temporal autocorrelation in the neural data, as nearby trials are inherently related both behaviorally and temporally within a continuous narrative. Although the authors carefully attempted to control for temporal distance and related confounds, fully disentangling representational similarity driven by social structure from similarity driven by temporal proximity remains challenging within a single-session task design.

While the findings of a two-dimensional representational structure is an important contribution, it remains an open question whether such a representation reflects an inherent property of how the human brain encodes social relationships, or whether it is partly driven by task constraints in which social interactions were limited to changes along two (affiliation and power) dimensions. Future studies that allow social relationships to vary along richer or higher-dimensional feature spaces will be necessary to determine the generality of low dimensional representations.

Reviewer #2 (Public review):

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

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

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

Strengths:

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

Weaknesses:

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

(1) Statistics:

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

Hypotheses:

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

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

Reviewer #2 (Public review):

Summary:

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

Strength:

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

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

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

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

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

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

Reviewer #3 (Public review):

Summary:

The study aims to investigate sensorimotor plasticity mechanisms by exposing a cohort of 20 subjects to manipulation activities while using wearable finger extensions. With a series of experiments involving localization and motor tasks, the authors provide evidence that the finger extensions are integrated into the body representation of the subjects.

Strengths:

The study deserves attention, and the psychophysical protocols are carefully designed, and the statistical analyses are solid.

Weaknesses:

However, the current version of the manuscript, in my opinion, makes an exaggerated use of the term plasticity, and this should be amended. This is because the authors support the plasticity claims with psychophysical experiments, without providing evidence of neural-plasticity mechanisms (e.g., neuroimaging methods are not used).

The authors are recommended to revise the wording of the manuscript and possibly perform additional experiments with brain imaging methods (e.g., EEG or fMRI).

Reviewer #1 (Public review):

Summary:

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

Strengths:

The group has strong expertise in Ewing sarcoma genetics and epigenetics and also in using and analyzing this model (Theisen et al., 2019; Boone et al., 2021; Showpnil et al., 2022).

They aim at better understanding how EWS-FLI mediated its oncogenic activity, which is critical to eventually identifying novel therapies against this aggressive cancer.

They use the most recent state-of-the-art omics methods to investigate transcriptome, epigenetics, and genome conformation methods. In particular, Micro-C enables achieving up to 1kb resolved 3D chromatin structures, making it possible to investigate a large number of TADs and sub-TADs structures where EWS-FLI1 mediates its oncogenic activity.

They performed all their experiments in an Ewing sarcoma genetic background (A673 cells) which circumvents bias from previously reported approaches when working in non-orthologous cell models using similar approaches.

Weaknesses:

The main weakness stems from the poor reproducibility of the Micro-C data. Indeed, the distances and clustering observed between replicates appear to be similar to, or even greater than, those observed between biological conditions. For instance, in Figure 1B, we do not observe any clear clustering among DBD1, DBD2, DBD+1, and DBD+2. Although no further experiments were performed, the authors tempered their claims by rephrasing aspects related to this issue and the reviewer also acknowledged that the transcriptomic data are convincing and support their findings.

Regarding DBD stability and the cycloheximide experiments requested to rule out any half-life bias of DBD (as higher stability of the re-expressed DBD+ could also partially explain the results independently of a 3D conformational change), the reviewer acknowledged that the WB, RNA-seq data and agar assays presented by the authors appear reproducible across experiments.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

Previously described weaknesses in the study's methods and interpretation were fully addressed during revision. Locomotor behavior of the mice during head-fixed imaging experiments was added and analysis of the correlation of locomotion with neural activity was also added.

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

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

Weaknesses:

The weaknesses noted in the primary review have all been addressed adequately.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The weaknesses noted in primary review have all been addressed adequately.

Reviewer #1 (Public review):

Summary:

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

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

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

Strengths:

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

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

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

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

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

Weaknesses:

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

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

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

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

Impact:

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #3 (Public review):

Summary:

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

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

Strengths:

This study demonstrates the following strengths:

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

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

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

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

Weaknesses:

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

How to interpret the work:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Major comments:

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

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

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

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

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

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

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

Reviewer #3 (Public review):

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

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

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

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

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

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

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

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

Reviewer #3 (Public review):

This work by Du et al. addresses a critical problem in cryo-electron microscopy. To date, there are few ways of generating phase contrast during cryo-EM imaging while remaining in focus. Cryo-EM practitioners today must generate contrast by collecting out-of-focus exposures, a process that introduces aberrations in the resulting image data. Recent work has shown that standing wave lasers are capable of using the ponderomotive effect to shift the phase of electrons in transmission electron microscopy to generate in-focus phase contrast imaging for cryo-EM. A limitation of this 'laser phase plate' is the high laser power required, which can damage optical mirrors and necessitate high laser safety. Thus, alternative approaches are needed for phase contrast imaging in cryo-EM.

In this manuscript, Du et al. exploit their expertise in ultrafast electron microscopy to explore the ability to shift the phase of electrons using pulsed electrons and lasers. The motivation for exploring pulsed laser phase plates stems from the fact that femtosecond pulses from 9W lasers can generate extremely high power (as much as the standing-wave laser phase plate, > 1 gigawatt) at the back focal plane. If successful, this type of instrument will likely be much more affordable and easier to deploy worldwide.

The work outlined here shows a proof of principle, highlighting that an ultrafast scanning electron microscopy beam at 30 kV can have the electron packets phase shift by 430 radians (24637 degrees), which is much greater than the required 1.5 radians (90 degrees) needed for phase contrast imaging. The data presented do not use any biological samples; instead, they measure the spread of the electron beam on a test sample to assess the ability to target pulsed lasers onto electron packets and the amount of electron spread (which relates to the phase shift). They were also able to take their system a step further to measure how changes to the system in terms of laser power affect performance, and show that the system can be stable for 10+ hours.

The only weaknesses relate to the broad readability of the text. Improved textual clarity will help ensure a wider readership.

Overall, this work is an important step toward developing lower-cost alternatives to the standing-wave laser phase plate.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors present the development and characterization of a pulsed ponderomotive phase plate for transmission electron microscopy (TEM). The primary goal is to overcome the long-standing challenge of generating stable, tunable phase contrast for weakly scattering biological specimens - a capability that has remained elusive despite decades of development. While the commercially available Volta Phase Plate offers phase enhancement, it suffers from a lack of control and stability. More recent efforts have focused on continuous-wave (CW) laser phase plates; however, these systems face significant practical hurdles, including extreme optical power requirements, thermal instability of mirrors, and the necessity for high-finesse optical cavities that act as diffraction gratings for the electron beam. The authors aim to demonstrate that a pulsed, free-space laser interaction can circumvent these limitations, offering a more robust path toward practically usable phase plates

Strengths:

The most significant strength of this work is the elegant use of a free-space pulsed interaction, which fundamentally simplifies the hardware requirements compared to cavity-based designs. By utilizing a high-intensity pulsed laser focus rather than a standing wave inside a resonator, the authors eliminate the need for complex locking feedback loops and avoid the thermal mirror deformation that currently limits CW systems.

Furthermore, this approach provides a critical theoretical advantage regarding image quality. Current CW cavity-based designs must grapple with the Kapitza-Dirac effect, where the standing wave creates a diffraction grating that generates unwanted "ghost images," delocalizing the signal. Recent proposals have had to resort to complex crossed-beam geometries to mitigate these artifacts. In contrast, the traveling-wave nature of the pulsed interaction described here inherently avoids the creation of a standing wave grating, thereby eliminating ghost images entirely without requiring elaborate compensation strategies.

The authors successfully demonstrate a proof-of-concept implementation, reporting a pronounced peak phase shift of approximately 430 radians and a stable angular deflection of the electron beam. The stability data, covering a 10-hour period, suggests that this approach is robust enough for data collection sessions typical in structural biology.

Weaknesses:

However, the strength of the evidence is modestly tempered by limitations in data presentation and analysis. The agreement between the experimental data and the theoretical simulation in Figure 2b is imperfect; the simulation underestimates the depth of the central signal trough. While the authors acknowledge this "muted" prediction, the discrepancy suggests that the theoretical model or the estimation of experimental parameters (such as electron beam size or laser intensity) requires refinement to fully describe the interaction.

While the authors claim stability over many hours, the data in Figure 3c reveal a significant drift in the baseline reference signal. Although attributed to a weakening electron beam, this drift complicates the reader's ability to assess the true stability of the laser-induced phase shift. A drift-corrected analysis would have provided more compelling evidence of the "stable angular kick" described.

Despite these specific weaknesses in data presentation, the work represents a fundamental step forward. The authors have effectively demonstrated that the trade-off between beam current and spatiotemporal resolution (driven by space-charge effects) can be managed to achieve significant phase modulation. By moving the field away from the tight constraints of optical cavities and toward free-space pulsed interactions, this work establishes a potentially more viable route for integrating laser phase plates into routine biological imaging workflows. This study will be of high value to biophysicists and microscopists seeking to push the boundaries of contrast in cryo-EM

Reviewer #1 (Public review):

Summary:

Du, Daniel X. et al studied the interaction of the ultrashort electron and laser pulses inside a scanning electron microscopy (SEM), aiming to build a foundation for pulsed laser phase plate electron microscopy, in which the contrast of cryo samples can be significantly increased. The author modified a commercial SEM to accommodate optics to introduce a laser beam inside the instrument to overlap with the electron beam and performed multiple experiments aimed to characterize the electron-light interaction, particularly reaching an extremely high phase shift of >400 rad. Moreover, the authors built a theoretical model for this interaction and estimated the laser beam parameters needed to reach 90 degrees phase shift in transmission electron microscopy (TEM).

Strengths:

The conclusion on the interaction of the electron pulses and laser pulses is well described and supported by the experiment.

The presented instrument can serve as a great tool for studying fundamental interactions of electrons with extremely intense light pulses.

Weaknesses:

The authors motivate the project by using the pulsed electron beam with a phase shift for improving the contrast in cryo-EM, and while they indicate the low current in UEM, they do not discuss the limitations of the laser beam properties.

Such, even for 1 ps electron pulses with the repetition rate of 100 GHz (duty cycle of 10%), they will need to use 100 GHz laser pulses with pulse energies of at least ~1 uJ a second (the lowest pulse energy reported in the simulations in Figure 4), which would mean that ~10 kW of optical power needs to enter the electron microscope and be dumped somewhere after leaving the instrument. This significantly complicates the system and, in my view, makes it harder to use a pulsed laser phase plate in cryo-EM due to either low acquisition rate at lower repetition rates or extreme difficulties to operate multi kW ultrafast laser system.

I would also expect the unscattered electron beam diameter to be <1 micron, which would significantly change the plot in 4b for the 300 keV electron beam.

Adding experimental parameters for a typical cryo-EM experiment with the pulsed phase plate, including the repetition rate, electron pulse duration, number of electrons per pulse, electron beam size, and the parameters of the laser beam (wavelength, laser pulse duration, pulse energy), will help readers better understand technical requirements for the proposed cryo-EM experiments.

Reviewer #2 (Public review):

Zhang et al. have developed an advanced three-dimensional culture system of human endometrial cells, termed a receptive endometrial assembloid, that models the uterine lining during the crucial window of implantation (WOI). During this mid-secretory phase of the menstrual cycle, the endometrium becomes receptive to an embryo, undergoing distinctive changes. In this work, endometrial cells (epithelial glands, stromal cells, and immune cells from patient samples) were grown into spheroid assembloids and treated with a sequence of hormones to mimic the natural cycle. Notably, the authors added pregnancy-related factors (such as hCG and placental lactogen) on top of estrogen and progesterone, pushing the tissue construct into a highly differentiated, receptive state. The resulting WOI assembloid closely resembles a natural receptive endometrium in both structure and function. The cultures form characteristic surface structures like pinopodes and exhibit abundant motile cilia on the epithelial cells, both known hallmarks of the mid-secretory phase. The assembloids also show signs of stromal cell decidualization and an epithelial mesenchymal transition, like process at the implantation interface, reflecting how real endometrial cells prepare for possible embryo invasion.

Although the WOI assembloid represents an important step forward, it still has limitations: the supportive stromal and immune cell populations decrease over time in culture, so only early-passage assembloids retain full complexity. Additionally, the differences between the WOI assembloid and a conventional secretory-phase organoid are more quantitative than absolute; both respond to hormones and develop secretory features, but the WOI assembloid achieves a higher degree of differentiation due to the addition of "pregnancy" signals. Overall, while it's a reinforced model (not an exact replica of the natural endometrium), it provides a valuable in vitro system for implantation studies and testing potential interventions, with opportunities to improve its long-term stability and biological fidelity in the future.

[Editors' note: the authors have responded to the previous round of recommendations.]

Reviewer #1 (Public review):

Summary:

This paper presents maRQup a Python pipeline for automating the quantitative analysis of preclinical cancer immunotherapy experiments using bioluminescent imaging in mice. maRQup processes images to quantify tumor burden over time and across anatomical regions, enabling large-scale analysis of over 1,000 mice. The study uses this tool to compare different CAR-T cell constructs and doses, identifying differences in initial tumor control and relapse rates, particularly noting that CD19.CD28 CAR-T cells show faster initial killing but higher relapse compared to CD19.4-1BB CAR-T cells. Furthermore, maRQup facilitates the spatiotemporal analysis of tumor dynamics, revealing differences in growth patterns based on anatomical location, such as the snout exhibiting more resistance to treatment than bone marrow.

Strengths:

(1) The maRQup pipeline enables the automatic processing of a large dataset of over 1,000 mice, providing investigators with a rapid and efficient method for analyzing extensive bioluminescent tumor image data.

(2) Through image processing steps like tail removal and vertical scaling, maRQup normalizes mouse dimensions to facilitate the alignment of anatomical regions across images. This process enables the reliable demarcation of nine distinct anatomical regions within each mouse image, serving as a basis for spatiotemporal analysis of tumor burden within these consistent regions by quantifying average radiance per pixel.

Weaknesses:

(1) While the pipeline aims to standardize images for regional assessment, the reliance on scaling primarily along the vertical axis after tail removal may introduce limitations to the quantitative robustness of the anatomically defined regions. This approach does not account for potential non-linear growth across dimensions in animals of different ages or sizes, which could result in relative stretching or shrinking of subjects compared to an average reference.

(2) Furthermore, despite excluding severely slanted images, the pipeline does not fully normalize for variations in animal pose during image acquisition (e.g., tucked body, leaning). This pose variability not only impacts the precise relative positioning of internal anatomical regions, potentially making their definition based on relative image coordinates more qualitative than truly quantitative for precise regional analysis, but it also means that the bioluminescent light signal from the tumor will not propagate equally to the camera as photons will travel differentially through the tissue. This differing light path through tissues due to variable positioning can introduce large variability in the measured radiance that was not accounted for in the analysis algorithm. Achieving more robust anatomical and quantitative normalization might require methods that control animal posture using a rigid structure during imaging.

Comments on revisions:

(1) Clarification of 2D Analysis. We strongly recommend that the authors explicitly define maRQup as a 2D spatiotemporal analysis technique. Since optical imaging quantification is inherently dependent on tissue type and signal depth, characterizing this as a 3D or volumetric method without tomographic correction is inaccurate. Please precede "spatiotemporal" with "2D" throughout the text to ensure precision regarding the method's capabilities.

(2) Data Validation and Scaling in Supplemental Figure g currently lacks the units necessary to support the assertion.

Non-Uniform Growth: The authors' method implies that mouse growth is linear and uniform in all directions (isotropic). However, murine growth is not akin to the inflation of a balloon; animals elongate and widen at different rates. The current scaling does not account for these physiological non-linearities.

Pose Variability: The scaling approach appears to neglect significant variability in animal positioning. Even under anesthesia, animal pose is rarely identical across subjects or time points.

Requirement for Evidence: Without quantitative data, there appears to be significant differences between the individual images and the merged image. If the authors assert that this is a "classical setting" where mouse positioning is 100% consistent and growth curves are identical in multiple dimensions, please provide specific references that validate these assumptions. Otherwise, the scaling must be corrected to account for anisotropic growth and pose differences or stated that scaling was only based on one dimension.

(3) Methodology of Spatial Regions The manuscript does not currently indicate how the nine distinct spatial regions were determined. Please expand the methods section to include the specific segmentation algorithms or anatomical criteria used to define these regions, as this is critical for reproducibility.

Reviewer #3 (Public review):

Summary:

The paper "The 1000+ mouse project: large-scale spatiotemporal parametrization and modeling of preclinical cancer immunotherapies" is focused on developing a novel methodology for automatic processing of bioluminescence imaging data. It provides quantitative and statistically robust insights on preclinical experiments that will contribute to optimizing cell-based therapies. There is an enormous demand for such methods and approaches that enable the spatiotemporal evaluation of cell monitoring in large cohorts of experimental animals.

Strengths:

The manuscript is generally well written, and the experiments are scientifically sound. The conclusions reflect the soundness of experimental data. This approach seems to be quite innovative and promising to improve the statistical accuracy of BLI data quantification.<br /> This methodology can be used as a universal quantification tool for BLI data for in vivo assessment of adoptively transferred cells due to the versatility of the technology.

Comments on revisions:

The critiques have been taken care of appropriately.

Reviewer #1 (Public review):

Summary:

This study presents a technically sophisticated intravital two-photon calcium imaging approach to characterize meningeal macrophage Ca²⁺ dynamics in awake mice. The development of a Pf4Cre:GCaMP6s reporter line and the integration of event-based Ca²⁺ analysis represent clear methodological strengths. The findings reveal niche-specific Ca²⁺ signaling patterns and heterogeneous macrophage responses to cortical spreading depolarization (CSD), with potential relevance to migraine and neuroinflammatory conditions. Despite these strengths, several conceptual, technical, and interpretational issues limit the impact and mechanistic depth of the study. Addressing the points below would substantially strengthen the manuscript.

Strengths:

The use of chronic two-photon Ca²⁺ imaging in awake, behaving mice represents a major technical strength, minimizing confounds introduced by anesthesia. The development of a Pf4Cre:GCaMP6s reporter line, combined with high-resolution intravital imaging, enables long-term and subcellular analysis of macrophage Ca²⁺ dynamics in the meninges.

The comparison between perivascular and non-perivascular macrophages reveals clear niche-dependent differences in Ca²⁺ signaling properties. The identification of macrophage Ca²⁺ activity temporally coupled to dural vasomotion is particularly intriguing and highlights a potential macrophage-vascular functional unit in the dura.

By linking macrophage Ca²⁺ responses to CSD and implicating CGRP/RAMP1 signaling in a subset of these responses, the study connects meningeal macrophage activity to clinically relevant neuroimmune pathways involved in migraine and other neurological disorders.

Weaknesses:

The manuscript relies heavily on Pf4Cre-driven GCaMP6s expression to selectively image meningeal macrophages. Although prior studies are cited to support Pf4 specificity, Pf4 is not an exclusively macrophage-restricted marker, and developmental recombination cannot be excluded. The authors should provide direct validation of reporter specificity in the adult meninges (e.g., co-labeling with established macrophage markers and exclusion of other Pf4-expressing lineages). At minimum, the limitations of Pf4Cre-based labeling should be discussed more explicitly, particularly regarding how off-target expression might affect Ca²⁺ signal interpretation.

The manuscript offers an extensive characterization of Ca²⁺ event features (frequency spectra, propagation patterns, synchrony), but the biological significance of these signals is largely speculative. There is no direct link established between Ca²⁺ activity patterns and macrophage function (e.g., activation state, motility, cytokine release, or interaction with other meningeal components). The discussion frequently implies functional specialization based on Ca²⁺ dynamics without experimental validation. To strengthen the conceptual impact, a clearer framing of the study as a foundational descriptive resource, rather than a functional dissection, would improve alignment between data and conclusions.

The GLM analysis revealing coupling between dural perivascular macrophage Ca²⁺ activity and vasomotion is technically sophisticated and intriguing. However, the directionality of this relationship remains unresolved. The current data do not distinguish whether macrophages actively regulate vasomotion, respond to mechanical or hemodynamic changes, or are co-modulated by neural activity. Statements suggesting that macrophages may "mediate" vasomotion are therefore premature. The authors should reframe these conclusions more cautiously, emphasizing correlation rather than causation, and expand the discussion to explicitly outline experimental strategies required to establish causality (e.g., macrophage-specific Ca²⁺ manipulation).

The authors conclude that synchronous Ca²⁺ events across macrophages are driven by extrinsic signals rather than intercellular communication, based primarily on distance-time analyses. This conclusion is not sufficiently supported, as spatial independence alone does not exclude paracrine signaling, vascular cues, or network-level coordination. No perturbation experiments are presented to test alternative mechanisms. The authors can either provide additional experimental evidence or rephrase the conclusion to acknowledge that the source of synchrony remains unresolved.

A major and potentially important finding is that the dominant macrophage response to CSD is a persistent decrease in Ca²⁺ activity, which is independent of CGRP/RAMP1 signaling. However, this phenomenon is not mechanistically explored. It remains unclear whether Ca²⁺ suppression reflects macrophage inhibition, altered viability, homeostatic resetting, or an anti-inflammatory program. Minimally, the discussion should be more deeply engaged with possible interpretations and implications of this finding.

The pharmacological blockade of RAMP1 supports a role for CGRP signaling in persistent Ca²⁺ increases after CSD, but the experiments are based on a relatively small number of cells and animals. The limited sample size constrains confidence in the generality of the conclusions. Pharmacological inhibition alone does not establish cell-autonomous effects in macrophages. The authors should acknowledge these limitations more explicitly and avoid overextension of the conclusions.

Reviewer #2 (Public review):

Using chronic intravital two-photon imaging of calcium dynamics in meningeal macrophages in Pf4Cre:TIGRE2.0-GCaMP6 mice, the study identified heterogeneous features of perivascular and non-perivascular meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD). Analyses of calcium dynamics and blood vessels revealed a subpopulation of perivascular meningeal macrophages whose activity is coupled to behaviorally driven diameter fluctuations of their associated vessels. The analyses also investigated synchrony between different macrophage populations and revealed a role for CGRP/RAMP1 signaling in the CSD-induced increase, but not the decrease, in calcium transients.

This is a timely study at both the technical and conceptual levels, examining calcium dynamics of meningeal macrophages in vivo. The conclusions are well supported by the findings and will provide an important foundation for future research on immune cell dynamics within the meninges in vivo. The paper is well written and clearly presented.

I have only minor comments.

(1) Please indicate the formal definition of perivascular versus non-perivascular macrophages in terms of distance from the blood vessel. This information is not provided in the main text or the Methods. In addition, please explain how the meningeal vasculature was imaged in the main text.

(2) Similarly, the method used to induce acute CSD (pin prick) is not described in the main text and is only mentioned in the figure legends and Methods. Additional background on the neurobiology of acute CSD, as well as the resulting brain activity and neuroinflammatory responses, could be helpful.

Reviewer #3 (Public review):

Summary:

The authors of this report wish to show that distinct populations of meningeal macrophages respond to cortical spreading depolarization (CSD) via unique calcium activity patterns depending on their location in the meningeal sub-compartments. Perivascular macrophages display calcium signaling properties that are sometimes in opposition to non-perivascular macrophages. Many of the meningeal macrophages also displayed synchronous activity at variable distances from one another. Other macrophages were found to display calcium signals in response to dural vasomotion. CSD could induce variable calcium responses in both perivascular and non-perivascular macrophages in the meninges, in part due to RAMP1-dependent effects. Results will inform future research on the calcium responses displayed by macrophages in the meninges under both normal and pathological conditions.

Strengths:

Sophisticated in vivo imaging of meningeal immune cells is employed in the study, which has not been performed previously. A detailed analysis of the distinct calcium dynamics in various subtypes of meningeal macrophages is provided. Functional relevance of the responses is also noted in relation to CSD events.

Weaknesses:

The specificity of the methods used to target both meningeal macrophages and RAMP1 is limited. Additional discussion points on the functional relevance of the two subtypes of meningeal macrophages and their calcium responses are warranted. A section on potential pitfalls should be included.

Reviewer #1 (Public review):

Summary:

In this study, the authors' aim was to determine whether hepatic palmitoylation is a physiologically relevant regulator of systemic metabolism. The data demonstrate that loss of DHHC7 in hepatocytes disrupts Gαi palmitoylation, enhances cAMP-PKA-CREB signaling, and drives transcriptional upregulation and secretion of Prg4. The KO mice display increased body weight, fat mass, and plasma cholesterol, but at 12 weeks on HFD, do not exhibit insulin resistance. The potential mechanism underlying the metabolic phenotype was examined by assessing adipocyte signaling and by exploring whether Prg4 acts through GPR146. Through this pathway, the authors intend to link DHHC7-dependent palmitoylation to the regulation of hepatokines that exert systemic metabolic effects.

Strengths:

(1) Hepatic palmitoylation in systemic metabolic regulation is largely unexplored. The authors demonstrate the role of DHHC7 in vivo using a successful liver-specific knockout mouse model that causes HFD-dependent obesity without insulin resistance.

(2) Several studies were performed on chow and HFD, as well as male and female mice.

(3) Plasma proteomics identified Prg4 as a circulating factor elevated in KO mice. Prg4 overexpression phenocopied the KO mice.

(4) There is solid mechanistic data supporting the hypothesis that hepatic DHHC7 loss selectively increases Prg4 secretion as a hepatokine.

(5) There is convincing evidence for the DHHC7 mechanism in liver: DHHC7 controls cAMP-PKA-CREB via Gαi palmitoylation. The authors recognize that the palmitoylation change is causative rather than correlated, and this needs to be more fully explored in the future.

(6) Strong in vitro data support that Prg4 acts through adipocyte GPR146 via its SMB domain

Weaknesses:

(1) The assessment of liver and adipose tissue responses to DHH7 loss is insufficient to support claims that it alters systemic lipolysis. In this new mouse model, liver histology is necessary, especially given the cholesterol increase in the KO. As this is a newly established mouse line, common assessments of the liver during HFD feeding would be important for interpreting the phenotype.

(2) The data show DHH7 loss causes adipose tissue dysfunction and alterations in lipid metabolism. Beyond that, I suggest not stating more regarding the phenotype of the DHH7 mice for this work. A thorough analysis would be needed to determine which factor drives the obesity and changes in energy balance in the mice. For example, the KO mice had lower oxygen consumption (but no change in CO2 production, which is also usually similarly altered), suggesting a CNS component could drive obesity. However, since the data are not normalized for lean mass and there is no information about locomotor activity, this analysis is incomplete. RER may be informative if available. A broad conservative description of the KO phenotype would be more accurate since Pgr4 has many paracrine targets and likely has autocrine signaling in the liver.

(3) Most references to lipolysis or lipolysis flux systemically would be inaccurate. To suggest a suppression of lipolysis, serum NEFA would need to be measured, and in vivo or in vitro lipolysis assays performed to test the effect of DHH7 loss or the specificity of PGR4 action on adipocytes in vivo. To demonstrate adipose tissue dysfunction, analysis of lipogenesis markers, canonical markers for insulin sensitivity, and mitochondrial dysfunction should be performed/measured.

(4) Line 179: The experiment was performed in brown adipocytes to show that Prg4 does not affect p-CREB Figure S8 under the heading: "DHHC7 controls hepatic PKA-CREB activity through Gαi palmitoylation to regulate Prg4 transcription." Unless repeated using liver lysate, the conclusions stated in the text throughout the paper should be revised.

(5) It appears that the serum and liver proteomics were only assessed for factors that increased in KO mice? Were proteins that were significantly decreased analyzed?

(6) The beige adipocyte culture method is unclear. The methods do not describe the fat pad used, and the protocol suggests the cells would be differentiated into mature white adipocytes. If they are beige cells, a reference for the method, gene expression, and cell images could support that claim.

(7) The use of tamoxifen can confound adipocyte studies, as it increases beigeing and weight gain even after a brief initiation period. Both groups were treated with Tam, but another way to induce Cre would be ideal.

(8) Evidence for the lack of the glucose phenotype is incomplete. One reason could be due to the IP route of glucose administration, which has a large impact on glucose handling during a GTT. To confirm the absence of a glucose tolerance phenotype, an OGTT should be performed, as it is more physiological. In addition, the mice should be fed for 16 weeks. Prg4 affects immune cells, changing how adipose tissue expands, and 12 weeks of HFD feeding is often not long enough to see the effects of adipose tissue inflammation spilling over into the system.

(9) There may be liver-adipose tissue crosstalk in KO mice, but this was not fully assessed in this study and would be difficult to determine in any setting, given the diverse cell types that are targets of Pdg4. The crosstalk claim is unnecessary to share the basic premises; there is the DHH7 mechanism/phenotype and the Pgr4 mechanism/phenotype, and while there is no Pgr4 adipose direct mechanism, the paper can be successfully reframed.

(10) Although the DHH7 loss on the chow diet did not result in a phenotype, did the Pgr4 increase in the KO mice on chow? This would determine whether either i) the expression of Pgr4 is dependent on HFD/obesity, or ii) circulating Pgr4 has effects only in an HFD condition. The receptors may also change on HFD, especially in adipocytes.

Impact:

This work would significantly contribute to the study of liver metabolism, provided it includes data describing the liver. The role of Pgr4 in adipocytes and other cell types is of substantial value to the field of metabolism. By reframing the paper and conducting some key experiments, its quality and impact can be increased.

Reviewer #2 (Public review):

In the current report, Sun and Colleagues sought to determine the liver-specific role that DHHC7, a DHHC palmitoyltransferase protein, plays in regulating whole-body energy balance and hepatic crosstalk with adipose tissues. The authors generated an inducible, liver-specific DHHC7 knockout mouse to determine how altered palmitoylation in hepatocytes alters hepatokine production/secretion, and in turn, systemic metabolism. The ablation of DHHC7 was found to alter the production of proteoglycan 4 (Prg4), a hepatokine previously linked to metabolic regulation. The authors propose that the change in Prg4 production is mediated by the loss of Gαi palmitoylation, due to DHHC7 ablation, thereby augmenting cAMP-PKA-CREB signaling in hepatocytes, which alleviates the 'brake' on Prg4 production. The authors further propose that Prg4 overexpression leads to excessive binding to GPR146 on adipocytes, which in turn suppresses PKA-mediated HSL activation, promoting impairments in lipolysis, leading to obesity. The report is interesting and generally well-written, but it appears to have some clear gaps in additional data that would aid in interpretation. The addition of confirmatory culture studies would be incredibly helpful for testing the hypotheses being explored. My comments, concerns, and/or suggestions are outlined below in no particular order.

(1) Figures: All data should be presented in dot-boxplot format so the reader knows how many samples were analyzed for each assay and group. n=3 for some assays/experiments is incredibly low, particularly when considering the heterogeneity in responsiveness to HFD, food intake, etc....

(2) Figure 1E-F: It is unclear when the food intake measure was performed. Mice can alter their feeding behavior based on a myriad of environmental and biological cues. It would also be interesting to show food intake data normalized to body mass over time. Mice can counterregulate anorexigenic cues by altering neuropeptide production over time. It is not clear if this is occurring in these mice, but the timing of measuring food intake is important. Additionally, the VO2 measure appears to be presented as being normalized to total body mass, when in fact, it would probably be more accurate to normalize this to lean body mass. Normalizing to total body mass provides a denominator effect due to excessive adiposity, but white fat is not as metabolically active as other high-glucose-consuming tissues. If my memory serves me right, several reports have discussed appropriate normalizations in circumstances such as this.

(3) Figure 1J-N: It is not all that surprising that fasting glucose and/or TGs were found to be similar between groups. It is well-established that mice have an incredible ability to become hyperinsulinemic in an effort to maintain euglycemia and lipid metabolism dynamics. A few relatively easy assays can be performed to glean better insights into the metabolic status of the authors' model. First, fasting insulin concentrations will be incredibly helpful. Secondly, if the authors want to tease out which adipose depot is most adversely affected by ablation, they could take an additional set of CON and KO mice, fast them for 5-6 hours, provide a bolus injection of insulin (similar to that provided during an insulin tolerance test), and then quickly harvest the animals ~15 minutes after insulin injections; followed by evaluating AKT phosphorylation. This will really tell them if these issues have impairments in insulin signaling. The gold-standard approach would be to perform a hyperinsulinemic-euglyemic clamp in the CON and KO mice. I now see GTT and ITT data, but the aforementioned assays could help provide insight.

(4) Figure 3A: This looks overexposed to me.

(5) Figures 3-4: It appears that several of these assays could be complemented with culture-based models, which would almost certainly be cleaner. The conditioned media could then be used from hepatocyte cultures to treat differentiated adipocytes.

(6) Figure 4: It is unclear how to interpret the phospho-HSL data because the fasting state can affect this readout. It needs to be made clear how the harvest was done. Moreover, insulin and glucagon were never measured, and these hormones have a significant influence over HSL activity. I suspect the KO mice have established hyperinsulinemia, which would likely affect HSL activity. This provides an example of why performing some of these experiments in a dish would make for cleaner outcomes that are easier to interpret.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Sun et al aimed to determine the metabolic function of hepatocyte DHHC7, one of the key enzymes in protein palmitoylation. They generated inducible liver-specific Dhhc7 knockout mice and discovered that Dhhc7-LKO mice are more prone to gain weight and develop adipose expansion and obesity. Via unbiased proteomic analysis, they identified PRG4 as one of the top secreted factors in the liver of Dhhc7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice. At the mechanistic level, PRG4, once secreted from the liver, can bind to GPR146 on adipocytes and inhibit PKA-HSL signaling and lipolysis. Taken together, their findings suggest a novel pathway by which the liver communicates with adipose tissue and impacts systemic metabolism.

Strengths:

(1) The systemic metabolic homeostasis depends on coordination among metabolically active tissues. Thus, active communication between the liver and adipose tissue when facing nutritional challenges (such as high-fat diet feeding) is crucial for achieving metabolic health. The concept that the liver can communicate with adipose tissue and impact the lipolysis process via secreted hepatokines is quite significant but remains poorly understood.

(2) Hepatocyte Dhhc7 knockout mice developed a significant obesity phenotype, which is associated with adipose expansion.

(3) Unbiased proteomic analysis identified PRG4 as one of the top secreted factors in the liver of Dhh7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice.

(4) In vitro cell-based assay showed that PRG4 can bind to adipocyte GPR146, inhibit PKA-mediated HSL phosphorylation, and subsequently, the lipolysis process.

Weaknesses:

(1) Lack of a causal-effect study to generate evidence directly linking hepatocyte DHH7 and PRG4 in driving adipose expansion and obesity upon HFD feeding.

(2) Lack of direct evidence to support that PRG4 inhibits adipocyte lipolysis via GPR146. A functional assay demonstrating adipocyte lipolysis is required.

(3) The conclusion is largely based on the correlation evidence.

Reviewer #1 (Public review):

Summary:

This manuscript by Lin et al. presents a timely, technically strong study that builds patient-specific midbrain-like organoids (MLOs) from hiPSCs carrying clinically relevant GBA1 mutations (L444P/P415R and L444P/RecNcil). The authors comprehensively characterize nGD phenotypes (GCase deficiency, GluCer/GluSph accumulation, altered transcriptome, impaired dopaminergic differentiation), perform CRISPR correction to produce an isogenic line, and test three therapeutic modalities (SapC-DOPS-fGCase nanoparticles, AAV9-GBA1, and SRT with GZ452). The model and multi-arm therapeutic evaluation are important advances with clear translational value.

My overall recommendation is that the work undergo a major revision to address the experimental and interpretive gaps listed below.

Strengths:

(1) Human, patient-specific midbrain model: Use of clinically relevant compound heterozygous GBA1 alleles (L444P/P415R and L444P/RecNcil) makes the model highly relevant to human nGD and captures patient genetic context that mouse models often miss.

(2) Robust multi-level phenotyping: Biochemical (GCase activity), lipidomic (GluCer/GluSph by UHPLC-MS/MS), molecular (bulk RNA-seq), and histological (TH/FOXA2, LAMP1, LC3) characterization are thorough and complementary.

(3) Use of isogenic CRISPR correction: Generating an isogenic line (WT/P415R) and demonstrating partial rescue strengthens causal inference that the GBA1 mutation drives many observed phenotypes.

(4) Parallel therapeutic testing in the same human platform: Comparing enzyme delivery (SapC-DOPS-fGCase), gene therapy (AAV9-GBA1), and substrate reduction (GZ452) within the same MLO system is an elegant demonstration of the platform's utility for preclinical evaluation.

(5) Good methodological transparency: Detailed protocols for MLO generation, editing, lipidomics, and assays allow reproducibility

Weaknesses:

(1) Limited genetic and biological replication

(a) Single primary disease line for core mechanistic claims. Most mechanistic data derive from GD2-1260 (L444P/P415R); GD2-10-257 (L444P/RecNcil) appears mainly in therapeutic experiments. Relying primarily on one patient line risks conflating patient-specific variation with general nGD mechanisms.

(b) Unclear biological replicate strategy. It is not always explicit how many independent differentiations and organoid batches were used (biological replicates vs. technical fields of view).

(c) A significant disadvantage of employing brain organoids is the heterogeneity during induction and potential low reproducibility. In this study, it is unclear how many independent differentiation batches were evaluated and, for each test (for example, immunofluorescent stain and bulk RNA-seq), how many organoids from each group were used. Please add a statement accordingly and show replicates to verify consistency in the supplementary data.

(d) Isogenic correction is partial. The corrected line is WT/P415R (single-allele correction); residual P415R complicates the interpretation of "full" rescue and leaves open whether the remaining pathology is due to incomplete correction or clonal/epigenetic effects.

(e) The authors tested week 3, 4, 8, 15, and 28 old organoids in different settings. However, systematic markers of maturation should be analyzed, and different maturation stages should be compared, for example, comparing week 8 organoids to week 28 organoids, with immunofluorescent marker staining and bulk RNAseq.

(f) The manuscript frequently refers to Wnt signaling dysregulation as a major finding. However, experimental validation is limited to transcriptomic data. Functional tests, such as the use of Wnt agonist/inhibitor, are needed to support this claim (see below).

(g) Suggested fixes/experiments

Add at least one more independent disease hiPSC line (or show expanded analysis from GD2-10-257) for key mechanistic endpoints (lipid accumulation, transcriptomics, DA markers)

Generate and analyze a fully corrected isogenic WT/WT clone (or a P415R-only line) if feasible; at minimum, acknowledge this limitation more explicitly and soften claims.

Report and increase independent differentiations (N = biological replicates) and present per-differentiation summary statistics.

(2) Mechanistic validation is insufficient

(a) RNA-seq pathways (Wnt, mTOR, lysosome) are not functionally probed. The manuscript shows pathway enrichment and some protein markers (p-4E-BP1) but lacks perturbation/rescue experiments to link these pathways causally to the DA phenotype.

(b) Autophagy analysis lacks flux assays. LC3-II and LAMP1 are informative, but without flux assays (e.g., bafilomycin A1 or chloroquine), one cannot distinguish increased autophagosome formation from decreased clearance.

(c) Dopaminergic dysfunction is superficially assessed. Dopamine in the medium and TH protein are shown, but no neuronal electrophysiology, synaptic marker co-localization, or viability measures are provided to demonstrate functional recovery after therapy.

(d) Suggested fixes/experiments

Perform targeted functional assays:

(i) Wnt reporter assays (TOP/FOP flash) and/or treat organoids with Wnt agonists/antagonists to test whether Wnt modulation rescues DA differentiation.

(ii)Test mTOR pathway causality using mTOR inhibitors (e.g., rapamycin) or 4E-BP1 perturbation and assay effects on DA markers and autophagy.

Include autophagy flux assessment (LC3 turnover with bafilomycin), and measure cathepsin activity where relevant.

Add at least one functional neuronal readout: calcium imaging, MEA recordings, or synaptic marker quantification (e.g., SYN1, PSD95) together with TH colocalization.

(3) Therapeutic evaluation needs greater depth and standardization

(a) Short windows and limited durability data. SapC-DOPS and AAV9 experiments range from 48 hours to 3 weeks; longer follow-up is needed to assess durability and whether biochemical rescue translates into restored neuronal function.

(b) Dose-response and biodistribution are under-characterized. AAV injection sites/volumes are described, but transduction efficiency, vg copies per organoid, cell-type tropism quantification, and SapC-DOPS penetration/distribution are not rigorously quantified.

(c) Specificity controls are missing. For SapC-DOPS, inclusion of a non-functional enzyme control (or heat-inactivated fGCase) would rule out non-specific nanoparticle effects. For AAV, assessment of off-target expression and potential cytotoxicity is needed.

(d) Comparative efficacy lacking. It remains unclear which modality is most effective in the long term and in which cellular compartments.

(e) Suggested fixes/experiments

Extend follow-up (e.g., 6+ weeks) after AAV/SapC dosing and evaluate DA markers, electrophysiology, and lipid levels over time.

Quantify AAV transduction by qPCR for vector genomes and by cell-type quantification of GFP+ cells (neurons vs astrocytes vs progenitors).

Include SapC-DOPS control nanoparticles loaded with an inert protein and/or fluorescent cargo quantitation to show distribution and uptake kinetics.

Provide head-to-head comparative graphs (activity, lipid clearance, DA restoration, and durability) with statistical tests.

(4) Model limitations not fully accounted for in interpretation

(a) Absence of microglia and vasculature limits recapitulation of neuroinflammatory responses and drug penetration, both of which are important in nGD. These absences could explain incomplete phenotypic rescues and must be emphasized when drawing conclusions about therapeutic translation.

(b) Developmental vs degenerative phenotype conflation. Many phenotypes appear during differentiation (patterning defects). The manuscript sometimes interprets these as degenerative mechanisms; the distinction must be clarified.

(c) Suggested fixes

Tone down the language throughout (Abstract/Results/Discussion) to avoid overstatement that MLOs fully recapitulate nGD neuropathology.

Add plans or pilot data (if available) for microglia incorporation or vascularization to indicate how future work will address these gaps.

(5) Statistical and presentation issues

(a) Missing or unclear sample sizes (n). For organoid-level assays, report the number of organoids and the number of independent differentiations.

(b) Statistical assumptions not justified. Tests assume normality; where sample sizes are small, consider non-parametric tests and report exact p-values.

(c) Quantification scope. Many image quantifications appear to be from selected fields of view, which are then averaged across organoids and differentiations.

(d) RNA-seq QC and deposition. Provide mapping rates, batch correction details, and ensure the GEO accession is active. Include these in Methods/Supplement.

(e) Suggested fixes

Add a table summarizing biological replicates, technical replicates, and statistical tests used for each figure panel.

Recompute statistics where appropriate (non-parametric if N is small) and report effect sizes and confidence intervals.

(6) Minor comments and clarifications

(a) The authors should validate midbrain identity further with additional regional markers (EN1, OTX2) and show absence/low expression of forebrain markers (FOXG1) across replicates.

(b) Extracellular dopamine ELISA should be complemented with intracellular dopamine or TH+ neuron counts normalized per organoid or per total neurons.

(c) For CRISPR editing: the authors should report off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus.

(d) It should be clarified as to whether lipidomics normalization is to total protein per organoid or per cell, and include representative LC-MS chromatograms or method QC.

(e) Figure legends should be improved in order to state the number of organoids, the number of differentiations, and the exact statistical tests used (including multiple-comparison corrections).

(f) In the title, the authors state "reveal disease mechanisms", but the studies mainly exhibit functional changes. They should consider toning down the statement.

(7) Recommendations

This reviewer recommends a major revision. The manuscript presents substantial novelty and strong potential impact but requires additional experimental validation and clearer, more conservative interpretation. Key items to address are:

(a) Strengthening genetic and biological replication (additional lines or replicate differentiations).

(b) Adding functional mechanistic validation for major pathways (Wnt/mTOR/autophagy) and providing autophagy flux data.

(c) Including at least one neuronal functional readout (calcium imaging/MEA/patch) to demonstrate functional rescue.

(d) Deepening therapeutic characterization (dose, biodistribution, durability) and including specificity controls.

(e) Improving statistical reporting and explicitly stating biological replicate structure.

Reviewer #2 (Public review):

Sun et al. have developed a midbrain-like organoid (MLO) model for neuronopathic Gaucher disease (nGD). The MLOs recapitulate several features of nGD molecular pathology, including reduced GCase activity, sphingolipid accumulation, and impaired dopaminergic neuron development. They also characterize the transcriptome in the MLO nGD model. CRISPR correction of one of the GBA1 mutant alleles rescues most of the nGD molecular phenotypes. The MLO model was further deployed in proof-of-principle studies of investigational nGD therapies, including SapC-DOPS nanovesicles, AAV9-mediated GBA1 gene delivery, and substrate-reduction therapy (GZ452). This patient-specific 3D model provides a new platform for studying nGD mechanisms and accelerating therapy development. Overall, only modest weaknesses are noted.

Reviewer #3 (Public review):

Summary:

In this study, the authors describe modeling of neuronopathic Gaucher disease (nGD) using midbrain-like organoids (MLOs) derived from hiPSCs carrying GBA1 L444P/P415R or L444P/RecNciI variants. These MLOs recapitulate several disease features, including GCase deficiency, reduced enzymatic activity, lipid substrate accumulation, and impaired dopaminergic neuron differentiation. Correction of the GBA1 L444P variant restored GCase activity, normalized lipid metabolism, and rescued dopaminergic neuronal defects, confirming its pathogenic role in the MLO model. The authors further leveraged this system to evaluate therapeutic strategies, including: (i) SapC-DOPS nanovesicles for GCase delivery, (ii) AAV9-mediated GBA1 gene therapy, and (iii) GZ452, a glucosylceramide synthase inhibitor. These treatments reduced lipid accumulation and ameliorated autophagic, lysosomal, and neurodevelopmental abnormalities.

Strengths:

This manuscript demonstrates that nGD patient-derived MLOs can serve as an additional platform for investigating nGD mechanisms and advancing therapeutic development.

Comments:

(1) It is interesting that GBA1 L444P/P415R MLOs show defects in midbrain patterning and dopaminergic neuron differentiation (Figure 3). One might wonder whether these abnormalities are specific to the combination of L444P and P415R variants or represent a general consequence of GBA1 loss. Do GBA1 L444P/RecNciI (GD2-10-257) MLOs also exhibit similar defects?

(2) In Supplementary Figure 3, the authors examined GCase localization in SapC-DOPS-fGCase-treated nGD MLOs. These data indicate that GCase is delivered to TH⁺ neurons, GFAP⁺ glia, and various other unidentified cell types. In fruit flies, the GBA1 ortholog, Gba1b, is only expressed in glia (PMID: 35857503; 35961319). Neuronally produced GluCer is transferred to glia for GBA1-mediated degradation. These findings raise an important question: in wild-type MLOs, which cell type(s) normally express GBA1? Are they dopaminergic neurons, astrocytes, or other cell types?

(3) The authors may consider switching Figures 2 and 3 so that the differentiation defects observed in nGD MLOs (Figure 3) are presented before the analysis of other phenotypic abnormalities, including the various transcriptional changes (Figure 2).

Reviewer #1 (Public review):

Summary:

This study uses data from a recent RVFV serosurvey among transhumant cattle in The Gambia to inform the development of an RVFV transmission model. The model incorporates several hypotheses that capture the seasonal nature of both vector-borne RVFV transmission and cattle migration. These natural phenomena are driven by contrasting wet and dry seasons in The Gambia's two main ecoregions and are purported to drive cyclical source-sink transmission dynamics. Although the Sahel is hypothesized to be unsuitable for year-long RVFV transmission, findings suggest that cattle returning from the Gambia River to the Sahel at the beginning of the wet season could drive repeated RVFV introductions and ensuing seasonal outbreaks. Upon review, the authors have removed an additional analysis evaluating the potential impacts of cattle movement bans on transmission dynamics, which was poorly supported by the methodological approach.

Strengths:

Like most infectious diseases in animal systems in low- and middle-income countries, the transmission dynamics of RVFV in cattle in The Gambia are poorly understood. This study harnesses important data on RVFV seroepidemiology to develop and parameterize a novel transmission model, providing plausible estimates of several epidemiological parameters and transmission dynamic patterns.

This study is well written and easy to follow.

The authors consider both deterministic and stochastic formulations of their model, demonstrating potential impacts of random events (e.g. extinctions) and providing confidence regarding model robustness.

The authors use well-established Bayesian estimation techniques for model fitting and confront their transmission model with a seroepidemiological model to assess model fit.

Elasticity analyses help to understand the relative importance of competing demographic and epidemiological drivers of transmission in this system.

Weaknesses:

The model does not include an impact of infection on cattle birth rates, but the authors justify that this parameter should have limited impact on dynamics given predicted low-level circulation patterns, as opposed to explosive outbreaks, in this region.

The importance of the LVFV positivity decay rate is highlighted but loss of immunity is not considered in the SIR model. The authors do discuss uncertainty regarding model structure and a need for future data collection to begin to answer this question.

The model's structure, including homogenous mixing within each ecoregion and step-change seasonality, allows for estimation of generalized transmission rates at a macro scale. However, it greatly simplifies the movement process itself and assumes that transhumant cattle movement is the only mechanism for RVF reintroduction into the Sahel region. The authors discuss that integration of more finely-scaled movement and contact data may help to address this limitation in future work.

This model seems well-suited to be exploited in future work to explore for e.g. impacts of cattle vaccination, and potential differential efficiency when targeting T herds relative to M or L.

Comments on revisions:

I thank the authors for thoughtfully and thoroughly addressing my concerns. I have no further comments.

Reviewer #1 (Public review):

Summary:

In this work by Mohite et al., they have used transcriptomic and metabolic profiling of H. armigera, muscle development, and S. frugiperda to link energy trehalose metabolism and muscle development. They further used several different bioinformatics tools for network analysis to converge upon transcriptional control as a potential mechanism of metabolite-regulated transcriptional programming for muscle development. The authors have also done rescue experiments where trehalose was provided externally by feeding, which rescues the phenotype. Though the study is exciting, there are several concerns and gaps that lead to the current results as purely speculative. It is difficult to perform any genetic experiments in non-model insects; the authors seem to suggest a similar mechanism could also be applicable in systems like Drosophila; it might be possible to perform experiments to fill some missing mechanistic details.

A few specific comments below:

The authors used N-(phenylthio) phthalimide (NPP), a trehalose-6-phosphate phosphatase (TPP) inhibitor. They also find several genes, including enzymes of trehalose metabolism, that change. Further, several myogenic genes are downregulated in bulk RNA sequencing. The major caveat of this experiment is that the NPP treatment leads to reduced muscle development, and so the proportion of the samples from the muscles in bulk RNA sequencing will be relatively lower, which might have led to the results. So, a confirmatory experiment has to be performed where the muscle tissues are dissected and sequenced, or some of the interesting targets could be validated by qRT-PCR. Further to overcome the off-target effects of NPP, trehalose rescue experiments could be useful.

Even the reduction in the levels of ADP, NAD, NADH, and NMN, all of which are essential for efficient energy production and utilization, could be due to the loss of muscles, which perform predominantly metabolic functions due to their mitochondria-rich environment. So it becomes difficult to judge if the levels of these energy molecules' reduction are due to a cause or effect.

The authors have used this transcriptomic data for pathway enrichment analysis, which led to the E2F family of transcription factors and a reduction in the level of when trehalose metabolism is perturbed. EMSA experiments, though, confirm a possibility of the E2F interaction with the HaTPS/TPP promoter, but it lacks proper controls and competition to test the actual specificity of this interaction. Several transcription factors have DNA-binding domains and could bind any given DNA weakly, and the specificity is ideally known only from competitive and non-competitive inhibition studies.

The work seems to have connected the trehalose metabolism with gene expression changes, though this is an interesting idea, there are no experiments that are conclusive in the current version of the manuscript. If the authors can search for domains in the E2F family of transcription factors that can bind to the metabolite, then, if not, a chip-seq is essential to conclusively suggest the role of E2F in regulating gene expression tuned by the metabolites.

Some of the above concerns are partially addressed in experiments where silencing of E2F/Dp shows similar phenotypes as with NPP and dsRNA. It is also notable that silencing any key transcription factor can have several indirect effects, and delayed pupation and lethality could not be definitely linked to trehalose-dependent regulation.

Trehalose rescue experiments that rescue phenotype and gene expression are interesting. But is it possible that the fed trehalose is metabolized in the gut and might not reach the target tissue? In which case, the role of trehalose in directly regulating transcription factors becomes questionable. So, a confirmatory experiment is needed to demonstrate that the fed trehalose reaches the target tissues. This could possibly be done by measuring the trehalose levels in muscles post-rescue feeding. Also, rescue experiments need to be done with appropriate control sugars.

No experiments are performed with non-target control dsRNA. All the experiments are done with an empty vector. But an appropriate control should be a non-target control.

Reviewer #2 (Public review):

Summary:

This study shows that the knockdown of the effects of TPS/TPP in Helicoverpa armigera and Spodoptera frugiperda can be rescued by trehalose treatment. This suggests that trehalose metabolism is necessary for development in the tissues that NPP and dsRNA can reach.

Strengths:

This study examines an important metabolic process beyond model organisms, providing a new perspective on our understanding of species-specific metabolism equilibria, whether conserved or divergent.

Weaknesses:

While the effects observed may be truly conserved across Lepidopterans and may be muscle-specific, the study largely relies on one species and perturbation methods that are not muscle-specific. The technical limitations arising from investigations outside model systems, where solid methods are available, limit the specificity of inferences that may be drawn from the data.

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

Finally, I would add that trehaolse metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

Reviewer #1 (Public review):

Summary:

This study evaluates the feasibility of using crispant founder mice, first-generation animals directly edited by CRISPR/Cas9, for initial phenotypic assessments. The authors target seven genes known to produce visible recessive traits to test whether mosaicism in founder animals prevents meaningful phenotype-genotype interpretation. Remarkably, they observe clear null phenotypes in founders for six of the seven genes, with high editing efficiencies. These results demonstrate that crispant mice can, under specific conditions, display recessive phenotypes that are readily interpretable. However, this conclusion should be moderated, as the study addresses only one biological context, visible Mendelian traits, and may not generalize to quantitative, subtle, or late-onset phenotypes. The report also examines attempts at multiplex CRISPR targeting, which reduce viability, underscoring limits for concurrent gene disruptions. Finally, the detailed description of diverse alleles generated by CRISPR provides valuable insight into how allelic series can be exploited to investigate protein function.

Strengths:

(1) The manuscript provides a comprehensive and technically rigorous description of CRISPR/Cas9‑induced mutations across several loci. The accompanying genotyping, sequencing, and analytical approaches are sound, complementary, and well-detailed, providing a resource that will be valuable to researchers using genome editing beyond the specific application of genetic screening.

(2) By documenting a wide diversity of alleles and mutation types, the study contributes to understanding how allelic series generated by CRISPR can be leveraged for dissecting protein function, a perspective that has been less systematically presented in prior literature and could be compared to targeted strategies such as those described by Cassidy et al. (2022, DOI: [10.1016/bs.mie.2022.03.053]) or other relevant studies addressing CRISPR-based allelic series generation.

(3) The work demonstrates technically solid editing and validation workflows, setting a methodological reference point for similar projects across species or trait categories.

Weaknesses:

(1) There is a disconnect between the abstract/introduction and the discussion. While both the abstract and introduction focus on the potential use of crispant founders for phenotypic assessment in the context of genetic screening, with the introduction notably emphasizing this framework through a detailed section on ENU-based screens, the discussion devotes relatively little attention to this aspect. Instead, it primarily examines CRISPR mutagenesis outcomes, mutation detection, and allele characterization. Overall, the study's aims are not clearly or explicitly defined, which contributes to the lack of alignment across sections.

(2) Important limitations of the approach are not sufficiently discussed. For instance, the paper does not address how applicable the findings are beyond visible Mendelian traits, such as for quantitative, late-onset, or more subtle phenotypes, including behavioral ones, or how to interpret wild-type appearing founders. There is little consideration of appropriate experimental controls (e.g., wild-type or mock-edited animals) or of how many animals might be required to robustly establish genotype-phenotype associations.

(3) The conclusion that this strategy will "dramatically reduce time, resources, and animal numbers" is not quantitatively supported by the data presented and should be expressed more cautiously.

Reviewer #2 (Public review):

Summary:

The authors sought to validate the use of genetic screening pipelines that assess phenotypes in founders (F0, referred to as "crispants") obtained from CRISPR/Cas9 gene editing in 1-cell zygotes. The application of this approach in mice has generally been avoided due to concerns that results would be confounded by genetic mosaicism, but benefits to this approach include reducing animal numbers needed to achieve goals of identifying knockout phenotypes, as well as improved efficiency in the use of time and resources. The authors targeted seven genes associated with visible recessive phenotypes and observed the expected null phenotype in up to 100% of founders for each gene. Although mosaicism was common in the crispants, the various alleles were generally all functional null alleles and, in fact, some in-frame deletions with null phenotypes revealed critical functional motifs within the gene products. The rigorous data presented support using crispants to assess knockout phenotypes when guide RNAs with strong on-target and low off-target scores are used for gene editing in 1-cell mouse embryos.

Strengths:

By targeting multiple genes with existing, well-characterized mutations, the authors established a robust system for validating the analysis of crispants to assess gene function.

Cutting-edge technologies were used to carefully assess the spectrum of mutations generated.

Weaknesses:

There could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).

Reviewer #3 (Public review):

Summary:

The study assesses whether CRISPR-generated founder (F0) "crispant" mice can be reliably used for initial phenotypic assessment and screening. By targeting seven genes with known visible recessive phenotypes, the authors show that, despite genetic mosaicism, the expected null phenotypes were observed in all targeted genes. These findings demonstrate that the phenotyping and screening of F0 "crispant" mice is a valid (and efficient) approach to selecting candidate alleles for follow-up studies, thereby streamlining mouse breeding and animal husbandry-related costs.

Strengths:

The study is comprehensive, carefully executed, and provides deep insight into the utility of F0 "crispant" mice for phenotypic screening. The authors evaluated the CRISPR/Cas9 editing outcomes across seven genes using multiple sequencing modalities, providing a robust framework for determining and interpreting complex founder genotypes. Importantly, the study examines/highlights the biological insight gained from compound heterozygous founders and naturally arising allelic series, enabling genotype-phenotype associations and functional inferences about protein domains.

More broadly, the authors' thorough evaluation of the CRISPR/Cas9-based gene editing events in the founders can serve as a benchmark for others in the field, engineering their own mouse "crispants."

Weaknesses:

The relationship between the sgRNA/Cas9 concentrations delivered to the zygotes and the resulting editing efficiencies are not explicitly investigated.

Reviewer #1 (Public review):

Summary:

Alveolar macrophages (AMs) are key sentinel cells in the lungs, representing the first line of defense against infections. There is growing interest within the scientific community in the metabolic and epigenetic reprogramming of innate immune cells following an initial stress, which alters their response upon exposure to a heterologous challenge. In this study, the authors show that exposure to extracellular ATP can shape AM functions by activating the P2X7 receptor. This activation triggers the relocation of the potassium channel TWIK2 to the cell surface, placing macrophages in a heightened state of responsiveness. This leads to the activation of the NLRP3 inflammasome and, upon bacterial internalization, to the translocation of TWIK2 to the phagosomal membrane, enhancing bacterial killing through pH modulation. Through these findings, the authors propose a mechanism by which ATP acts as a danger signal to boost the antimicrobial capacity of AMs.

Strengths:

This is a fundamental study in a field of great interest to the scientific community. A growing body of evidence has highlighted the importance of metabolic and epigenetic reprogramming in innate immune cells, which can have long-term effects on their responses to various inflammatory contexts. Exploring the role of ATP in this process represents an important and timely question in basic research. The study combines both in vitro and in vivo investigations and proposes a mechanistic hypothesis to explain the observed phenotype.

Weaknesses:

The authors have revised the manuscript to address the comments raised during the first round of review. However, several figures, figure legends, and methodological sections still require additional adjustments and clarification.

The interpretation of CFU from lysates as 'killing' is unclear; lysate CFUs typically reflect intracellular surviving bacteria and are confounded by differences in uptake. Please include an uptake control (early time point) or time-course to distinguish phagocytosis from intracellular killing. Also, clarify how bacterial burden was calculated (supernatant vs cell-associated vs total). Supernatant alone may not capture adherent bacteria. The normalization as 'fold killing' (mean negative control / sample) is non-standard; please report absolute CFU (log scale) and specify the exact definition of killing/survival.

The Methods section is largely incomplete and requires substantial revision. For instance, the authors report quantification of cytokine concentrations, yet no information is provided regarding how these measurements were performed. It is unclear whether cytokines were measured in BALF by ELISA, or assessed at the mRNA level by qPCR from total lung lysates, or by another method. This information must be clearly specified. In addition, the rationale for selecting the measured cytokines should be justified. While the choice of IL-1β and IL-6 is relatively straightforward, the focus on IL-18 requires explicit justification.

Similarly, the methodology used to quantify immune cell populations presented in Figure 2 is not described. It is not stated how immune cells were isolated and identified (e.g. flow cytometry from lung tissue). No information is provided regarding tissue digestion, cell isolation procedures, or gating strategy (presumably by flow cytometry). These details are essential and should be included, together with the corresponding gating strategy and absolute cell numbers.

Moreover, immune cell quantification would be expected in the context of the challenge experiment as well. Reporting unchanged percentages of lung immune cells following ATP exposure does not support the conclusion of a training effect, particularly one that is specific to alveolar macrophages (AMs). In addition, AMs are not considered recruited immune cells; this should be corrected in the figure legend and throughout the manuscript where applicable.

There are inconsistencies throughout the manuscript. For example, the authors report n = 5 for the survival curves in the figure legend, whereas n = 7 is stated in the Methods section. This discrepancy is unclear and should be clarified.

Supplementary Fig. 1 contains major conceptual errors. The volcano plot represents ATAC-seq peaks (differentially accessible chromatin regions), yet the figure, legend, and color scale repeatedly refer to 'genes' and 'differentially expressed genes'. This conflates chromatin accessibility with gene expression and is misleading. Peaks are secondarily annotated to nearby genes, which should be clearly described as an annotation step rather than the unit of analysis. The figure should be revised to explicitly present peak-level statistics (DARs), with gene names shown only as optional annotations. Additionally, the use of simultaneous P < 0.05 and Q < 0.05 thresholds is non-standard, and the absence of down-regulated regions in the plot requires explanation.

In Figure 7, trained WT and Nlrp3⁻/⁻ mice display similar levels of bacterial clearance. How should this result be interpreted?

Overall, while the study addresses an interesting biological question, the manuscript would benefit from substantial revision prior to publication. In particular, clarifications and improvements regarding the methodology, data presentation, and interpretation are required to strengthen the rigor and reproducibility of the conclusions.

Reviewer #2 (Public review):

Summary:

In this manuscript, Thompson et al. investigate the impact of prior ATP exposure on later macrophage functions as a mechanism of immune training. They describe that ATP training enhances bactericidal functions which they connect to the P2x7 ATP receptor, Nlrp3 inflammasome activation, and TWIK2 K+ movement at the cell surface and subsequently at phagosomes during bacterial engulfment. This is an incremental addition to existing literature, which has previously explored how ATP alters TWIK2 and K+, and linked it to Nlrp3 activation. The novelty here is in discovering the persistence of TWIK2 change and exploring the impact this biology may have on bacterial clearance. Additional experiments could strengthen their hypothesis that the in vivo protective effect of ATP-training on bacterial clearance is mediated by alveolar macrophages.

Strengths:

The authors demonstrate three novel findings: 1) prolonged persistence of TWIK2 at the macrophage plasma membrane following ATP that can translocate to the phagosome during particle engulfment, 2) a persistent impact of ATP exposure on remodeling chromatin around nlrp3, and 3) administering mice intra-nasal ATP to 'train' lungs protects mice from otherwise fatal bacterial infection.

Weaknesses:

(1) Some methods remain unclear including the timing and method by which lung cellularity was assessed in Figure 2. It is also difficult to understand how many mice were used in experiments 1, 2 and 6 and thus how rigorous the design was. A specific number is only provided for 1D and the number of mice stated in legend and methods do not match.

(2) The study design is not entirely ideal for the authors' in vivo question. Overall, the discussion would benefit from a clear summary of study caveats, which are primarily that that 1) in vitro studies attributing ATP training-mediated bacterial killing to persistent TWIK2 relocation, K+ influx, a glycolytic metabolic shift , and epigenetic nlrp3 reprogramming were performed in BMDM or RAW cells and not primary AMs, 2) data does not eliminate the possibility that non-AM immune or non-immune cells in the lung are "trained" and responsible for ATP-mediated protection in vivo; flow data examined total lung digest which may obscure important changes in alveolar recruitment, and 3) in vivo work shows data on acute bacterial clearance but does not explore potential risks that "training" for a more responsive inflammasome may have for the severity of lung injury during infection.

(3) The is some lack of transparency on data and rigor of methods. Clear data is not provided regarding the RNA-sequencing results. Specific identities of DEGs is not provided, only one high-level pathway enrichment figure. It would also be ideal if controls were included for subcellular fractionating to confirm pure fractions and for dye microscopy to show negative background.

(4) In results describing 5A, the text states that "ATP-induced macrophage training effects, as measured by augmented bactericidal activity, were diminished in macrophages treated with protease inhibitors". However, these data are not identified significant in the figure; protease dependence can be described as a trend that supports the authors' hypothesis but should not be stated as significant data in text.

In summary, this work contains some useful data showing how ATP can train macrophages via TWIK2/Nlrp3. Revisions have significantly improved methods reporting, added some data to strengthen the conclusions, and toned down on overstatements to bring conclusions more in line with data presented. The title still overstates what the authors have actually tested, since no macrophage-specific targeting in vivo (no conditional gene deletion, macrophage depletion etc) was performed in infection studies. However, in vitro data provide clear evidence that macrophages can be trained by ATP, and through caveats remain, it is plausible that macrophage training is a key mechanism for the protection observed here in the lung.

Reviewer #1 (Public review):

Summary

The authors set out to define the genomic distribution and potential functional associations of acetylation of histone H3 lysine 115 (H3K115ac), a poorly characterized modification located in the globular domain of histone H3. Using native ChIP-seq and complementary genomic approaches in mouse embryonic stem cells and during differentiation to neural progenitor cells, they report that H3K115ac is enriched at CpG island promoters, active enhancers, and CTCF binding sites, where it preferentially localizes to regions containing fragile or subnucleosomal particles. These observations suggest that H3K115ac marks destabilized nucleosomes at key regulatory elements and may serve as an informative indicator of chromatin accessibility and regulatory activity.

Strengths

A major strength of this study is its focus on a histone post-translational modification in the globular domain, an area that has received far less attention than histone tail modifications despite strong evidence from structural and in vitro studies that such marks can directly influence nucleosome stability. The authors employ a wide range of complementary genomic analyses-including paired-end ChIP-seq, fragment size-resolved analyses, contour (V-) plots, and sucrose gradient fractionation-to consistently support the association of H3K115ac with fragile nucleosomes across promoters, enhancers, and architectural elements. The revised manuscript is careful in its interpretation and provides a coherent and internally consistent picture of how H3K115ac differs from other acetylation marks such as H3K27ac and H3K122ac. The datasets generated will be valuable to the chromatin community as a resource for further exploration of nucleosome dynamics at regulatory elements.

Weaknesses

The conclusions are largely correlative. While the authors provide strong evidence for the localization of H3K115ac to fragile nucleosomes, the work does not directly test whether this modification causally contributes to nucleosome destabilization or regulatory function in vivo. Questions regarding the enzymes responsible for depositing or removing H3K115ac and its direct functional consequences therefore remain open.

Overall assessment and impact

Overall, the authors largely achieve their stated aims by providing a detailed and well-supported characterization of H3K115ac distribution in mammalian chromatin and its association with fragile nucleosomes at regulatory elements. While mechanistic insight remains to be established, the study introduces a compelling new perspective on globular-domain histone acetylation and highlights H3K115ac as a potentially useful marker for identifying functionally important regulatory regions. The work is likely to stimulate further mechanistic studies and will be of broad interest to researchers studying chromatin structure, transcriptional regulation, and genome organization.

Reviewer #2 (Public review):

Summary:

Kumar et al. aimed to assess the role of the understudied H3K115 acetylation mark, which is located in the nucleosomal core. To this end, the authors performed ChIP-seq experiments of H3K115ac in mouse embryonic stem cells as well as during differentiation into neuronal progenitor cells. Subsequent bioinformatic analyses revealed an association of H3K115ac with fragile nucleosomes at CpG island promoters, as well as with enhancers and CTCF binding sites. This is an interesting study, which provides important novel insights into the potential function of H3K115ac. However, the study is mainly descriptive, and functional experiments are missing.

Strengths:

(1) The authors present the first genome-wide profiling of H3K115ac and link this poorly characterized modification to fragile nucleosomes, CpG island promoters, enhancers, and CTCF binding sites.

(2) The study provides a valuable descriptive resource and raises intriguing hypotheses about the role of H3K115ac in chromatin regulation.

(3) The breadth of the bioinformatic analyses adds to the value of the dataset

Comments on revisions:

The authors sufficiently addressed my concerns.

Reviewer #3 (Public review):

Summary:

Kumar et al. examine the H3K115 epigenetic mark located on the lateral surface of the histone core domain and present evidence that it may serve as a marker enriched at transcription start sites (TSSs) of active CpG island promoters and at polycomb-repressed promoters. They also note enrichment of the H3K115ac mark is found on fragile nucleosomes within nucleosome-depleted regions, on active enhancers and CTCF bound sites. They propose that these observations suggest that H3K115ac contributes to nucleosome destabilization and so may servers a marker of functionally important regulatory elements in mammalian genomes.

Strengths:

The authors present novel observations suggesting that acetylation of a histone residue in a core (versus on a histone tail) domain may serve a functional role in promoting transcription in CPG islands and polycomb-repressed promoters. They present a solid amount of confirmatory in silico data using appropriate methodology that supports the idea that H3K115ac mark may function to destabilize nucleosomes and contribute to regulating ESC differentiation. These findings are quite novel.

Weaknesses:

Additional experiments to confirm specificity of the antibodies used have been done, improving confidence in the study.

Reviewer #1 (Public Review):

This study by Ryu et al, provides compelling evidence to demonstrate the distributions of Oxt and Oxtr in the murine brain using an advanced RNAscope technique. Detailed information on the distributions was provided, revealing differences in Oxt and Oxtr expressions between males and females. This study will provide a new platform for investigators to study previously unknown roles of brain-region specific Oxt and Oxtr neurons and signaling in animal behaviors and metabolism, and others.

Reviewer #2 (Public Review):

This an exciting study investigating the role of OXT in central nervous system (CNS) regulation of different behaviors and physiological processes. The study clearly shows the expression level of Oxt and Oxtr in different brain nuclei and regions.

Sex differences in Oxt expression are also well demonstrated.

Extensions of OXT's function in CNS regulation are sufficiently discussed.

Overall, this provides a good direction for further investigate OXT's role in CNS's regulation on different behaviors and physiological processes.

Reviewer #1 (Public Review):

This study extends a previous study by the same group on the generalization of odor discrimination from one nostril to the other. In their earlier study, the group showed that learning to discriminate between two enantiomers does not generalize across nostrils. This was surprising given the Mainland & Sobel 2001 study that found that detecting androstenone in people who do not detect it can generalize across the two nostrils. In this study, they confirmed their previous results and reported that, unlike enantiomers, learning to discriminate odor mixtures generalizes across nostrils, generalizes to other odor mixtures, and is persistent over at least two weeks. This interesting and important result extends our knowledge of this phenomenon and will likely steer more research. It may also help develop new training protocols for people with impairments in their sense of smell.

The main weakness of this study is its scope, as it does not provide substantial insight into why the results differ for enantiomers and why training on odor mixtures generalizes to other odor mixtures.

Reviewer #2 (Public Review):

The manuscript from Chang et al. taps on an important issue in olfactory perceptual plasticity, named the generalization of perceptual learning effect by training using odors. They employed a discrimination training/learning task with either binary odor mixture or odor enantiomers, and tested for post-training effect at several time intervals. Their results showed contrasting patterns of specificity (enantiomers) and transfer (odor mixtures), and the learning effect persisted at 2 weeks post-training. They demonstrated that the effect was independent of task difficulty, olfactory adaptation and gender.

Overall this was a well-controlled study and shows novel results. The strength of the study includes the consideration of odor structure and perceptual (dis)similarity and the control training condition. I have two minor issues that hope the authors could address in the next version of the manuscript.

1) The author used a binary odor mixture with a ration 7:9 or 9:11, why is this ratio chosen and used for the experiment?

2) Over the course of training, has the valence of odor (odor mixture) changed, it would be helpful to include these results in the supplements. As the author indicated in the discussion, the potential site underlying the transfer effect is the OFC, which has been found to represent odor valence previously (Anderson, Christoff et al. 2003). It would be nice to see the author replicate the results with odor/odor mixture valence (change) controlled.

Anderson, A. K., K. Christoff, I. Stappen, D. Panitz, D. G. Ghahremani, G. Glover, J. D. Gabrieli and N. Sobel (2003). "Dissociated neural representations of intensity and valence in human olfaction." Nat Neurosci 6(2): 196-202.

Reviewer #1 (Public Review):

The authors tested the hypothesis that at high elevations avian eggs will be adapted to prevent desiccation that might arise from loss of water to surrounding drier air. They used a combination of gas diffusion experiments and scanning electron microscopy to examine water vapour conductance rates and eggshell structure, including thickness, pore size, and pore density among 197 bird species distributed along an elevational gradient in the Andes. While there was a correlation between water vapour conductance and elevation among species, a decrease in water vapour conductance with elevation was not associated with eggshell thickness, pore size, and pore density, suggesting the variation in the structure of the eggshells is unlikely to do with among species differences in water loss along elevational gradients. This study is very interesting and timely, especially with increasing water vapour pressure due to climate warming. It is a very well-written study and easy to read. However, I have some concerns about the conclusions drawn from the results.

There are more than twice as many species in low and medium-elevation sites compared to high-elevation sites, so the amount of variation in low and medium-elevation should be expected to be higher by default. The argument for a wider range of variation in low-elevation species will be stronger if the comparison was a similar sample size. Moreover, the pattern clearly breaks down within families. Note also that for Low and medium elevation there is no difference in the amount of variation in conductance residuals possibly because the sample sizes are similar. The seemingly strong positive correlation between eggshell conductance and egg mass may be driven by the five high and two medium-elevation species with large eggs. There seem to be hardly any high-elevation species with egg mass greater than 12g whereas species in low elevation egg size seem to be as high as 80g (Figure 2a). Since larger eggs (and thus eggs of larger birds) lose more water compared to smaller eggs, the correlation between water vapour conductance and elevation may be more strongly associated with body size distribution along elevational gradients rather than egg structure and function.

Authors argue that the observed variation in the relationship between water vapour conductance and elevation among and within bird families suggests potential differences in the adaptive response to common selective pressures in terms of eggshell thickness and pore density, and size. The evidence for this is generally weak from the data analyses because the decrease in water vapour conductance with elevation was not consistent across taxonomic groups nor were differences associated with specific patterns in eggshell thickness and pore density, and size.

It is not clear how the authors expected the relationship between water vapour conductance and elevation to differ among taxonomic groups and there was no attempt to explain the biological implication of these differences among taxonomic groups based on the specific traits of the species or their families. This missing piece of information is crucial to justify the argument that differences among taxonomic groups may be due to differences in adaptive response.

Reviewer #2 (Public Review):

Many tropical montane species live only within narrow elevational ranges. Rapid climate change has led to considerable interest in determining whether these narrow elevational ranges are the result of physiological specialization: if so, then warming temperatures will have direct fitness consequences. Thus far, studies of tropical montane ectotherms have often found patterns consistent with physiological specialization, while the few field studies of tropical montane birds (endotherms) have not. However, these few studies measured the thermal physiology of adult birds. The early life stages of birds may show physiological specialization, as eggs and nestlings function as ectotherms.

In this paper, Ocampo and colleagues provide the first test of the hypothesis that bird eggs are physiologically specialized to the climatic conditions of certain elevational zones. They use experiments and observations to measure water vapor conductance rates and eggshell traits in a diverse set of 197 species that live from the lowland Amazon to the high Andes. Ocampo and colleagues present two principal results: (1) High-elevation eggs lose less water over time than do low-elevation eggs, high elevations tend to be less humid than low elevations and (2) Eggshell traits do not show consistent patterns along the elevational gradient. The pattern in water loss is consistent with the hypothesis that high-elevation eggs are physiologically specialized for the slightly drier environments they experience. The finding that eggshell traits did not vary with elevation, however, means that the pattern of water loss is not driven by single eggshell traits (thicker eggshells could reduce water loss rates, as could fewer or smaller eggshell pores).

This paper represents a strong advance for two main reasons. First, it provides evidence that egg physiology varies with elevation as predicted by the hypothesis that eggs are physiologically adapted to certain climatic conditions. This means egg physiological adaptation is a factor that could influence species' elevational ranges. Second, it is a proof-of-concept study that shows it is possible to measure eggshell physiology for a large number of species in the field in order to test hypotheses. As such, it should inspire many further tests that examine adaptation in egg physiology in the context of species' distributions along environmental gradients.

There are two caveats that readers should be aware of. First, measuring these traits is difficult, and there remain questions about the efficacy of different methods. For example, the authors note that quantifying eggshell structures is very difficult, with several unresolved questions about their method of using scanning electron microscopy images to measure eggshell pores. Similarly, the authors mention that temperature variation may partially influence their main result that high-elevation eggs lose water at slower rates than low-elevation eggs (temperatures were colder for experiments at high elevations than for low elevations). Second, I regard the analyses of eggshell traits for specific families as exploratory. There are no a priori expectations for how different families might be expected to differ in their patterns. These analyses are fruitful in that they generate additional hypotheses that future work can test. However, it does mean that the statistical significance of eggshell trait relationships with elevation for specific families should be interpreted with caution.

Reviewer #1 (Public Review):

This study aims to develop a new system to analyze genetic determinants of neutrophil function by large-scale genetic screens. For that, the authors use a genetically-engineered ER-Hoxb8 neutrophil progenitor line that expresses Cas9 to perform CRISPR screens and to identify genes required for neutrophil survival and differentiation.

A main strength of this study is that the authors develop a pooled CRISPR sgRNA library applicable to neutrophils and show potential determinants of neutrophil differentiation in vitro using this screening methodology.

A main weakness of this study is that identified candidates associated with neutrophil differentiation, as those indicated in Fig. 4A, were not validated in vivo using neutrophil-specific K.O. models or further characterized in vitro (e.g. transcriptional or epigenetic changes during maturation when compared to non-targeting sgRNA controls).

As secondary strengths, the authors provide evidence of efficient gene editing in Cas9+ER-Hoxb8 neutrophils both in vivo and in vitro and provide evidence of the specificity of this methodology using a Cas9+ER-Hoxb8 immortalized cell line that differentiates into macrophages.

In terms of methodology, this study provides a useful tool to explore gene regulatory networks in neutrophils in large-scale genetic screens. However, it falls short in identifying and validating the true potential of this kind of methodology in the biology of the neutrophil.

Moreover, the technical advances in the field are only incremental as several studies, including those using CRISPR/Cas9 technology in Hoxb-8 immortalized neutrophil progenitor cell lines have been already performed.

Reviewer #2 (Public Review):

In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function. The major strength of the paper lies in its careful validation of many aspects of the Hoxb8-immortalized progenitor cells, including their differentiation capacity, their ability to clear bacteria, and their capacity to differentiate in vivo. The authors succeed at this, with results correctly supporting their conclusions. The major weaknesses are its presentation and writing, some of which are poorly organized. Finally, while the potential impact of this resource in the field could be very large, the CRISPR screening results appear half-baked, almost preliminary, and could be better validated, or at least presented. A few other points that warrant revision are included below:

• The introduction should be better constructed and organized. It should be written with more connectors to present facts in a stream that flows naturally, from the broad general facts to the experimental details implemented in the manuscript. It should also discuss other similar approaches used in the literature, such as LaFleur et al. 2019, and relate in which ways these presented methods could be better.

• The scheme in Figure 4A should more clearly indicate the timings, doublings, numbers of cells, and other aspects of the experimental design.

• The volcano plot in Figure 4B is poorly informative and almost redundant. What does one make of it?

• The representation (normalized reads) of each sgRNA in the library and across multiple experiments, including their correlation, should be checked and plotted, to visualize how robust these replicates are.

• In Figure 4E, the distribution of the hit sgRNAs should be compared to all other targeting guides (instead of just to non-targeting controls). Linear density distribution plots or scatter plots of all guides are usually the best way, but there are others (for example, see Figure 4 of LaFleur et al. 2019). Ideally, each independent sgRNA for each gene in the library, as well as biological replicates, should be separately shown, with hits clearly highlighted.

• While in vivo differentiation is shown as possible with these cell lines, it is unclear whether CRISPR screenings could be performed in vivo too. Would sgRNA representation suffice for genome-wide? At least some of the new hits could be validated by testing differentiation in vivo (i.e. WASH complex).

• In the methods section, the RNA-seq analysis pipeline details are missing (versions, software for alignment, quantification, differential gene expression, and enrichment). Also, parameters for MAGeCK and MAGeCKFlute should be explicit and detailed.

• The discussion is mostly a summary of the results. It is lacking in detail and thoughtful discussion regarding novelty and impact beyond the validation of the cell line. What about potential applications? What about extending screenings to test bacterial-killing, as suggested in Figure 2? What about limitations compared to other similar methods out there? There is little discussion of such important potential matters. Also, a large part of the discussion is dedicated to discussing details about Cebpe that are all well known in the literature and add little value.

• Figure legends are typically too succinct and hard to interpret, especially for non-experts. The text should enable the figure reader to correctly interpret what is shown in each panel.

Reviewer #3 (Public Review):

Primary neutrophils are difficult to modify genetically, whereas the generation of knockout mice to study the role of specific proteins is time-consuming and expensive. CRISPR-Cas 9 genetic modification of neutrophil progenitors in vitro offers a platform to study neutrophil biology. Hoxb8 cells are immortalized neutrophil progenitors that differentiate into neutrophils when cultured in the presence of G-CSF, and have been shown to recapitulate the stages of murine neutrophil differentiation. They have also been shown to be amendable to CRISPR-Cas 9 genetic editing with successful deletion of key transcriptional regulators of neutrophil maturation and function. The authors of this manuscript offer an extension to this technique, by generating Hoxb8 cells that constitutively express Cas9. This may reduce the variation between the generated knock-out cells by avoiding the introduction of Cas9 in a plasmid every time together with a guide RNA.

The first part of the manuscript is dedicated to the characterisation of Cas9+HoxB8 cells throughout their differentiation. Considering the existing body of literature on HoxB8 progenitors and their differentiation into neutrophils ex vivo, it does not significantly further our understanding of these cells, but rather provides a good validation to their Cas9+ modified version of them. Gene editing using Cas9+ Hoxb8 progenitors seems to be highly efficient, which is an important technical point, however, it is hard to assess the degree of improvement in efficiency compared to the published protocols with Cas9 delivery in a plasmid.

As a test, the authors use Cas9+HoxB8 progenitor to generate a knockout of CEBPE, known for its important role in neutrophil development. They convincingly demonstrate its impact on HoxB8 cell differentiation, with in vivo reconstitution of wild-type and CEBPE-deficient HoxB8 progenitors into irradiated mice being especially elegant. However, the transfer into different recipient mice assumed no differences in the recipient environment, while immunophenotyping for mature neutrophils within the HoxB8 progenitor-derived cells did not account for possible differences in numbers of wt and CEBPE KO surviving cells, limiting the conclusions.

Finally, the authors put the system to the test by screening a library of Brie gRNA library of ~80K mouse sgRNAs, targeting almost 20K genes with 4 gRNA per gene coverage, to identify genes that are needed for the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils. They identify a number of hits, amongst which the WASH complex and CEBPE are highlighted. A comparison of cell numbers prior to differentiation and at 4 days post differentiation indicates that they are indeed required for neutrophil survival. To validate the role of these hits in neutrophil maturation itself, as they stated in the aims, i.e. "to identify genes that modulate the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils", phenotypic, functional, and morphological characterization of these cell lines could have been appropriate.

Overall, this study has the potential to improve on the established lentiviral CRISPR-Cas9 editing of Hoxb8 cells and be valuable for library-screening approaches for neutrophil modulators, which will benefit the community.

Reviewer #4 (Public review):

I maintain that the images in Figure 12 (new Figure 14) do not support the authors' interpretation that 2-cell embryos resulted from in vitro fertilization (IVF) of Amrc-/- rescued sperm. They are clearly not normal 2-cell embryos and instead look very much like fragmented eggs that can be seen occasionally following in vitro fertilization procedures even when that is done with wild type eggs and sperm. The only portion of current Figure 14 that has normal looking 2-cell embryos is in panel 14A4, where wild type B6D2 sperm were used. Even in that panel, there are some fragmented eggs that the authors identify as 2-cell embryos.

The authors offer the explanation that CD1 eggs fertilized by B6D2F1 hybrid male sperm do not develop beyond the 2-cell stage, citing a 2008 paper published in Biology of Reproduction by Fernandez-Goonzalez et al. I read through that paper very carefully and even had a colleague read through it in case I missed something, but that paper says nothing at all about strain incompatibilities, much less 2-cell arrest due to them. The only crosses done in that paper are CD1 eggs x CD1 sperm and B6D2 eggs x B6D2 sperm, all by intracytoplasmic sperm injection and not standard in vitro fertilization. [Note that the paper does mention performing in vitro fertilization but says nothing about how it was done or what mouse strains were used.] I even searched the literature for information regarding incompatibility between these strains and could find nothing relevant. But even if the authors are correct and there happens to be a strain incompatibility and 2-cell arrest is expected, what the authors are calling 2-cell embryos are clearly not.

A second explanation offered by the authors is that they used collagenase to remove the cumulus cells and that this may have affected the appearance of the embryos. This technique is actually used to remove both the cumulus cells and the zona pellucida and has been described as a gentler way to do so than other standard methods (hyaluronidase treatment followed by acid Tyrodes to remove the zona pellucida) (Yamatoya et al., Reprod Med Biol 2011, DOI 10.1007/s12522-011-0075-8). I think it is highly relevant to the current study that the method they used to remove cumulus cells also dissolves the zona, either partially or completely. Given that many of the eggs, fragmented eggs, and 2-cell embryos (from the WT sperm) in Figure 14A are lacking a zona pellucida, it seems very likely that many of the eggs were either zona-free or had partial zona dissolution from the start. In fact, the authors state in the Methods section that "Cumulus-free and zona-free eggs were collected..." for how IVF was done. Partial zona dissolution is standard in some protocols for performing IVF using frozen mouse sperm, which usually have much lower motility and overall efficacy than fresh sperm. In any case, it would improve transparency if the manuscript made clear somewhere other than buried in the Methods that the IVF procedure was done on eggs with partially or fully removed zonas, to allow proper interpretation.

In the rebuttal, the authors go on to state: "To provide additional functional evidence, we complemented the IVF experiments with ICSI using rescued Armc2-/- sperm and B6D2 oocytes, which allowed embryos to develop to the blastocyst stage. In these experiments, 25% of injected oocytes reached the blastocyst stage with rescued sperm compared to 13% for untreated Armc2-/- sperm (Supplementary Fig. 9) These results support the functional competence of rescued sperm and demonstrate partial recovery of fertilization ability following Armc2 mRNA electroporation."

Their conclusion that the data support partial recovery of fertilization ability following Armc2 mRNA electroporation in my opinion has no basis. This experiment was done only once, and no information is provided regarding how many eggs underwent ICSI or how many reached the blastocyst stage. The authors claim that the rescued sperm were better than the Armc2-/- sperm in producing blastocysts, but this is based on a simple percentage report of 25% vs 13% without any statistical analysis, even on the results from the single experiment presented.

Overall, the paper shows rescue of some sperm motility by the new method they use, and the new title is therefore appropriate. The authors have also dealt reasonably with many of the original concerns regarding documenting that their methodology was effective in producing protein (at least the GFP marker) in spermatogenic cells. In my view the authors have, however, not shown any indication of functional recovery over what is already known for the knockout sperm, that ICSI can support blastocyst stage embryo development. They also have not, in my view, justified the claims at the end of the abstract "These motile sperm were able to produce embryos by IVF..." and that "...mRNA electroporation can restore...partially fertilizing ability..."

Reviewer #5 (Public review):

While the study presents an innovative and potentially impactful mRNA-based approach for addressing monogenic causes of male infertility, several significant weaknesses limit the strength of the authors' central conclusions.

First, the functional evidence for true fertility restoration remains incomplete. Although the authors convincingly demonstrate partial recovery of sperm motility, the downstream reproductive outcomes, particularly for IVF, are weak. Importantly, these concerns are shared by all three reviewers and the former Reviewing Editor, and to my eye they are both thoughtfully articulated and well warranted. The ICSI data show modest improvement, but this rescue is difficult to interpret.

In parallel, significant mechanistic questions persist regarding the unusually prolonged persistence of naked mRNA and reporter protein expression in germ cells, which is not fully reconciled with established mRNA and protein half-life biology and is supported largely by inference rather than by direct decay measurements.

Finally, although the authors have conducted additional cellular analyses, concerns about the extent and specificity of germ-cell targeting versus Sertoli-cell expression remain unresolved. Together, these issues do not negate the technical novelty of the work, but they do constrain the confidence with which the current dataset can support the authors' strongest therapeutic claims.

Reviewer #1 (Public Review):

The heterogeneity within the neutrophil population is becoming clear. However, it was not clear if neutrophil progenitors are also heterogenous. Because neutrophils are short-lived, it is technically challenging to tackle the question. This study used a system to isolate and expand clonal neutrophil progenitors (granulocyte-monocyte progenitors; GMPs) to achieve molecular and functional profiling. In the study, transcriptional profiling was performed by RNAseq and ATACseq. Functional assays were performed ex vivo to examine phagocytosis, ROS production, NET formation, and neutrophil swarming using Candida albicans, as well as C. glabrata and C. auris. The strengths of this study include the use of the neutrophil clone system to track GMPs developing into neutrophils. The clone-based approach made it possible to evaluate the functions of multiple neutrophil subpopulations. Limitations of this study include the dependency on ex vivo approaches and the modest degree of heterogeneity within presented neutrophils. Nevertheless, the finding - the heterogeneity of neutrophils can be traced back to the GMP stage - is significant.

Reviewer #2 (Public Review):

The stated goal of the authors is to establish and characterize an experimental system to study neutrophil heterogeneity in a manner that allows for functional outcomes to be probed. To do so, they start with murine GMPs that are conditionally immortalized by ER-HoxB8 expression and make single-cell clonal populations to ask whether those GMPs or neutrophils derived by differentiating such clonal GMPs harbor heterogeneity. At a conceptual level, this is an innovative approach that could shed light on mechanisms of neutrophil heterogeneity that have been described in both health and disease. They perform bulk multi-omics and functional analyses of both the clonal GMPs and neutrophil-like cells, including transcriptional and epigenetic profiling. However, the major weakness of the study is that the authors do not provide rigorous or convincing data that the cells they derive are truly mature neutrophils. To the contrary, the neutrophil-like cells lack Ly6G expression and so the authors fall back on using CD11b as the primary marker for delineating neutrophils; however, CD11b is expressed by both myeloid progenitors and some premature and mature myeloid lineages that are not neutrophils. They acknowledge this shortcoming, but they make an assumption that Ly6G expression is the only way in which the cells they derive are different from primary neutrophils without presenting any evidence indicating such. The authors use only SCF during the maturation of ER-HoxB8 GMPs into leukocytes, rather than including other cytokines such as G-CSF (or use in vivo maturation) that could have better-induced differentiation and maturation into granulocytes/neutrophils. The authors did not use their transcriptional analyses to further establish that the cells they derive from ER-HoxB8 GMPs are similar/different from primary murine neutrophils. Unfortunately, this shortcoming means that all of the analyses of neutrophil-like cells derived from clonal GMPs may or may not represent the transcriptional, epigenetic, etc. profile of a true mature neutrophil. It is also not rigorously addressed whether what they call PMNs derived from clonal GMPs are a transcriptionally uniform population or if they harbor heterogeneity within the bulk population. Overall, while conceptually intriguing and in pursuit of an experimental system that would be impactful for the field, the study as performed has critical flaws.

Reviewer #1 (Public review):

Summary:

In their study the authors investigated the F. graminearum homologue of the Drosophila Misato-Like Protein DML1 for a function in secondary metabolism and sensitivity to fungicides.

Strengths:

Generally, the topic of the study is interesting and timely and the manuscript is well written, albeit in some cases details on methods or controls are missing.

Weaknesses:

However, a major problem I see is with the core result of the study, the decrease of the DON content associated with deletion of FgDML1: Although some growth data are shown in figure 6 - indicating a severe growth defect - the DON production presented in figure 3 is not related to biomass. Also, the method and conditions for measuring DON are not described. Consequently, it could well be concluded that the decreased amount of DON detected is simply due to a decreased growth and specific DON production of the mutant remains more or less the same.

To alleviate this concern, it is crucial to show the details on the DON measurement and growth conditions and to relate the biomass formation on the same conditions to the DON amount detected. Only then a conclusion as to an altered production in the mutant strains can be drawn.

Comments to the revised manuscript:

The authors carefully revised the manuscript and provided explanations for methods in several cases. However, there are still some problems - probably due to misunderstanding - that need revision.

(1) A major problem of the first version of the manuscript was the lack of appropriate description of biomass analysis and the consideration of the respective results for evaluation of production of DON and other metabolites. Although the authors provide some explanation in the response to reviews, I could not find a corresponding explanation or description in the manuscript. It is not sufficient to explain the problem to me, but a detailed explanation and description of the method has to be provided in the manuscript along with the definition of one "unit of mycelium". It is still not entirely clear to me what such a "unit of mycelium" is.

Please clarify this and any other uncertainties that were commented on by me and other reviewers in the manuscript, not only in the response to reviews. Also adjust the reference list accordingly.

(2) Another problem was, that the authors considered FgDML1 a regulator of DON production. As mentioned by me and reviewer 3, FgDML1 is crucial to numerous functions in F. graminearum and its lack causes a plethora of problems for fungal physiology. Hence, although it is clear that the lack of FgDML1 causes alterations in DON production, it is not appropriate to designate this factor as a "regulator".<br /> It seems to me that the authors are afraid that if FgDML1 would not be a "regulator" that this would decrease the value of their study, which is not the case. This is a matter of correct wording. Therefore, please revise the wording accordingly, starting with the title:

...FgDML1 impacts DON toxin biosynthesis...

Moreover, for sure the manuscript might benefit from more detailed description of the whole cascade leading from FgDML1 to DON biosynthesis and production of the other metabolites that change upon deletion. Such explanation can help the reader grasp the relevance of FgDML for regulatory processes as well as on more general versus specific effects.

Reviewer #2 (Public review):

Summary:

The manuscript entitled "Mitochondrial Protein FgDML1 Regulates DON Toxin Biosynthesis and Cyazofamid Sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" identified the regulatory effect of FgDML1 in DON toxin biosynthesis and sensitivity of Fusarium graminearum to cyazofamid. The manuscript provides a theoretical framework for understanding the regulatory mechanisms of DON toxin biosynthesis in F. graminearum and identifies potential molecular targets for Fusarium head blight control. The paper in innovative, but there are issues in the writing that need to be added and corrected.

Comments on revisions:

The author has addressed my questions.

Reviewer #2 (Public review):

This paper proposes two changes to classic RSA, a popular method to probe neural representation in neuroimaging experiments: computing RSA at row/column level of RDM, and using linear mixed modeling to compute second level statistics, using the individual row/columns to estimate a random effect of stimulus. The benefit of the new method is demonstrated using simulations and a re-analysis of a prior fMRI dataset on object perception and memory encoding.

The author's claim that tRSA is a promising approach to perform more complete modeling of cogneuro data, and to conceptualize representation at the single trial/event level (cf Discussion section on P42), is appealing.

In their revised manuscript, the authors have addressed some previous concerns, now referencing more literature aiming to improve RSA and its associated statistical inferences, and providing more guidance on methodological considerations in the Discussion. However, I wish the authors had more extensively edited the Introduction to better contextualize the work and clarify the specific settings in which they see the method as being beneficial over classic RSA. For example, some of the limitations of cRSA mentioned on page 6, e.g. related to presenting the same stimuli to multiple subjects, seem to be quite specific to settings where the researcher expects differential responses across subjects to fundamentally alter the interpretation, rather than something that will just average out by repeatedly offering the same stimulus, or combining data across subjects. It's not clear to me how the switch from 'matrix-level' to 'row-level' analysis in tRSA necessarily addresses this problem. I would be very helpful if the authors would more explicitly outline what problem the row-level aspect of tRSA is solving; what problem statistical inference via LMM is solving; and walk the reader through a very specific use case (perhaps a toy version of the real-data experiment which is now at the end of the paper). Explaining the utility of tRSA for experimental settings in which assessing representational strength for a single-events is crucial would clarify the contribution of this new method better.

A few weaknesses mentioned in my previous review were not adequately addressed. To demonstrate the utility of the method on real neural recordings, only a single dataset is used with a quite complicated experimental design; it's not clear if there is any benefit of using tRSA on a simpler real dataset. Moreover, the cells of an RDM/RSM reflect pairwise comparisons between response patterns. Because the response patterns are repeatedly compared, the cells of this matrix are not independent of one another. While the authors show examples that failure to meet independence assumptions do not affect results in their specific dataset, it does not get acknowledged as a problem at a more fundamental level. Finally, while the paper now states that 'simulations and example tRSA code' are publicly available, the link points to the lab's general github page containing many lab repositories, in which I could not identify a specific repository related to this paper. This is disappointing given that the main goal of this manuscript is to provide a new method that they encourage others to use; a clear pointer to available code is only a minimal requirement to achieve that goal. A dedicated repository, including documentation, READMEs and tutorials/demo's to run simulations, compare methods, etc. would greatly enhance the paper's contribution.

Reviewer #1 (Public review):

In this manuscript, Dillard and colleagues integrate cross-species genomic data with a systems approach to identify potential driver genes underlying human GWAS loci and establish the cell type(s) within which these genes act and potentially drive disease.

Specifically, they utilize a large single cell RNA-seq (scRNA-seq) dataset from an osteogenic cell culture model - bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) - from a genetically diverse outbred mouse population called the Diversity Outbred (DO) stock to discover network driver genes that likely underlie human bone mineral density (BMD) GWAS loci. The DO mice segregate over 40M single nucleotide variants, many of which affect gene expression levels, therefore making this an ideal population for systems genetic and co-expression analyses.

The current study builds on previous published work from the same group that used co-expression analysis to identify co-expressed "modules" of genes that were enriched for BMD GWAS associations. In this study, the authors utilized a much larger scRNA-seq dataset from 80 DO BMSC-OBs, inferred co-expression based on Bayesian networks for each identified mesenchymal cell type, focused on networks with dynamic expression trajectories that are most likely driving differentiation of BMSC-OBs, and then prioritized genes ("differentiation driver genes" or DDGs) in these osteogenic differentation networks that had known expression or splicing QTLs (eQTL/sQTLs) in any GTEx tissue that co-localized with human BMD GWAS loci. The systems analysis is impressive, the experimental methods are described in detail, and the experiments appear to be carefully done. The computational analysis of the single cell data is comprehensive and thorough, and the evidence presented in support of the identified DDGs, including Tpx2 and Fgfrl1, is for the most part convincing. Some limitations in the data resources and methods hamper enthusiasm somewhat and are discussed below.

Overall, while this study will no doubt be valuable to the BMD community, the cross-species data integration and analytical framework may be more valuable and generally applicable to the study of other diseases, especially for diseases with robust human GWAS data but for which robust human genomic data in relevant cell types is lacking.

Specific strengths of the study include the large scRNA-seq dataset on BMSC-OBs from 80 DO mice, the clustering analysis to identify specific cell types and sub-types, the comparison of cell type frequencies across the DO mice, and the CELLECT analysis to prioritize cell clusters that are enriched for BMD heritability (Figure 1). The network analysis pipeline outlined in Figure 2 is also a strength, as is the pseudotime trajectory analysis (results in Figure 3).

Potential drawbacks of the authors' approach include their focus on genes that were previously identified as having an eQTL or sQTL in any GTEx tissue. The authors rightly point out that the GTEx database does not contain data for bone tissue, but reason that eQTLs can be shared across many tissues - this assumption is valid for many cis-eQTLs, but it could also exclude many genes as potential DDGs with effects that are specific to bone/osteoblasts. Indeed, the authors show that important BMD driver genes have cell-type specific eQTLs. Another issue concerns potential model overfitting in the iterativeWGCNA analysis of mesenchymal cell type-specific co-expression, which identified an average of 76 co-expression modules per cell cluster (range 26-153). Based on the limited number of genes that are detected as expressed in a given cell due to sparse per cell read depth (400-6200 reads/cell) and drop outs, it's surprising that as many as 153 co-expression modules could be distinguished within any cell cluster. I would suspect some degree of model overfitting is responsible for these results.

Overall, though, these concerns are minor relative to the many strengths of the study design and results. Indeed, I expect the analytical framework employed by the authors here will be valuable to -- and replicated by -- researchers in other disease areas.

Comments on revisions:

Thank you for addressing my concerns. This is an impressive study and manuscript that you should be proud of.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single cell RNAseq analysis on bone marrow derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is, in parts, hard to read due to the use of acronyms and there are some questions about data analysis that still need to be addressed.

Comments on revisions:

Dillard et al have made several improvements to their manuscript.

(1) We previously asked the authors to determine whether any cell types were enriched for BMD-related traits since the premise of the paper is that 'many genes impacting BMD do so by influencing osteogenic differentiation or ... adipogenic differentiation'. Given the potential for the cell culture method to skew the cell type distribution non-physiologically, it is important to establish which cell types in their assay are most closely associated with BMD traits. The new CELLECT analysis and Figure 1E address this point nicely. However, it would still be nice to see the correlations between these cell types and BMD traits in the mice as this would provide independent evidence to support their physiological importance more broadly.

(2) Shortening the introduction.

(3) Addressing limitations that arise from not accounting for founder genome SNPs when aligning scRNA-seq data.

(4) The main take-away of this paper is, to us, the development of a single cell approach to studying BMD-related traits. It is encouraging that the cells post-culture appear to be representative of those pre-culture (supplemental figure 3).

However, the authors seem to have neglected several comments made by both reviewers. While we share the authors' enthusiasm for the single cell analytical approach, we do not understand their reluctance to perform further statistical tests. We feel that the following comments have still not been addressed:

(1) The manuscript still contains the following:

"To provide further support that tradeSeq-identified genes are involved in differentiation, we performed a cell type-specific expression quantitative trait locus (eQTL) analysis for each mesenchymal cell type from the 80 DO mice. We identified 563 genes (eGenes) regulated by a significant cis-eQTL in specific cell types of the BMSC-OB scRNA-seq data (Supplementary Table S14). In total, 73 eGenes were also tradeSeq-identified genes in one or more cell type boundaries along their respective trajectories (Supplementary Table S9)."

The purpose of this paragraph is to convince readers that the eGenes approach aligns with the tradeSeq approach (and that their approach can therefore be trusted). It is essential that such claims are supported by statistical reasoning. Given that it would be very simple to perform permutation/enrichment analyses to address this point, and both reviewers requested similar analyses, we do not understand the author's reluctance here. Otherwise, this section should be rewritten so that it does not imply that the identification of these genes provides support for their approach.

(2) Given that a central purpose of this manuscript is to establish a systematic workflow for identifying candidate genes, the manuscript could still benefit from more explanation as to why the authors chose to highlight Tpx2 and Fgfrl1. Tpx2 does already have a role in bone physiology through the IMPC. The authors should comment on why they did not explore Kremen1, for instance, as this gene seems important for the transition to both OB1 and 2.

A final minor comment is that it would be very helpful if the authors could indicate if the DDGs in Table 1 are also eGenes for the relevant cell type. This is much more meaningful than looking through GTEx.

Reviewer #1 (Public review):

Summary:

In this fMRI study, the authors wished to assess neural mechanisms supporting flexible temporal construals. For this, human participants learned a story consisting of fifteen events. During fMRI, events were shown to them, and participants were instructed to consider the event from "an internal" or from "an external" perspective. The authors found distinct patterns of brain activity in the posterior parietal cortex (PPC) and anterior hippocampus for the internal and the external viewpoint. Specifically, activation in the posterior parietal cortex positively correlated with distance during the external-perspective task, but negatively during the internal-perspective task. The anterior hippocampus positively correlated with distance in both perspectives. The authors conclude that allocentric sequences are stored in the hippocampus, whereas egocentric sequences are supported by the parietal cortex.

Strengths:

The research topic is fascinating, and very few labs in the world are asking the question of how time is represented in the human brain. Working hypotheses have been recently formulated, and the work tackles them from the perspective of construals theory.

Weaknesses:

Although the work uses two distinct psychological tasks, the authors do not elaborate on the cognitive operationalization the tasks entail, nor the implication of the task design for the observed neural activation.

Reviewer #2 (Public review):

Summary:

Xu et al. used fMRI to examine the neural correlates associated with retrieving temporal information from an external compared to internal perspective ('mental time watching' vs. 'mental time travel'). Participants first learned a fictional religious ritual composed of 15 sequential events of varying durations. They were then scanned while they either (1) judged whether a target event happened in the same part of the day as a reference event (external condition); or (2) imagined themselves carrying out the reference event and judged whether the target event occurred in the past or will occur in the future (internal condition). Behavioural data suggested that the perspective manipulation was successful: RT was positively correlated with sequential distance in the external perspective task, while a negative correlation was observed between RT and sequential distance for the internal perspective task. Neurally, the two tasks activated different regions, with the external task associated with greater activity in the supplementary motor area and supramarginal gyrus, and the internal condition with greater activity in default mode network regions. Of particular interest, only a cluster in the posterior parietal cortex demonstrated a significant interaction between perspective and sequential distance, with increased activity in this region for longer sequential distances in the external task but increased activity for shorter sequential distances in the internal task. Only a main effect of sequential distance was observed in the hippocampus head, with activity being positively correlated with sequential distance in both tasks. No regions exhibited a significant interaction between perspective and duration, although there was a main effect of duration in the hippocampus body with greater activity for longer durations, which appeared to be driven by the internal perspective condition. On the basis of these findings, the authors suggest that the hippocampus may represent event sequences allocentrically, whereas the posterior parietal cortex may process event sequences egocentrically.

Strengths:

The topic of egocentric vs. allocentric processing has been relatively under-investigated with respect to time, having traditionally been studied in the domain of space. As such, the current study is timely and has the potential to be important for our understanding of how time is represented in the brain in the service of memory. The study is well thought out and the behavioural paradigm is, in my opinion, a creative approach to tackling the authors' research question. A particular strength is the implementation of an imagination phase for the participants while learning the fictional religious ritual. This moves the paradigm beyond semantic/schema learning and is probably the best approach besides asking the participants to arduously enact and learn the different events with their exact timings in person. Importantly, the behavioural data point towards successful manipulation of internal vs. external perspective in participants, which is critical for the interpretation of the fMRI data. The use of syllable length as a sanity check for RT analyses as well as neuroimaging analyses is also much appreciated.

Suggestions:

The authors have done a commendable job addressing my previous comments. In particular, the additional analyses elucidating the potential contribution of boundary effects to the behavioural data, the impact of incorporating RT into the fMRI GLMs, and the differential contributions of RT and sequential distance to neural activity (i.e., in PPC) are valuable and strengthen the authors' interpretation of their findings.

My one remaining suggestion pertains to the potential contribution of boundary effects. While the new analyses suggest that the RT findings are driven by sequential distance and duration independent of a boundary effect (i.e., Same vs. Different factor), I'm wondering whether the same applies to the neural findings? In other words, have the authors run a GLM in which the Same vs. Different factor is incorporated alongside distance and duration?

Reviewer #1 (Public review):

In this study, the authors provide an integrated proteogenomics pipeline to enable the discovery of novel peptides in an Ewing sarcoma cell line (A673). To identify novel full-length resolved isoforms, they performed long-read RNA sequencing (Oxford Nanopore Technology). Then, to increase the chance of detecting Ewing-specific neopeptides, the authors combined two approaches: a multi-protease digestion and a multi-dimensional proteomics approach.

Given the importance of novel isoforms and cryptic sites in neoantigen discovery and its putative applications in immunotherapy, this method and resource paper are of interest for the Ewing community and potentially for a broader cancer audience. The originality of this paper relies mostly on this optimized method to discover novel peptides (long-read sequencing with multiprotease, multi-dimensional trapped ion mobility spectrometry parallel accumulation-serial fragmentation mass spectrometry). Although, to my knowledge, no study combining long-read sequencing and proteomics methods has been published on Ewing Sarcoma, this study appears limited by a few aspects:

(1) The study is restricted to the analysis of a single cell line (A673). The authors should consider extending the analysis to other Ewing cell lines.

(2) The characterization of the 1121 non-canonical transcripts can be improved. How many are just splice variants of known genes, and how many are bona fide neogenes? In this respect, the definition of what the authors call neogene is quite unclear. Is a transcript with a new exon reported as a neogene? Is a transcript with a new start site reported as a neogene? It should be clearly indicated which categories of Figure 4B are reported on Figure 4D. A general flow chart would be very useful to help follow the analysis process.

(3) Similarly, the authors detect 3216 A673 specific proteins with no match in SwissProt. This number decreases to 72 "putative non-canonical proteoforms with unique peptides after BLASTp" against Uniprot. Again, a flow chart would conveniently enable one to follow the step-by-step analysis.

(4) Finally, only 17 spectral matches are suggested to be derived from non-canonical proteoforms. It would be important to compare the spectrum of these detected peptides with that of synthetic peptides. Such an analysis would enable us to assess the number of reliably detected proteoforms that can be expected in an Ewing sarcoma cell line.

(5) It is very unclear what the authors want to highlight in Supplementary Figure 5. Is it that non-canonical transcripts are broadly expressed in normal tissue? Which again raises the question of definitions of neogenes, non-canonical... Apparently, this figure shows that these non-canonical transcripts contain a large part of canonical sequences, which account for the strong signal in many normal tissues. A similar heatmap could be presented, including only the non-canonical sequences of the non-canonical transcripts. This figure should also include Ewing sarcoma samples.

Reviewer #2 (Public review):

The paper from Kulej et al. reports a set of tools for proteogenomic analysis of cancer proteomes. Their approach utilizes modern methods in long-read RNA sequencing to assemble a proteome database that is specific to Ewing sarcoma-derived A673 cells. To maximize proteome coverage and therefore increase the odds of detecting cancer-specific alterations at the protein level, the authors use multiple enzymes (trypsin, gluC, etc.) to digest cellular proteins and then perform multidimensional peptide fractionation. Peptide samples are then analyzed by LC-MS/MS using data-dependent and data-independent schemes on a timstof mass spectrometer. Proteogenomics is an important area of investigation for cancer research and does require new informatics tools.

The authors describe an end-to-end workflow where they claim to have optimized four different steps:

(1) Assembly of a sample-specific protein database using long-read transcriptomic data.

(2) Use of 8 different proteolytic enzymes to maximize diversity of peptides.

(3) Multiple stages of peptide fractionation using SCX and high pH rp chromatography.

(4) Utilize acquisition methods on the timstof mass spec to provide MS/MS data from single-charged peptides and multiply-charged peptides.

The authors published two earlier versions of ProteomeGenerator (versions 1 and 2) in the Journal of Proteome Research. In these earlier versions, 'ProteomeGenerator' was the set of software tools designed to integrate DNA and RNA sequencing to create a sample-specific protein database. To test the performance of each ProteomeGenerator version, the authors generated LC-MS/MS data using a combination of trypsin and LysC, then in the other paper, trypsin, LysC, and GluC. In both papers, they performed some levelof peptide fractionation prior to LC-MS/MS. They acquired LC-MS/MS data on a Thermo Q-Exactive in one paper and a Thermo Orbitrap mass spec in the other paper.

In the current paper, the primary innovation is the use of long-read sequencing to potentially improve the quality of the sample specific protein database. The other three components noted above are incremental compared to the authors' previous two papers and generally accepted practices in the field of proteomics. To note one example, the authors previously digested proteins using three enzymes and now use eight. Similarly, they are now using a timstof Bruker mass spec instead of one from Thermo. The detailed descriptions around the use of many enzymes and peptide fractionation, etc., create a very technically oriented paper, similar to or more so than the authors' earlier papers in J. Proteome Research. So, while there is enthusiasm for the use of long-read sequencing across biomedical research, the impact here for proteogenomic applications is somewhat lost with all of the technical description for experimental details that are not particularly innovative. In this respect, the report is not well matched to a broad readership.

Reviewer #1 (Public review):

Summary:

This manuscript investigates how herbivorous insects, specifically whiteflies and planthoppers, utilize salivary effectors to overcome plant immunity by targeting the RLP4 receptor.

Strengths:

The authors present a strong case for the independent evolution of these effectors and provide compelling evidence for their functional roles.

Reviewer #2 (Public review):

Summary:

The authors tested an interesting hypothesis that white flies and planthoppers independently evolved salivary proteins to dampen plant immunity by targeting a receptor-like protein. Unlike previously reported receptor-like proteins with large ligand-binding domains, the NtRLP4 here has a malectin LRR domain. Interestingly, it also associates with the adaptor SOBIR1. While the function of this protein remains to be further explored, the authors provide strong evidence showing it's the target of salivary proteins as the insects' survival strategy.

Major points:

The authors mixed the concepts of LRR-RLPs with malectin LRR-RLPs. These are two different type of receptors. While LRR-RLPs are well studied, little is known about malectin LRR-RLPs. The authors should not simply apply the mode of function of LRR-RLPs to RLP4 which is a malectin LRR-RLP. In addition, LRR-RLPs that function as ligand-binding receptors typically possess >20 LRRs, whereas RLP4 in this work has a rather small ectodomain. It remains unclear whether it will function as a PRR.

I can't agree with the author's logic of testing uninfested plants for proving a PRR's function. The function of a pattern recognition receptor depends on perceiving the corresponding ligand. As shown by the data provided, RLP4-OE plants have altered transcriptional profile indicating activated defense, suggesting it's unlikely a PRR. An alternative explanation is needed.

More work on BAK1 will also help to clarify the ideas proposed by the authors.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al., investigate how herbivorous insects overcome plant receptor-mediated immunity by targeting plant receptor-like proteins. The authors identify two independently evolved salivary effectors, BtRDP in whiteflies and NlSP694 in brown planthoppers, that promote the degradation of plant RLP4 through the ubiquitin-dependent proteasome pathway. NtRLP4 from tobacco and OsRLP4 from rice are shown to confer resistance against herbivores by activating defense signaling, while BtRDP and NlSP694 suppress these defenses by destabilizing RLP4 proteins.

Strengths:

This work highlights a convergent evolutionary strategy in distinct insect lineages and advances our understanding of insect-plant coevolution at the molecular level.

Two minor comments:

In line 140, yeast two-hybrid (Y2H) was used to screen for interacting proteins in plants. However, it is generally difficult to identify membrane receptors using Y2H. Please provide more methodological details to justify this approach, or alternatively, include a discussion explaining this.

In Figure S12C, the interaction between the two proteins appears to be present in the nucleus as well. Please provide a possible explanation for this observation.

Reviewer #1 (Public review):

Summary:

In this manuscript, Seegren and colleagues demonstrate that in a mouse model of neonatal E. coli meningitis, loss of endothelial toll-like receptor 4 (TLR4) leads to a marked decrease in transcriptional dysregulation across multiple leptomeningeal cell types, a decrease in vascular permeability, and a decrease in macrophage abundance. In contrast, loss of macrophage TLR4 had less pronounced effects. Using cultured wild-type and TLR4-knockout endothelial cells, the authors further demonstrate that TLR4-NF-κB signaling leads to reversible internalization of the tight junction protein claudin-5, establishing a potential mechanism of increased vascular permeability. Finally, the authors use RNA-sequencing of wild-type and TLR4-knockout endothelial cells to define the TLR4-dependent cell-autonomous transcriptional response to E. coli.

Strengths:

(1) The authors address an important, well-motivated hypothesis related to the cellular and molecular mechanisms of leptomeningeal inflammation.

(2) The authors use model systems (mouse conditional knockouts and cultured endothelial cells) that are appropriate to address their hypotheses. The data are of high quality.

Weaknesses:

(1) The authors perform single-nucleus RNA-seq on dissected leptomeninges from control and E. coli-infected mice across three genotypes (WT, Tlr4MKO, and Tlr4ECKO). A major discovery from this experiment, as summarized by the authors, is: "Tlr4ECKO mice exhibited a global attenuation of infection-induced transcriptional responses across all major leptomeningeal cell types, as judged by the positions of cell clusters in the UMAP." This conclusion could be considerably strengthened by improving the qualitative and quantitative analysis.

(2) The authors interpret E. coli infection-induced increases in leptomeningeal sulfo-NHS-biotin as evidence of compromised BBB integrity (i.e., extravasation from the vasculature) (Results, page 7), but another possible route in this context is sulfo-NHS-biotin entry from the dura across a compromised arachnoid barrier. The complete rescue in Tlr4ECKOs is strongly suggestive that the vascular route dominates, but it would strengthen the work if the authors could assess arachnoid barrier fidelity (e.g. via immunohistochemistry). At a minimum, authors should mention that the sulfo-NHS-biotin signal in this context may represent both vascular and arachnoid barrier extravasation.

(3) The authors state that "deletion of TLR4 prevented both NF-κB nuclear translocation and Cldn5 internalization in response to E. coli (Figure 4A-D)" (Results, page 9). In Figures 4C and D, however, there is no indicator of a statistical test directly comparing the two genotypes. A comparison of within-genotype P-values should not be used to support a genotype difference (PMID: 34726155).

(4) In the first paragraph of the Results, the authors summarize the meningeal layers as (1) pia, (2) subarachnoid space, (3) arachnoid, and (4) dura, and then state "The second and third layers constitute the leptomeninges." This definition of leptomeninges seems to omit the pia, which is widely considered part of the leptomeninges (PMID: 37776854).

(5) The Cdh5-CreER/+;Tlr4 fl/- mouse lacks TLR4 in all endothelial cells (i.e., in peripheral organs as well as CNS/leptomeninges), and, as the authors note, the periphery is exposed to E. coli. It would be helpful if the authors could comment in the Discussion on the possibility that peripheral effects (e.g., peripheral endothelial cytokine production, changes to blood composition as a result of changes to peripheral endothelial permeability) may contribute to the observed leptomeningeal phenotypes.

Reviewer #2 (Public review):

Summary:

The authors use a postnatal mouse model of E. coli bacterial meningitis and a mouse brain endothelioma cell line combined with cell-type-specific gene deletion to study the function of endothelial TLR4, a cell surface receptor that recognizes gram positive bacterial wall components, in the local leptomeningeal (LPM) response with a focus on endothelial barrier breakdown mediated by TLR4. Single-cell transcriptional profiling and imaging studies using whole-mount preps of the LPM support that LPM endothelial, CD206+ local macrophage and LPM fibroblast and arachnoid barrier cell inflammatory response and is abrogated in endothelial-specific KO of TLR4, pointing to a role for endothelial TLR4 in local LPM response. Culture studies using Bend3.1 cells (a mouse brain endothelioma cell line) support a direct role for TLR4 in the bacteria-mediated inflammatory response and in internalization of Cldn5 via the endosomal-lysosomal pathway, resulting in loss of barrier integrity

Strengths:

The local LPM cell response in meningitis and the role of specific LPM cells in inflammation and CNS barrier breakdown have not been extensively studied, despite ample evidence for primary immune response in the meninges in human patients and in animal models. The authors employ a robust, multi-model approach using both in vivo and in vitro models with cell-type-specific knockout to study the function of TLR4 in brain endothelial cell response. The authors nicely combine functional barrier assays with IF for junctional localization in their experimental design, and they delve into potential mechanisms of Cldn5 internalization using markers of endosomal-lysosomal pathway localization. The authors also describe a new type of barrier assay using a streptavidin-coated plate upon which barrier-forming cell cultures can be placted, this could be a very useful alternative or complement to other size-selective barrier assays and presumably could work for other barrier forming cells types, likely epithelial cells.

Weaknesses:

(1) There are no measures of bacterial burden in peripheral organs, blood, in the LPM or brain in the TLR4 endothelial cKO mice. Lack of TLR4 in endothelial cells could prevent bacterial 'access' into the LPM and brain, essentially preventing meningitis and leading to a lack of inflammatory responses in the LPM-located cells simply because there is no bacteria present. Bacteremia may also be reduced, as might inflammatory responses in peripheral organs with TLR4-deficient peripheral endothelium. Bacterial counts and inflammatory measures in peripheral organs and blood are important to better understand the mechanism(s) underlying the reduced inflammatory profile in LPM cells and no LPM endothelial breakdown in the Tlr4 endothelial cKO mice. In other words, does deleting TLR4 in EC protect against the development of meningitis by somehow blocking bacteria access to the LPM (this would be supported by low or no CFU counts in infected Tlr4 endothelial cKO) or is it what the authors appear to propose in Figure 1J that TLF4 in EC is the only cell responding to the bacteria to trigger the immune cascade in the LPM? More data is needed to resolve this, as this is a major claim of the paper.

(2) The authors look at the underlying cortical response (cerebral vasculature for ICAM and immune cells) but do not use markers that could identify microglia (Iba1), the primary resident immune cell (CD206 is not useful, at this stage, in perivascular macrophages that are extremely sparse in the postnatal brain). This would be important to better study the impact on CNS resident immune cell morphological activation.

(3) The authors suggest that Cldn5 junctional localization is selectively disrupted upon bacterial exposure, mediated by TLR4 - they suggest this based on studying PECAM, GLUT-1, ZO-1 and B-catenin (all normally junction or cell surface located in cultured Bend3.1) in relationship to Cldn5 localization (normally high) - it is possibly these are also impact by bacteria exposure (maybe through different mechanisms?) - a better measure would be to use the similar cyto/PM measure they do for Cldn5 in Fig. 4D and to evaluate this or to use intensity measurements.

(4) The discussion could benefit from delving more into the prior literature on E.coli-mediated breakdown of junctions in cultured human microvascular brain endothelial cell model and critical host-pathogen interactions of the bacteria with ECs (PMID: 14593586), and how this might involve TLR4.

(5) It would be important to discuss how their results relate to earlier studies on TLR4-/- and TLR2-/- global knockout mice and protection vs vulnerability to development of meningitis (see PMCID: PMC3524395) - this paper showed that TLR4 global KO mice have increased susceptibility to die from meningitis and have much higher CFU counts in the CNS. In this manuscript and their prior work (Wang et al., 2023), this group shown that both global TLR4-/- mutants and their EC-specific KO have reduced barrier permeability, but we don't have any information about CFU or susceptibility to death from meningitis in their models.

Reviewer #3 (Public review):

Summary:

This study investigates the molecular underpinnings of immune responses in the leptomeninges in neonatal bacterial meningitis. Bacterial meningitis is a major disease burden, particularly for neonates, and it has previously been noted that the meningeal immune environment in infants is permissive to opportunistic infection (Kim et al., Sci Immunol, 2023). There is less known about the contribution of the stromal compartment to meningeal immune responses. Seegren et al. interrogate the role of leptomeningeal endothelium in host defence in E. coli infected neonatal mice using mouse genetic tools to delete the LPS receptor Tlr4 from either endothelial cells (using Cdh5-CreER) or macrophages (using LysM-Cre). The authors use snRNAseq, cleared cortical mounts, and in vitro work to define the impact of E. coli infection on leptomeningeal endothelial cells. This study uses a range of innovative techniques to probe the role of the stromal compartment in meningitis.

Strengths:

This study makes excellent use of cleared cortical mounts to examine the biology of the leptomeninges, in particular, changes to the endothelium, with unprecedented detail. In combination with high-quality sequencing data provide new insights into the impact of meningitis on the leptomeninges. The data presented by the authors is of very high quality.

Weaknesses:

The weaknesses of the study were in terms of interpretation and perhaps study design.

(1) Most importantly, the authors need to provide additional validation of their conditional knockout models. The authors need to confirm that the Cdh5-CreER does not impact leptomeningeal fibroblasts and to confirm gene deletion in macrophages.

(2) The authors could also strengthen the paper by providing data on the impact of these conditional knockout models on the course of meningitis and bacterial burden.

(3) Finally, it is perhaps not surprising that Tlr4 is required for meningitis responses with E. coli. However, it is unclear if these findings can be generalised to other, more common, meningitis infections (streptococcal/pneumococcal).

(4) There are additional minor issues; for instance, the arachnoid fibroblast 2 population appears to closely resemble dural border cells.

(5) The cell line model (bEnd.3) is a relatively low-fidelity model of BBB endothelial cells, and this should be acknowledged.

With these caveats, it is difficult to be certain that the endothelium alone is the driver of meningeal immune responses in meningitis, and what the impact of these is.

Reviewer #1 (Public review):

Summary:

In brief, this manuscript addresses a very interesting topic, namely, the impact of the Mediterranean diet on the development of cancer. Using one mouse model and three tumor cell lines, the data show that a Mediterranean diet is sufficient to promote an anti-tumor response mediated by the microbiota, metabolites, and the immune system. Mechanistically, the Mediterranean diet promotes the expansion of Bacteroides thetaiotaomicron (B. theta for short), which converts tryptophan into 3-IAA. Both B. theta and the metabolite are sufficient to phenocopy the effect of the Mediterranean diet on cancer growth in vivo. The manuscript also shows that this effect is mediated by CD8 T cells and suggests, by way of in vitro assays, that 3-IAA sustains the functionality of CD8 T cells, preserving their exhaustion and blocking the ISR pathway.

Strengths:

The conclusions of this manuscript are potentially interesting and of potential clinical relevance.

Weaknesses:

For a full technical evaluation of the strength of the data, I am missing important technical and experimental details (e.g., number of independent experiments, statistics), and found some legends with potential labelling inaccuracies.

Reviewer #2 (Public review):

Summary:

The authors aimed to investigate the mechanistic link between a Mediterranean-mimicking diet (MedDiet)-specifically the synergy between high fiber and fish oil-and its ability to suppress tumor growth. They successfully identify that this dietary combination alters the gut microbiome to favor the expansion of Bacteroides thetaiotaomicron. This bacterium metabolizes dietary tryptophan into indole-3-acetic acid (3-IAA), which then acts systemically to prevent CD8+ T-cell exhaustion.

Strengths:

The study integrates controlled dietary interventions, microbiome perturbation, metabolite profiling, and immune functional analyses into a coherent and well-organized framework, making the overall logic of the work easy to follow. The dietary design is carefully controlled, allowing clear interpretation of which broad dietary features are associated with the observed antitumor effects. The immune dependence of the phenotype is addressed using appropriate experimental approaches, and the results broadly support a role for gut microbiota-derived metabolites in shaping immune cell function. In addition, analyses of human datasets provide important context and enhance the potential relevance and usefulness of the findings for a broader research community.

Weaknesses:

While the manuscript provides strong support for a role of the microbial metabolite indole-3-acetic acid and downstream stress signaling in shaping immune cell function, the upstream mechanism by which this metabolite exerts its effects remains unresolved. In particular, the specific molecular sensor or binding target through which the metabolite acts has not been identified, and this uncertainty limits mechanistic precision. Framing this point more explicitly as an open question would help align the interpretation with the current data.

In addition, at several points, the presentation may imply that a single microbial species is uniquely responsible for the observed effects. However, the experimental evidence more directly demonstrates sufficiency under the tested conditions rather than necessity. A clearer distinction between "sufficient" and "necessary" claims would help readers better assess the generality of the findings and their applicability to more complex microbial communities.

The interpretation of the human data also warrants some caution. The diet-associated score applied to human datasets is derived from gene-expression signatures identified in mouse models and therefore represents an indirect proxy rather than a direct measure of dietary intake. Although the score correlates with clinical outcomes, it does not establish that patient survival is driven by consumption of specific dietary components such as fiber and fish oil.

Reviewer #1 (Public review):

Summary:

In this manuscript, Zhang et al. demonstrate that depletion of the 18S rRNA m6A methyltransferase Mettl5 compromises translation fidelity and consequently increases neoantigen generation, thereby uncovering an unexpected role for Mettl5 in tumor immunity. Mettl5-KO tumors exhibit enhanced CD8⁺ T-cell infiltration and show improved responses to immune checkpoint blockade. Mechanistically, loss of Mettl5 perturbs the local structure of 18S rRNA and disrupts the ribosome's ability to perform accurate translation. Subsequent ribosome profiling and mass spectrometry analyses provide compelling evidence that Mettl5 functions as a previously unrecognized regulator of translation to participate in tumor immune evasion.

Strengths:

This study presents a comprehensive set of experimental data supporting a mechanistic link between rRNA modification, translation fidelity, and neoantigen generation. The observed synergistic effect of Mettl5 depletion and anti-PD-1 therapy highlights the potential translational relevance of targeting rRNA modifications in cancer immunotherapy.

Weaknesses:

(1) In light of the principal function of Mettl5, which is to methylate 18S rRNA within the small ribosomal subunit, the authors focus primarily on translation fidelity, largely associated with elongation, but provide limited exploration of potential effects on translation initiation. Loss of Mettl5 may alter the initiation landscape, potentially promoting alternative or noncanonical initiation events (e.g., initiation at CUG codons), which could also contribute to the observed neoantigen repertoire changes. Further investigation into initiation-level alterations would strengthen the mechanistic interpretation.

(2) Given the broad involvement of rRNA methyltransferases in ribosome function, the authors should incorporate a parallel analysis using another enzyme (e.g., Zcchc4 or Nsun5) as a negative control. Such an experiment is essential to demonstrate that the tumor immunity phenotype observed is specific to Mettl5 rather than a general consequence of perturbing rRNA modification.

Reviewer #2 (Public review):

Summary:

This study demonstrates that METTL5-mediated rRNA m⁶A1832 modification regulates tumor neoantigen generation by maintaining translational fidelity. Loss of METTL5 in tumor cells promotes immune cell infiltration into the tumor microenvironment and enhances the therapeutic efficacy of anti-PD-1 treatment, identifying a novel and potentially important target for cancer immunotherapy.

Strengths:

In murine tumor models, the authors found that Mettl5 depletion increases CD8⁺T cell infiltration and T cell receptor (TCR) repertoire diversity, and revealed a novel mechanism by which reduced ribosomal translation fidelity enhances non-canonical translation, thereby promoting the production of tumor neoantigens.

Weaknesses:

(1) While Mettl5 knockout enhances T-cell infiltration into tumors, it remains unclear whether loss of Mettl5 affects the expression of chemokines involved in immune cell recruitment.

(2) Although the authors report a significant reduction in tumor cell growth as well as tumor volume and weight, direct evidence demonstrating T-cell-mediated cytotoxicity is lacking.

Reviewer #1 (Public review):

This is an excellent paper from Dr. Yokoyama and colleagues. The experiments are technically demanding, given the very low cell numbers and the challenges of working with implantation sites at gestational days 6.5, 10.5, and 14.5. Overall, the impact of TGF-β receptor II deficiency in the NK lineage on uterine trNK cell numbers and litter size is convincing, and the authors' conclusions are well supported by the data. Less convincing, however, is the claim that the decrease in trNK cells is compensated by an increase in cNK cells; rather, the absence of TGF-β receptor II appears to result in an overall reduction of NK/ILC1 cells.

Major Points:

(1) Figure 1A and B

Although a trend is evident, it does not appear that the absolute number of cNK cells at day 14 is significantly changed from day 6.5?

(2) Figure 2E

The authors state, "This reduction of uterine trNK cells was accompanied by a concomitant increase in the absolute number and frequency of CD49b+Eomes+ cNK cells within the pregnant uterus of TGF-βRIINcr1Δ dams (Figure 2 D, E). The number of cNK cells appears relatively low (visually ~1,000-1,300), and although the difference is statistically significant, its physiological relevance is unclear. More importantly, this modest increase does not correlate with the marked decrease in trNK and ILC1 populations, as cNK cells do not appear to accumulate. In my opinion, the conclusion "Collectively, these findings indicate that a TGF-β-driven differentiation pathway directs the conversion of peripheral cNK cells into uterine trNK cells during murine pregnancy" should be slightly toned down.

(3) Figures 2-4

It is unclear whether the littermate controls are floxed mice or floxhet-Ncr1iCre mice? This distinction is important, as Ncr1iCre expression itself could potentially lead to a phenotype.

Reviewer #2 (Public review):

In their manuscript "TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy", Yokoyama and colleagues investigate the role of Tgfbr2 expression by NK cells in the formation of tissue-resident uterine NK cells and subsequent importance in murine pregnancy. By transferring congenic splenic conventional NK cells into pregnant mice, they show conversion of circulating NK cells into uterine ivCD45 negative tissue-resident NK cells. When interfering with the formation of uterine trNK cells, spiral artery remodelling was impaired, fetal resorption rates were increased, and litter sizes were reduced.

Generally, this is a research topic of high interest, yet the manuscript is lacking detailed mechanistic insights, and some questions remain open. At the current state, the data represent an interesting characterisation of the Tgfbr2-fl/fl Ncr1-Cre mice in pregnancy, but considering (a) the recent publication by the group (Reference 17) on the role of Eomes+ cNK cells during pregnancy, (b) the previously described role of Tgfbr2 and autocrine TGFb expression for uterine NK cell differentiation in virgin mice (also cited by the authors), and (c) the well-known relevance of uterine NK cells during pregnancy, additional experiments addressing the specific role of Tgfb during pregnancy would help to improve novelty and significance of the manuscript. To this end, the following aspects should be discussed and, where applicable, experimentally addressed by the authors:

(1) The authors suggest cNK extravasation and local differentiation into iv- trNK.

Can it be estimated how much this process contributes to the trNK pool vs. a potential local proliferation of already existing trNK? How do absolute numbers of CD49a+ Eomes+ trNK change during pregnancies? (In Figure 1A, the cell numbers of CD49a+ Eomes+ trNK seem to go down dramatically between gd 6.5 and 14.5). The plot in 1B could also include absolute numbers of ILC1s and trNKs. Would recruited cNK cells compensate for a potential loss of CD49a+ Eomes+ trNK?

(2) Figure 1C: 2.5

Mio cNK cells have been transferred, but only very few cells can be detected within the uterus (concatenated FACS plot shown). What may represent the limit to generate uterine trNK out of cNK? Is the niche supporting cNK-trNK differentiation limited? Is it only a specific subset of (splenic) cNK capable of differentiating into trNK? Is gd 0.5 the optimal timepoint for the transfer? Is there continuous recruitment of cNK into the uterus and differentiation into trNK, or is it enhanced at specific timepoints of pregnancy? Could there be local proliferation of cNK-derived trNK? This could be studied by proliferation dye dilution of WT cNK cells in this transfer-setup.

(3) The authors should consider inducible Tgfbr2 deletion (e.g. with Tamoxifen-inducible Cre) to enable development of the uterine NK compartment in virgin mice and only ablate trNK differentiation during pregnancy. This could help to estimate the turnover of cNK into trNK, or to understand if constant cNK recruitment is required to form the uterine trNK compartment during pregnancy.

(4) Did the authors consider transfer of Tgfbr2-floxed Ncr1-Cre cNK in the same setup as in Fig. 1C? This experiment could confirm the requirement of Tgfbr-dependent signalling for cNK to trNK conversion during pregnancy versus effects of Tgfb signals on trNK numbers in the uterus at steady state (before pregnancy).

(5) Figures 2D/E

The authors should state that ILC1s are reduced in the virgin uterus of female Tgfbr2-floxed or Tgfb1-floxed Ncr1-Cre mice and cite the relevant work (the Ref #29 discussed in this context did not show that?). It would be helpful to include an analysis of all three uterine ILC subsets in steady state. This could help to answer the question if the cNK cell changes are pregnancy-specific or a general phenomenon in Tgfbr2-floxed Ncr1-Cre mice.

(6) Figure 2E

Please phrase more carefully about the "concomitant increase" of cNKs, since this increase is much less pronounced compared to the very strong reduction (absence) of trNKs in Tgfbr2-floxed Ncr1-Cre mice. Do the authors suggest that cNKs are halted at this stage and cannot differentiate into trNK, based on these data?

(7) Figure 3/4

Can the reduced litter size and the abnormal spiral artery formation be rescued by transfer of WT cNK into Tgfbr2-floxed Ncr1-Cre mice?

Reviewer #1 (Public review):

The manuscript titled "The distinct role of human PIT in attention control" by Huang et al. investigates the role of the human posterior inferotemporal cortex (hPIT) in spatial attention. Using fMRI experiments and resting-state connectivity analyses, the authors present compelling evidence that hPIT is not merely an object-processing area, but also functions as an attentional priority map, integrating both top-down and bottom-up attentional processes. This challenges the traditional view that attentional control is localized primarily in frontoparietal networks.

The manuscript is strong and of high potential interest to the cognitive neuroscience community. Below, I raise questions and suggestions to help with the reliability, methodology, and interpretation of the findings.

(1) The authors argue that hPIT satisfies the criteria for a priority map, but a clearer justification would strengthen this claim. For example, how does hPIT meet all four widely recognized criteria, such as spatial selectivity, attentional modulation, feature invariance, and input integration, when compared to classical regions such as LIP or FEF? A more systematic summary of how hPIT meets these benchmarks would be helpful. Additionally, to what extent are the observed attentional modulations in hPIT independent of general task difficulty or behavioral performance?

(2) The authors report that hPIT modulation is invariant to stimulus category, but there appear to be subtle category-related effects in the data. Were the face, scene, and scrambled images matched not only in terms of luminance and spatial frequency, but also in terms of factors such as semantic familiarity and emotional salience? This may influence attentional engagement and bias interpretation.

(3) The result that attentional load modulates hPIT is important and adds depth to the main conclusions. However, some clarifications would help with the interpretation. For example, were there observable individual differences in the strength of attentional modulation? How consistent were these effects across participants?

(4) The resting-state data reveal strong connections between hPIT and both dorsal and ventral attention networks. However, the analysis is correlational. Are there any complementary insights from task-based functional connectivity or latency analyses that support a directional flow of information involving hPIT? In addition, do the authors interpret hPIT primarily as a convergence hub receiving input from both DAN and VAN, or as a potential control node capable of influencing activity in these networks? Also, were there any notable differences between hemispheres in either the connectivity patterns or attentional modulation?

(5) A few additional questions arise regarding the anatomical characteristics of hPIT: How consistent were its location and size across participants? Were there any cases where hPIT could not be reliably defined? Given the proximity of hPIT to FFA and LOp, how was overlap avoided in ROI definition? Were the functional boundaries confirmed using independent contrasts?

Comments on revisions:

The authors have successfully addressed my previous questions and concerns. The public comments above reflect my views on the initial submission and, in my opinion, will remain helpful for general readers. Given this, I do not have additional public comments and will keep my previous public review unchanged.

Reviewer #2 (Public review):

Summary

This study investigates the role of the human posterior inferotemporal cortex (hPIT) in attentional control, proposing that hPIT serves as an attentional priority map that integrates both top-down (endogenous) and bottom-up (exogenous) attentional processes. The authors conducted three types of fMRI experiments and collected resting-state data from 15 participants. In Experiment 1, using three different spatial attention tasks, they identified the hPIT region and demonstrated that this area is modulated by attention across tasks. In Experiment 2, by manipulating the presence or absence of visual stimuli, they showed that hPIT exhibits strong attentional modulation in both conditions, suggesting its involvement in both bottom-up and top-down attention. Experiment 3 examined the sensitivity of hPIT to stimulus features and attentional load, revealing that hPIT is insensitive to stimulus category but responsive to task load - further supporting its role as an attentional priority map. Finally, resting-state functional connectivity analyses showed that hPIT is connected to both dorsal and ventral attention networks, suggesting its potential role as a bridge between the two systems. These findings extend prior work on monkey PITd and provide new insights into the integration of endogenous and exogenous attention.

Strength

(1) The study is innovative in its use of specially designed spatial attention tasks to localize and validate hPIT, and in exploring the region's role in integrating both endogenous and exogenous attention, as prior works focus primarily on its involvement in endogenous attention.

(2) The authors provided very comprehensive experiment designs with clear figures and detailed descriptions.

(3) A broad range of analyses was conducted to support the hypothesis that hPIT functions as an attentional priority map -- including experiments of attentional modulation under both top-down and bottom-up conditions, sensitivity to stimulus features and task load, and resting-state functional connectivity. These analyses showed consistent results.

(4) Multiple appropriate statistical analyses - including t-tests, ANOVAs, and post-hoc tests-were conducted, and the results are clearly reported.

Comments on revisions:

The authors have addressed our comments in their revised manuscript and in their response to the reviewers. We don't have any further suggestions or comments.

Reviewer #1 (Public review):

Summary:

The article investigates how the Japanese macaque makes gait transitions between quadruped and biped gaits. It presents a compelling neuromechanical simulation that replicates the transition and an interesting analysis based on an inverted pendulum that can explain why some transition strategies are successful and others are not.

Strengths:

I enjoyed reading this article. I think it presents an interesting study and elegant modeling approaches (musculoskeletal + inverted pendulum). The study is well conducted, and the results are interesting. I particularly liked how the success of gait transitions could be predicted based on the inverted pendulum and its saddle node stability. I think it makes a useful and interesting contribution to the state of the art.

Weaknesses:

The article is already in great shape, but could be improved a bit by:

(1) Strengthening the comparison to animal data. In particular, videos of the real animal should be included + snapshots of their gaits (quadruped, biped, and transitions).

(2) Exploring and testing a broader range of conditions. I think it would be very interesting to test gaits and gait transitions on up and down slopes (both with the musculoskeletal model and with the inverted pendulum model). This could be used to make predictions on how the real animal adapts to those conditions. Ideally, this should be tested on the animal as well. I think this could increase (even more) the impact of this work.

(3) Better explaining several aspects of the PSO optimization.

(4) (Ideally) performing a sensitivity analysis on the optimized parameters (e.g. variations of +-5, 10, 20%) in order to determine their respective importance and how much their instantiated values have influenced the results.

(5) Running a spell checker, as there are quite a few typos.

Reviewer #2 (Public review):

Summary:

This article presents a neuromusculoskeletal (NMS) model of the Japanese Macaque. This model is added with a neural feedforward controller based on CPG and synergy that allows for reproducing quadrupedal and bipedal gait as well as the transition between quadrupedal and bipedal gait. The model and controller were validated using experimental data. Results were also compared to an inverted pendulum model to show that the transition between quadrupedal and bipedal in macaque is using this kind of representation for transition and stability. Overall, the article is very interesting, but it sometimes lacks clarity.

Strengths:

The results of the model present impressive results for quadrupedal, bipedal, and transition, validated by experimental data. NMS controllers based on feedforward controllers are very difficult to fine-tune.

Weaknesses:

(1) The movement regulator is not clear and should be better explained. At first, it seems that it is just a new CPG/synergy (feedforward) added, but in the methods, it seems to be a feedback controller.

(2) It is also not clear what is meant by discretizing the weight for the trigger limb from 0 to 1 (page 8).

(3) The controller is mainly using a feedforward controller, allowing only anticipatory movement. Animals are also using a reflex-based feedback controller. A controller with feedback/reflex could reduce failed attempts in training and better represent the transition.

(4) There are small typos throughout the article that should be corrected.

Reviewer #3 (Public review):

Summary:

The purpose of this study was to test the hypothesis that the inverted pendulum mechanism contributes to the gait transition from quadrupedal to bipedal gait in Japanese macaques. The author uses a neuromusculoskeletal model to generate different motor tasks by varying motor command parameters during forward dynamics simulations. After simulations were done, the authors used dynamical system analysis of the inverted pendulum model to reveal the underlying principles of gait transition control. The authors showed that successful gait transition from quadrupedal to bipedal gait mostly depends on increased step length of a hindlimb.

Strengths:

This study is important not only for understanding gait transition, but also to understand stability control of bipedal gaits. Another advantage of this study is that it allows us to estimate the effect of one control mechanism and find its effect and limits. In animal studies, we also have a combination of compensatory stability control mechanisms.

Weaknesses:

Any simulation is not perfect, so discrepancies from experimental data are expected. A 2D model is used, but the advantage of using a 3D model is not clear, and it is much more complicated.

Reviewer #1 (Public review):

Summary:

Hoverflies are known for a striking sexual dimorphism in eye morphology and early visual system physiology. Surprisingly, the male and female flight behaviors show only subtle differences. Nicholas et al. investigate the sensori-motor transformation of sexually dimorphic visual information to flight steering commands via descending neurons. The authors combined intra- and extracellular recordings, neuroanatomy, and behavioral analysis. They convincingly demonstrate that descending neurons show sexual dimorphisms - in particular at high optic flow velocities - while wing steering responses seem relatively monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1, which receives inputs from so-called HS cells, and OFS DN2, which receives input from a set of VS cells. Their likely counterparts in Drosophila connect to the neck, wing, and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude, and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - are complex and less straightforward than one might expect.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known about hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled the hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship, while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver a comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Weaknesses:

Cell-typing using receptive field preferred directions (RFPDs): if I understood correctly, this classification method mostly relies on the LPDs near the center of the receptive field (median within the contour in Fig.1). I have two concerns here. First, this method is great if we are certain there are only two types of visual DNs as described in the manuscript. But how certain is this? Given the importance of vision in flight control, I would expect many DNs that transmit optic flow information to the motor center. I'd also like to point out that there are other lobula plate tangential cells (LPTCs) than HS and VS cells, which are much less studied and could potentially contribute to dimorphic behaviors. Second, this method feels somewhat impoverished given the richness of the data. The authors have nicely mapped out the directional tuning for almost the entire visual field. Instead of reducing this measurement to 2 values (center and direction), I was wondering if there is a better method to fully utilize the data at hand to get a better characterization of these DNs. As the authors are aware, local features alone can be ambiguous in characterizing optic flows. What's more, taking into account more global features can be useful for discovering potentially new cell types.

Line 131, it wasn't clear to me why full-screen stimuli were used for comparison here, instead of the full receptive field maps. Male flies exhibit sexual dimorphic behaviors only during courtship, which would suggest that small-sized visual stimuli (mimicking an intruder or female conspecific) would be better suited to elicit dimorphic neuronal responses. A similar comment applies to the later results as well. Based on the receptive field mapping in Figure 1, I'm under the impression that these 2 DN types are more suited to detect wide-field optic flows, those induced by self-motion as mentioned in the manuscript. The results are still very interesting, but it's good to make this point clear early on to help set appropriate expectations. Conversely, this would also suggest that there are other visual DN types that are responsible for the courtship-related sexually dimorphic behaviors.

Reviewer #1 (Public review):

Summary:

The dysgranular retrosplenial cortex (RSD) and hippocampus both encode information related to an animal's navigation through space. Here, the authors study the different ways in which these two brain regions represent spatial information when animals navigate through interconnected rooms. Most importantly, they find that the RSD contains a small fraction of neurons that encode properties of interconnected rooms by firing in different head directions within each room. This direction is shifted by 180 degrees in 2-room environments, and by 90 degrees in 4-room environments. While it cannot be definitively proven that this encoding is not just related to the presence of exits (doors) in each room, this is a noteworthy finding and will motivate further study in more complex and well-controlled environments to understand this coding scheme in the RSD. The recordings and analyses used to identify these multi-directional cells are mostly solid. Additional conclusions regarding the rotational symmetry across rooms seen in the RSD neurons that do not encode direction (representing the majority of RSD neurons) remain incomplete, given the evidence presented thus far. The differences between RSD and hippocampus encoding of space are clear and consistent with prior observations.

Strengths:

(1) Use of tetrode recordings from the RSD to identify multi-direction cells that only encode one direction in each room, but shift the preferred direction by either 180 or 90 degrees depending on the number of rooms in the environment.

(2) Solid controls to show that this multi-direction encoding is stable over time and across some environmental manipulations.

(3) Convincing evidence that these multi-direction cells can co-exist with single-direction head direction cells in the RSD (as both cell types can be simultaneously recorded).

(4) Convincing evidence for clear differences between directional and spatial encoding in the RSD versus hippocampus, consistent with prior observations.

Weaknesses:

(1) The paper mostly uses the term "retrosplenial cortex", but it is important to clarify that the study is only focused on the dysgranular retrosplenial cortex (RSD; Brodmann Area 30) and not the granular retrosplenial cortex (Brodmann Area 29). These are two distinct regions (despite the similar names), each with distinct connectivity and distinct behavioral encoding and function, so it is important to clarify in the abstract and title that the present study is solely about the RSD to prevent confusion in the literature.

(2) The proportion of each observed cell type is not clearly stated, although it is clear that the multi-directional cells are in the minority. Having the proportion of well-isolated neurons in distinct sessions that encode each type of information (e.g., multi vs single direction encoding) would greatly aid the interpretation of the result and help the field know how common each cell type is in the RSD.

(3) The authors state that "MDCs [multi-directional cells] never exhibited multidirectional activity within a single room" - but many of the single room examples from the 4-room environment (shown in Figures 2E and 2F) reveal multi-peaked directional encoding. This suggests that the multi-direction encoding may be more compatible with encoding some property of the number of exits rather than relative room orientations.

(4) The spatial rotation analyses of non-directional cell analyses are considered incomplete. This is impacted by the slower speed at the doors and hence altered firing rates (as evidenced in spatial rate plots). The population rate is not relevant as the correlational analyses are done on a single cell level. Since some cells fire more with increasing speed and others fire less, that will necessarily result in a population rate map that minimizes firing rate differences near the doorway, where the animals move more slowly. But on a single cell level, that reduced speed is having a big effect, as evidenced by individual rate map examples, and the rooms will need to be rotated to obtain a higher correlation by overlapping the doorway regions. This does not necessarily say anything about spatial coding across the two or four interconnected rooms being rotationally symmetric, and it would appear difficult to draw any conclusions related to spatial encoding from those analyses.

Reviewer #2 (Public review):

Summary:

Laurent et al. perform in vivo electrophysiological recordings in the retrosplenial cortex of rats foraging in multi-compartment environments with either identical or unique visual features. The authors characterize two types of directional signals in the area that they have previously reported: classic head direction cells anchored to the global allocentric reference frame and multi-direction cells (MDCs), which have a rotationally preserved directional field anchored to local compartments. The primary finding of this work is that MDCs seem sensitive to local environmental geometry rather than visual context. They also show that MDC tuning persists in the absence of hippocampal place field repetition, further dissociating the RSC local directional signal from the broader allocentric representation of space. A novel observation is that RSC non-directional spatial signals are anchored to the local environment, which could and should be explored further. While the data is solid and the analyses are mostly appropriate, the primary findings are incremental, and more interesting novel claims are not explored in detail or not explicitly tested.

Strengths:

The environmental manipulations clearly demonstrate that tuning is not modulated by complex visual information.

The finding that RSC two-dimensional spatial responses are stable and anchored to environmental features is novel and can be further explored in future work.

Weaknesses:

The observation that BDCs and MDCs are insensitive to visual context builds upon the author's previous work (and replicates aspects of Zhang et al., 2022) but leaves many open questions that are not addressed with the current set of experiments. Specifically, what exactly are MDCs anchoring to? The primary theory is that they anchor to environmental geometry, but there are no explicit experimental manipulations to test this theory. It is important to note that 2- and 4-compartment environments share many features, including the same cardinal axes, making any differences/similarities in these two conditions difficult to interpret.

The main finding presented with respect to BDC/MDs tuning is that they are not sensitive to visual context as manipulated by distinct visual patterns on the wall and floor in multicompartment environments. One could argue that the individual rooms are, in actuality, quite similar in low-level visual features - each possesses a large white background square visual feature on a single wall with a fixed relationship to the door(s). How can the authors rule out that i) BDC/MDC responses are modulated by these low-level features rather than geometry and/or ii) that the rats are not paying attention to any visual features at all? There is no task requiring them to indicate which room they are in. Furthermore, the doorways themselves are prominent visual features that are present in each context. It would be interesting to see if MDC/BDC tuning persisted in a square room where the number of doorways was manipulated to rule out this possibility.

A strong possibility is that the rotational symmetry of both MDCs and non-directional spatial neurons is related to i) door-related firing, 2) stereotyped movement, and 3) stereotyped directional sampling. In Supplemental Figure 8, the authors begin to address this by comparing a 'population ratemap' to a 'population speed map.' I do not think this is sufficient and is difficult to interpret. Instead, the authors should assess whether MDC and BDCs fire more at doorways and what the overlap is with the speed-modulated cells they report. Moreover, they should assess whether the spatial speed profile itself is rotationally symmetric within each session. It would also be useful to look at the confluence of the variables simultaneously using some form of regression analysis. The authors could generate a directional predictor that captures the main response property of these cells and see if it accounts for greater variability in spiking than speed or x,y position. Finally, rotationally symmetric directional sampling biases could arise from the doors being present on the same two walls in each room. The authors should assess whether MDC tuning is still present if directional sampling is randomly downsampled to match directional observations in each compartment.

Recent work has demonstrated that neurons with egocentric corner or boundary tuning are observed in RSC. The authors do not address whether egocentric tuning contributes to MDC signals. An explicit analysis of the relationship and potential overlap of MDC and egocentric populations is warranted.

Many of the MDCs presented in the main figures are not especially compelling. This includes alterations to MDC tuning in Figure 2, which is a key datapoint. The authors should show significantly more (if not all) examples of MDCs in each environment. It would similarly be useful to see all/more examples of non-directional spatially tuned neurons with rotationally symmetric firing patterns.

"One might hypothesize that specific environmental cues, such as door orientation or landmark positioning, drive these tuning shifts. However, our results argue against this interpretation. In four-room environments, each room had multiple entry points, yet MDCs never exhibited multidirectional activity within a single room."

I do not understand the logic here. Can the authors unpack this? Also, it is clear that some of the example cells have more than one peak in individual compartments. How is this quantified?

Reviewer #3 (Public review):

Summary:

The authors examine firing of dysgranular retrosplenial cortex (dRSC) neurons in relation to head orientation and location for rats exploring open-field environments. One environment utilized was a square arena with high walls that is split into two rectangular spaces connected by a doorway. Another environment is a square arena split into quadrants connected by doors near the center. For each, the different sub-spaces of the environments are either identical in terms of visual and tactile cues or different. For head direction neurons, the authors present one population where each neuron maintains a single tuning direction for the two or four sub-compartments of the two environments. A second population exhibits what is termed multi-directional firing, wherein neurons exhibit (overall) two or four head direction peaks in firing. For such neurons, firing in each of the sub-compartments is associated with only a single preferred direction, but the directions across compartments are shown to be at 180-degree (two-compartment environment) or 90-degree offsets. The offsets evidence tuning to the "same" orientation for the sub-compartments that are, in the global reference frame, oriented at 180 or 90 degree offsets. The results are similar whether or not the sub-compartments have the same or different tactile and visual cues. Thus, the first population is said to be global in its head direction tuning, while the second relates to each local environment in a way that is systematic across sub-compartments. Spatially-specific activity of another population of non-direction-tuned RSC neurons is examined, and comparisons of sub-compartment spatial firing maps suggest that spatial tuning in RSC also repeats across compartments when the firing maps for the compartments are rotated to match each other (as in physical space). Finally, a population of hippocampal "place" cells exhibited different location mapping across sub-compartments. The findings are interpreted to indicate that RSC can simultaneously map orientation in both local and global reference frames, possibly forming a mechanism whereby the sub-compartments' shared geometry (given by the boundary shapes and the door locations) can be related to each other and to the global space they share.

Strengths:

This paper addresses an interesting problem and expands how the field will think about directional tuning.

Weaknesses:

It is not clear that the experimental design allows for a clear interpretation of the data. Rates for preferred turning are low, as are ratemap correlations for spatially-tuned neurons.

(1) It is concerning that the neurons with head direction tuning have fairly low peak firing rates (mean close to 5 Hz), where prior studies examining head direction tuning in dRSC found head direction-tuned neurons with peak rates more than an order of magnitude higher (100 Hz or more). Under circumstances where neurons are tuned well to variables other than head direction (for example, angular velocity of movement), weak head direction tuning may be observed if those other variables are not sampled equally across head directions. The manuscript contains no rigorous control for this possibility. One place to start to address this issue would be to map out variables such as angular velocity by head orientation, and to test whether such relationships also carry 90 and 180 degree offsets.

(2) There is some question as to whether dRSC neurons (spatial or directional) following the sub-compartment "geometry" is appropriate in terms of interpreting the data. In the condition with sub-compartments carrying different tactile and visual cues, it seems that such cues pertain only to the floor of the environments. The distal visual space of the boundaries appears to be identical. One is left to wonder whether distinguishing environments according to boundary wall visual cues would lead to different results. The CA1 data does not help to rule this possibility out. A second reason to doubt the "shared geometry" interpretation is that there is no condition where sub-compartment geometry is varied. It is also the case that the sub-compartment doorways may stand as the only salient distal visual cue linking the environments. Local sensory cues and geometry seem not so disentangled in this study, but this is a major claim in the abstract.

(3) There is some concern with the interpretation that the spatial tuning of some dRSC neurons repeats in rotated form across sub-compartments. The firing rate map correlations are very low on average (~0.2), and far lower than the population of CA1 having repeating fields across the same vs different visual/tactile cue conditions. The authors should define the chance level of ratemap correlation by shuffling neuron identities. Apologies if this is indeed the current approach, but it seems not to be (I was left a bit lost by the description in the methods). For any population of hippocampal place cells, the cross-neuron correlations of firing rate maps are typically not zero, and correlations at 0.2 would normally be evidence for remapping.

(4) A somewhat picky point here that is not meant to claim that multi-compartment studies are not useful - the introduction states that real-world environments typically consist of multi-compartment rooms. This is certainly not true for rodents and is only sometimes true in humans.

(5) The discussion lacks a consideration of how such dRSC output might impact the target structures of dRSC.

(6) The discussion speaks to the idea that multi-directional neurons may aid in transitioning between contexts (sub-compartments). But it is notable that none of the multidirectional neurons have multi-directional tuning in all sub-compartments, but such firing was seen in the 2017 Nature Neuroscience study by Jacob/Jeffery. The discussion should address this difference and perhaps posit a means by which the firing of global and local head direction neurons can be related to each other to yield navigation that depends on both scales.

(7) The authors should provide the size of the smoothing function for spatial firing rate maps.

(8) The authors should devise a measure to define directional tuning in 4 directions (with 90-degree offsets).

(9) Figures 2D and 2H - The offsets in preferred tuning across sub-compartments are rather variable.

Reviewer #1 (Public review):

Summary:

This manuscript presents a tunable Bessel-beam two-photon fluorescence microscopy (tBessel-TPFM) platform that enables high-speed volumetric imaging with stable axial focus. The work is technically strong and broadly significant, as it substantially improves the flexibility and practicality of Bessel-beam-based two-photon microscopy. The demonstrations are generally strong and bridge a wide range of neuroimaging applications, namely vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. These convincingly show that the approach enables biological measurements that are difficult or impractical with existing methods.

The evidence supporting the technical and biological claims is generally strong. The optical design is carefully motivated, clearly described, and validated through a combination of simulations and experimental characterization. The biological applications are diverse and well chosen to highlight the strengths of the proposed method, and the data are of high quality, with appropriate controls and comparative measurements where relevant.

Strengths:

(1) The optical innovation addresses a well-recognized limitation of existing Bessel-TPFM implementations, namely axial focus drift during tuning, and does so using a relatively simple, light-efficient, and cost-effective design.

(2) The manuscript provides convincing experimental evidence for this being a versatile platform to map flow dynamics across diverse vessel sizes and orientations in both healthy and pathological states.

(3) Biological demonstrations are comprehensive and span multiple domains such as hemodynamics, neurovascular coupling, and neuroimmune responses.

(4) Quantitative analyses of blood flow across vessel sizes and orientations, including kilohertz line scanning, are particularly compelling and clearly beyond the reach of standard Gaussian TPFM.

(5) Particular advantages are that higher blood slow speeds become measurable up to 23mm/sec (20x more than conventional frame scanning), and that simultaneous (Bessel-)imaging and (Gaussian-)perturbation are possible because of the stable axial focus.

Weaknesses:

(1) At present, the paper does not properly position the new Bessel-beam method against previous work, and fails to compare it to alternative fast volumetric imaging methods without Bessel beams.

(2) The cost-effectiveness of the proposed method is not well described or supported by evidence; it would be useful to include more detail or remove this claim.

(3) Some biological conclusions, e.g., regarding novel features of microglial dynamics (i.e., the observed two-wave responses and coordinated extension-retraction), are based on relatively limited sample size and would benefit from clearer discussion of variability across animals and fields of view.

(4) The use of neural network-based denoising for microglial imaging is reasonable but introduces potential concerns about trustworthiness; additional clarification of validation or failure modes would strengthen confidence in these results.

To conclude, most of the authors' claims are well supported by the data. The central conclusion, namely that tBessel-TPFM provides tunable volumetric imaging enabling experiments not feasible with existing two-photon approaches, is justified. Some biological interpretations would benefit from a more cautious framing, but they do not undermine the main technical and methodological contributions of the study. This is a strong and technically rigorous manuscript that makes a substantial methodological advance with clear relevance to neuroscience and intravital imaging. Minor clarifications and a slightly more measured discussion of certain biological findings are recommended.

Reviewer #2 (Public review):

Summary:

The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.

Strengths:

A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across several in vivo applications, including neurovascular imaging, blood flow measurements, optogenetic stimulation, and microglial dynamics.

Weaknesses:

In the in vivo demonstrations, the authors employ different Bessel beam configurations across experiments, but the beam parameters are not dynamically tuned during live imaging. A video example showing continuous or interactive tuning of the Bessel beam within a single in vivo imaging sequence would further highlight the practical advantages of this platform and strengthen the case for its potential applications. In addition, while excitation powers are reported, the manuscript does not place these values in the broader context of known photodamage thresholds for two-photon microscopy, which would be helpful to the readers. Denoising/image restoration are applied in one of the in vivo examples, but it is unclear why this step was used specifically for this dataset and whether it was necessary to achieve adequate SNR or primarily included as an additional demonstration.

Reviewer #3 (Public review):

Summary:

The manuscript presents an elegant and cost-effective approach for generating a tunable Bessel beam on a conventional two-photon microscope. The authors assemble a compact optical module comprising three axicons and a series of lenses that permits rapid adjustment of both lateral resolution and axial extent without modifying the focal plane. This flexibility enables the system to be readily adapted to a variety of biological preparations. As a proof of concept, the authors employ the device to record blood flow velocities in cortical microcapillaries, arterioles, and venules, thereby directly visualizing vasodilatation and vasoconstriction dynamics and permitting quantitative analysis of neurovascular coupling across cortical layers in awake mice.

The authors demonstrate that the tunability of the Bessel beam can be exploited to match the numerical aperture to the vessel type: a high NA configuration, albeit slower scan, is optimal for resolving flow in capillaries, whereas a low NA setting provides faster acquisition suitable for arterioles and venules. By implementing a one-dimensional line scan with the Bessel beam, they achieve an imaging speed that is twentyfold faster than conventional frame-by-frame scanning, which proves sufficient to capture hemodynamic transients before and after an induced ischemic stroke.

In addition to pure observation, the authors integrate a co-propagating Gaussian line to the system, allowing simultaneous imaging and photostimulation within the same focal plane. This capability addresses a common limitation of other Bessel beam implementations, in which the observation and perturbation planes often become misaligned when the Bessel beam is altered. The manuscript also emphasizes the advantage of Bessel beam excitation for calcium imaging after a perturbation, because it captures neuronal activity in planes both above and below the nominal focal plane, signals that would be missed with a standard Gaussian focus. Finally, the authors apply the technique to investigate the neuroimmune response following targeted microglial ablation; they report that adjacent microglia extend processes toward the injury site while retracting processes in the opposite direction.

Overall, the work offers a technically straightforward yet powerful extension to existing two-photon platforms, providing high-speed, volumetric imaging and stimulation capabilities that are well-suited to a broad range of neurovascular and neuroimmune studies. The experimental validation is quite thorough, and the presented data convincingly illustrates the benefits of the approach.

Strengths:

The authors present a truly clever and inexpensive optical module that can be integrated into almost any two-photon microscope, providing a tunable Bessel beam with a minimal modification of the existing system. The experimental data and accompanying quantitative analysis convincingly demonstrate that the system can reveal physiological events, such as capillary flow, calcium transients across multiple axial planes, and microglial process dynamics, that are difficult or impossible to capture with a conventional Gaussian beam. The breadth of experiments chosen for the manuscript illustrates the practical utility of the device and supports the authors' conclusions that it extends the functional repertoire of standard two-photon microscopy.

Weaknesses:

The manuscript would benefit from a more detailed contextualisation of the claimed speed advantage. Although the authors mention other techniques in the introduction, they do not provide any direct comparison with other state-of-the-art high-speed two-photon approaches such as light beads microscopy (Demas et al., Nat. Methods 2021), temporal multiplexing schemes (Weisenburger et al., Cell 2019), or random access microscopy (Villette et al., Cell 2019). A brief comparison of imaging speed, spatial resolution, and instrumental complexity would enable readers to assess the relative merits of the present method.

A second limitation that warrants discussion is the inherent trade off between volumetric coverage and image specificity. Because the Bessel beam excites fluorescence throughout an extended axial range, the detector inevitably integrates signal from a three dimensional volume into a two dimensional image. In densely labelled tissue, this can lead to significant signal crosstalk, reducing contrast and complicating quantitative interpretation. A brief analysis of how labeling density affects the fidelity of flow or calcium measurements, or suggestions for mitigating crosstalk (e.g., computational deconvolution, adaptive excitation shaping, or combinatorial sparse labeling), would broaden the applicability of the technique.

Reviewer #1 (Public review):

Summary:

The study investigates the Drosophila non-visual light receptor rhodopsin7 with regard to its role in light information processing and resulting consequences for behavioral patterns and circadian clock function. Using behavioral, in situ staining, and receptor activation assays together with different fly mutants, the authors show that rhodopsin7 is an important determinant of activity under and response to darkness, which likely signals via a pathway distinct from other, visual Drosophila rhodopsins. Based on phylogenetic analysis, the authors further discuss a potentially conserved functional role of non-visual photoreceptors like rhodopsin7 and the mammalian melanopsin light information processing and circadian clock modulation.

Strengths:

The manuscript follows a very clear structure with all investigations logically building onto each other. Background information and methodology are provided in appropriate detail so that readers can fully understand why and how experiments were conducted. It is further praiseworthy that the authors provide the details that allow also non-experts in the field to fully understand their approaches. Experimental work was conducted in a highly standardized manner, and also considered potential "side-aspects" like the consequences of temperature cycles and changed photoperiods. The detailed and clear description of the obtained results makes them very convincing, with (almost) all observable patterns being addressed.

By highlighting the evolutionary old phylogenetic position of rhodopsin7 and its conservation across numerous clades, the authors provide strong reasoning for the relevance of their work, also pointing out the similarities to the mammalian melanopsin. The postulated hypothesis regarding protein structure and functioning, as well as the role in light information processing and behavioral and circadian clock modulation are well based on the authors' observations, and speculative aspects are correctly pointed out.

Weaknesses:

Where the manuscript still has potential for improvement is the discussion, which in its current form does seem slightly self-contained and does not fully integrate the findings of previous studies on Drosophila rhodopsin7. As the introduction specifically points out that previous findings have been contradictory, this seems like a missed opportunity. Further details on this are provided in the recommendations below.

Similarly, the manuscript currently lacks a discussion of the possible relevance of rhodopsin7 (and other non-visual light receptors in other organisms) in the context of a species' environment and lifestyle, i.e., what is the relevance/benefit of having rhodopsin7 in the fly's everyday life? While this clearly involves speculation, when done carefully, it can elevate the paper's relevance from a primarily academic to a societal one.

An additional point concerns the title and abstract, which postulate rhodopsin7 roles in contrast vision as well as motion and brightness perception. Contrast remains poorly defined in the text, leaving it ambiguous whether it refers to bright/dark contrasts, e.g., along edges, or the temporal contrast that results from dark pulses (startle response). While the latter seems to apply here, the former is likely more intuitive. Thus, this aspect should be rephrased (also in the title) or properly clarified early on. Regarding motion detection, this is backed up by the optomotor response results, but the findings stand somewhat isolated from the other results, lacking a clear connection aside from general visual processing. Lastly, brightness perception is mentioned in the abstract, but never again, possibly due to inconsistent phrasing throughout the manuscript.

Reviewer #2 (Public review):

Summary:

This is a very interesting paper bringing new and important information about the poorly understood rhodopsin 7 photoreceptive molecule. The very ancient origin of the gene is revealed in addition to data supporting a signaling pathway that is different from the one known for the canonical rhodopsins. Precise expression data, particularly in the optic lobe of the fly, as well as clear behavioral phenotypes in responses to light changes, make this study a strong contribution to the understanding of the still-debated function of rhodopsin 7.

Specific comments

(1) Title and abstract: Contribution of Rh7 to circadian clock regulation

(a) It is not that clear to me what rhodopsin does in terms of circadian regulation (even though its function might be circadianly regulated). The clear role in the light/dark distribution of activity might not be circadian per se, but mostly light/dark-driven, and there is no evidence here for a role in the entrainment of the clock.

(b) The authors should cite Lazopulo, which nicely shows that Rh7 has an important role in peripheral neurons to allow flies to escape from blue light (see below).

(2) Figure 2 C

The finding showing that Galphaz but not Galphaq can trigger signaling from light-excited Rh7 is a very intriguing finding to better understand Rh7 function. Since Galphaz is related to Gi/o, it would be interesting to test those, for example, by expressing RNAi with Rh7-gal4 and testing the Light-dark or light-off response behavior.

(3) Figures 3-4

The change in the locomotor activity distribution between light and dark in LD conditions provides a nice assay for Rh7 function. Since Lazopulo et al. (2019) have shown that wild-type but not Rh7 mutants do escape from blue light, it would be important to compare and discuss these LD behavior data with the Lazopulo results. Precisely, is this nighttime preference linked to blue light?

The expression data are really nice and show that Rh7 is mostly a non-retinal photoreceptor. However, the paper would be strongly reinforced by correlating this with the LD behavior. The LD phenotype should be tested in flies with Rh7 expression rescued under Rh7gal4 control (as done for the startle response). This is important to show whether the expression pattern is likely responsible for the described Rh7 function in LD. If L5 and or M11 drivers are available, they should be used to rescue Rh7? Since expression in some clock neurons is shown, the rescue experiment should also be done with a clock neuron driver.

In the same line, can the LD phenotype (or startle response phenotype of Figure 4) be restored by expressing Rh7 under ppk control, as shown for the blue light avoidance phenotype by Lazopulo et al?

Finally, the Rh7 "darkfly" rescued flies should be tested in LD.

Reviewer #3 (Public review):

Summary:

While our knowledge regarding visual opsins is largely very good, a lot more uncertainty exists around the role of non-visual opsins. Using the power of the Drosophila melanogaster model system, Kirsh et al. investigate the role of the non-visual opsin Rhodopsin7 (Rh7). Expression analysis, based on Rh7-Gal4>UAS-GFP and HRC in situ staining, reveals strong expression in the optic lobes and somewhat weaker, but nevertheless extensive expression in the brain. An investigation of motor activity reveals that loss of function leads to an altered day and night rhythm, specifically decreasing activity during the dark phase. These flies were also less sensitive, but still responsive to a light-induced startle response and showed deficiencies in the optomotor response. To further investigate how Rh7 may modulate these responses, inspired by the Dark line of flies (which were kept in the dark for ~1400 generations) and which has accumulated C-terminal related losses, the authors conducted rescues with an intact and a C-terminal-deficient Rh7 and were able to pinpoint that region as an important driver of related behavioral shifts. These findings are particularly intriguing as Rh7 represents an ancient opsin with phylogenetic and mechanistic parallels to mammalian melanopsin.

Strengths:

The paper is well-written and contains high-quality data with appropriate sample sizes, and the conclusions are well supported.

Weaknesses:

No weaknesses were identified by this reviewer, but the following recommendations are made:

(1) The authors should clarify exactly what tissues were taken for the comparative qPCR. This is particularly interesting in terms of the retina. Since Rh7 appears not to be expressed within the photoreceptor cells of the retina, this raises the important question as to which cells it is expressed in. To address this important question, it would also be helpful to include an expression analysis of the retina itself (by extending the RH7-GFP expression patterns and/or adding HCR in situ of the ommatidia array). The cell types of the retina are very well classified, and some evidence already exists for Rh7 expression in support cells (e.g., Charlton-Perkins et al., (2017); PMID: 28562601). This study has a unique opportunity to investigate this further by adding these critical data for a more complete picture of Rh7.

(2) Mammalian opsins should be included in the phylogenetic analysis illustrated in Figure 2A and indicate their position on the tree. This will allow readers to better put the authors' statements regarding the intermediate position of Rh7 into perspective. In addition, note that the distinction between red and deep red is easy to miss regarding the Rh7 cluster. Perhaps the authors could use a more distinct colour scheme, for example, orange and deep red.

(3) More details should be provided on the optomotor response experiments. Specifically, specifications of the frequencies used for the optomotor response are needed. Results show a relatively large level of variation, which may be due to different angular perspectives that flies may have had while viewing the stimulus. If possible, provide videos as examples, as they will make it clearer to viewers how much flies could move around in the setup (from the methods, it seems they could move within the 2.2 of the 3 cm diameter of the arena, which would lead to substantial differences in the visual angle of the viewed grating.

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the Drift and Diffusion Model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options in individuals with bulimia nervosa (BN) and healthy participants (2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has potential to improve the understanding of pathological food choices.

Weaknesses:

I thank the authors for revising their manuscript.

I still notice that the authors did not go through their manuscript to look for wordings refering to a prediction interpretation of their results while I already highlighted the inappropriateness of this wording in my two first rounds of reviews: e.g. there is still "we used zero-inflated negative binomial models to predict the three-month frequency" and I can find other statements like this. The design of their study does not allow such claims.

The authors answered my major concern regarding the experimental induction towards a negative or a neutral state before running the food decision task. My concern is: BN patients already seemed to be already in a high negative state before undergoing the neutral induction, while these patients are in a lower negative state before undergoing the negative induction. It is therefore not surprising that patients seem to report a similar level of negative state after the two inductions (according to the figure of the authors' previous article). Of note is that the additional analysis the authors ran within the BN group only provides a significant result: this result shows that there has been an induction but does not rule out that patients were in the exact same magnitude of negative state to perform the task as the figure in their previously published article suggests it. The major issue is to show that:

(1) As compared to the neutral induction, there has been a higher variation in negative state after as compared to before the negative induction.

(2) The magnitude of the negative state after the negative induction is higher than the magnitude of the negative state after the neutral induction.

The first point shows that the induction worked. The second point shows that the participants are in two distinct states. Without showing the second point, it may be possible that one induction increases the negative state of participants to the same level as the one of the second induction that has not increased anything.

Within this context, how is it possible to associate, in patients, a difference in the DDM between the two sessions to a negative state (which is one of the main focus of the article) rather than to another parameter that has not been captured? A similar situation would be in an experiment studying the consequence of stress, a stressfull induction over relaxed participants attending the lab has high chances to raise the level of stress of those participants to the same level as the one that the same participants would experience after a neutral induction when these participants attend the lab with an already high level of stress. In that case, would it be approrpiate to claim that a difference at a task performed after the induction would be related to stress while the participants would be at the same level of stress when performing the task despite the fact that the induction worked ?

In the experiment performed by the authors, the additional analysis to perform would be a paired sample t-test (or the appropriate non-parametric test) to check whether the magnitude of negative state of BN patients was different between the negative and neutral conditions after the induction only. If not, associating the difference at the DDM with negative states in BN is highly misleading.

I read carefully the authors' answer related to mixed models: they claim that mixed models take into account correlations within their repeated data. The specification of the structure of the covariance matrix allows to control only partly for that. I notice that the authors did not specify the structure of that matrix: the article they refer to to justify the appropriatness of their analyses is not adapted. The specification of the structure of the covariance matrix needs to address, in a mixed model, the difference in handling 4 repeated data per participants that cannot be paired as compared to 4 repeated data that can be paired (two per session with one before and one after the neutral or negative priming sessions, if I count right). Of note is that a covariance structure that is left free of constraint for the fit of the model does not capture appropriately the pairing of the data: it has all chances to capture the covariance in a different way. And a covariance structure that has constraints has more chances to lead to a model that cannot be estimated because of an absence of convergence of the algorithms.

By the way, a single two-sample t-test (or a Mann-Whitney test if appropriate), and not a set of multiple paired-sample t-test as the authors suggest, would answer the goal of the authors to test for what they call the three-way interaction in their comment. This test would be performed between the two groups of participants (BN/controls) with the computation for each participant separately: (assessment after neutral induction-assessment before neutral induction)-(assessment after negative induction-assessment before negative induction). This analysis answers points 1, 2 and 4 they raise together with my point of controlling for the paired data. I would have agreed with their choice of a mixed model if they had an unbalanced dataset within each participant.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well-understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

Sample size was relatively small, and participants were all women with BN, which limits generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. These limitations are adequately noted in the discussion.

Reviewer #1 (Public review):

Summary:

This useful study provides incomplete evidence of an association between atovaquone-proguanil use (as well as toxoplasmosis seropositivity) and reduced Alzheimer's dementia risk. The study reinforces findings that VZ vaccine lowers AD risk and suggests that this vaccine may be an effect modifier of A-P's protective effect. Strengths of the study include two extremely large cohorts, including a massive validation cohort in the US. Statistical analyses are sound, and the effect sizes are significant and meaningful. The CI curves are certainly impressive.

Weaknesses include the inability to control for potentially important confounding variables. In my view, the findings are intriguing but remain correlative / hypothesis generating rather than causative. Significant mechanistic work needs to be done to link interventions which limit the impact of Toxoplasmosis and VZV reactivation on AD.

Weaknesses:

Major:

(1) Most of the individuals in the study received A-P for malaria prophylaxis as it is not first line for Toxo treatment. Many (probably most) of these individuals were likely to be Toxo negative (~15% seropositive in the US), thereby eliminating a potential benefit of the drug in most people in the cohort. Finally, A-P is not a first line treatment for Toxo because of lower efficacy.

(2) A-P exposure may be a marker of subtle demographic features not captured in the dataset such as wealth allowing for global travel and/or genetic predisposition to AD. This raises my suspicion of correlative rather than casual relationships between A-P exposure and AD reduction. The size of the cohort does not eliminate this issue, but rather narrows confidence intervals around potentially misleading odds ratios which have not been adjusted for the multitude of other variables driving incident AD.

(3) The relationship between herpes virus reactivation and Toxo reactivation seems speculative.

(4) A direct effect on A-P on AD lesions independent on infection is not considered as a hypothesis. Given the limitations above and effects on metabolic pathways, it probably should be. The Toxo hypothesis would be more convincing if the authors could demonstrate an enhanced effect of the drug in Toxo positive individuals without no effect in Toxo negative individuals.

Minor:

(5) "Clinically meaningful" should be eliminated from the discussion given that this is correlative evidence.

Reviewer #2 (Public review):

Summary:

This manuscript examines the association between atovaquone/proguanil use, zoster vaccination, toxoplasmosis serostatus and Alzheimer's Disease, using 2 databases of claims data. The manuscript is well written and concise. The major concerns about the manuscript center around the indications of atovaquone/proguanil use, which would not typically be active against toxoplasmosis at doses given, and the lack of control for potential confounders in the analysis.

Strengths:

(1) Use of 2 databases of claims data.

(2) Unbiased review of medications associated with AD, which identified zoster vaccination associated with decreased risk of AD, replicating findings from other studies.

Weaknesses:

(1) Given that atovaquone/proguanil is likely to be given to a healthy population who is able to travel, concern that there are unmeasured confounders driving the association.

(2) The dose of atovaquone in atovaquone/proguanil is unlikely to be adequate suppression of toxo (much less for treatment/elimination of toxo), raising questions about the mechanism.

(3) Unmeasured bias in the small number of people who had toxoplasma serology in the TriNetX cohort.

Reviewer #1 (Public review):

The current manuscript investigates a regulatory axis containing Prmt1, which methylates RNA binding proteins and alters intron splicing outcomes and expression of matrix genes. Authors test the effects of deficient Prmt1, Sfpq, and various other factors, using a combination of bioinformatic analyses and wet-lab validation approaches. Authors show that intron retention often triggers NMD, contributing to aberrant gene expression regulation and craniofacial development. The revised manuscript introduces several complementary experiments that help to strengthen conclusions. For example, authors directly investigate NMD-mediated transcript turnover to better understand how retention contributes to expression changes in genes of interest, and they assess several additional factors downstream of Prmt1 to justify a centralized interested in the PRMT1/SFPQ axis.

Weaknesses:

However, some points remain unaddressed or unexplored, which could bolster conclusions. For example, the transcriptome data from knockdown experiments indicate robust exon skipping, suggesting that analysis of these patterns in parallel with intron retention could provide additional insights into the responsive gene programs. Given that SFPQ is known to have multiple regulatory roles, a more thorough investigation of its possible mechanisms of action during craniofacial development would allow for definitive conclusions about the isolated impact of SFPQ-dependent splicing. Although authors employ CUT&Tag analysis of Pol II binding at the promoters and across the gene body, at the current scope, no change in Pol II association (i.e., absence of transcriptional repression) does not directly indicate a lack of transcriptional regulation by other means (pause release, elongation rate or processivity, transcription termination, etc.). Without a more thorough investigation of these mechanisms, this confounds definitive claims about their relative contributions to the gene expression landscape.

Reviewer #2 (Public review):

Summary:

The manuscript by Lima et al examines the role of Prmt1 and SFPQ in craniofacial development. Specifically, the authors test the idea that Prmt1 directly methylates specific proteins that results in intron retention in matrix proteins. The protein SFPQ is methylated by Prmt1 and functions downstream to mediate Prmt1 activity. The genes with retained introns activate the NMD pathway to reduce the RNA levels. This paper describes an interesting mechanism for the regulation of RNA levels during development.

Strengths:

The phenotypes support what the authors claim that Prmt1 is involved in craniofacial development and splicing. They use of state of the art sequencing to determine the specific genes that have intron retention and changes in gene expression is a strength.

Weaknesses: