Reviewer #2 (Public review):

Summary:

The authors derive an integrate-and-fire model to describe the dynamics of a more complex Wang-Buzsaki model and compare the two models. A detailed discussion of bifurcation schemes in both models is convincing and allows us to evaluate the simpler model.

Strengths:

The idea is interesting, and the mathematical approach appears to be convincing. In addition, differences between the simple and original models are also discussed.

Reviewer #2 (Public review):

Summary:

The authors generated two novel aphid-symbiont associations and examined the impact of these new symbiotic associations on plant-insect-symbiont interactions. The authors notably provide detailed phenotypic assessments of the insect hosts and host plants. They show that one introduced symbiont, Rickettsiella, increases aphid-induced damage to host plants, while the other, Regiella, ameliorates aphid damage. The authors suggest that such novel insect-symbiont pairings may be used as tools to mitigate crop damage in the future.

Strengths:

Although a few experiments seem to have limited sample sizes and limited statistical power, these are often complemented with highly replicated smaller-scale experiments. The combination of larger mesocosm and population-level experiments along with assessments of individual insects generally provides a comprehensive depiction of the effects of these symbionts on their hosts. The opposing impacts of Regiella and Rickettsiella infection on the aphid host plant are of broad interest. It is also surprising that the host plants did not exhibit strong differences in canonical defensive signalling, despite these differences.

Weaknesses:

It is a little surprising that mesocosm-dispersal experiments were not also conducted using Regiella-infected lines. At several points throughout the manuscript, the idea of using symbiont transfections to reduce plant harm is raised. I can understand that these experiments are likely time-, space-, and resource-intensive, but that seems like these would have been relevant experiments, especially in the context of controlling damage to plants.

Reviewer #2 (Public review):

Summary:

This compelling study proposes a framework to implement latent variable models using population level calcium imaging data. The study incorporates autoregressive dynamics and latent Poisson spiking to improve inference of latent states across different model classes including HMMs, Gaussian Process Factor Analysis and nonlinear dynamical systems models. This approach allows for a more seamless integration of existing methods typically used with spiking data to apply on calcium imaging data. The authors test the model on piriform cortex recordings as well as a biophysical simulator to validate their methods. This approach promises to have wide usability for neuroscientists using large population level calcium imaging.

Strengths:

The strength of this study is the flexibility in the choice of models and relatively easy adaptation to user-specific use cases.

Weaknesses:

The weakness of the study lies in its limited validation of biological calcium imaging data. Calcium dynamics in a task-specific context in a sensory brain region might be very different from slower dynamics in a region of integration.

Reviewer #2 (Public review):

Summary:

Chang et al. attempted to analyze a large number of ribo-seq datasets through a standardized pipeline, identifying novel non-canonical ORFs and elucidating their evolutionary and expression characteristics.

Strengths:

(1) The datasets analyzed by the authors are sufficiently comprehensive, and the use of standardized pipelines ensures excellent analytical consistency.

(2) Their analyses of ORF evolution and co-expression further deepen our understanding of these ORFs.

Weaknesses:

(1) The authors primarily conducted analyses through bioinformatics, lacking sufficient wet-lab experimental evidence.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors set out to evaluate the role of hypothalamic pituitary axis hyperactivity on cardiac and autonomic changes during epileptogenesis and following seizures in a mouse model of temporal lobe epilepsy. Epilepsy is very common. It can frequently result in death from sudden unexpected death in epilepsy, or SUDEP. SUDEP is thought to be at least in part due to seizure related cardiac and autonomic instability. Increased stress states are well known to be comorbid with epilepsy. This comorbidity is thought to increase the risk of SUDEP. Here the authors hypothesized that a mouse model of heightened stress in which there is hyperactivity of the CRH neurons in the hypothalamus would demonstrate exaggerated cardiac and autonomic effects of seizures and epilepsy.

Strengths:

For the chronic stress model, they employed the Kcc2/Crh mice that have a genetic deletion of the potassium chloride cotransporter in CRH neurons. They treated these mice and their wild type littermates with intra hippocampal kainic acid or saline, as epileptic and sham-treated animals respectively. The assessed cardiac activity, blood pressure, baroreflex, and the Bezold-Jerisch reflex during epileptogenesis. This in general is an interesting study. They make some interesting and potentially important observations regarding heart rate and blood pressure in seizures and epilepsy.

Weaknesses:

While the revised manuscript is much improved, there are still some concerns that should be addressed.

(1) The low-pressure baroreceptor responses they show in Figure 4 are somewhat confusing. Should they not be seeing a reflex increase in heart rate when blood pressure is lowered with sodium nitroprusside? It would be helpful if they could describe whether the control responses were as expected or not, and if not, why? This makes it difficult to assess changes seen in the different genotypes and conditions.

(2) It does not seem appropriate to label the assessments associated with Figure 5 as the Bezold Jarisch Reflex. This reflex involves bradycardia, hypotension, vasoconstriction, and hypopnea. They seem to be only looking at the cardiac component, which is likely mediated though peripheral 5HT3 receptors. Did they measure blood pressure and breathing? Can they include these? If they can only comment on HR, then the discussion should reflect this.

(3) In Figure 1B, it would be helpful to show some short (e.g., 0.5 sec) snippets of ECG traces that exemplify the changes in HR (spikes/second).

(4) The day 21 examples given in Figure 1B, do not seem to be representative of the data depicted in Figure 1C.

(5) From the top panel examples in Figure 2A it looks like there might be greater EEG suppression following seizures in the Kcc2/CRH mice. Was this consistent? It might be worth looking into.

(6) Can the authors include scale bars for the top panels in Figure 2A?

Reviewer #2 (Public review):

Summary:

Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Parasite invasion of host cells, intracellular replication, and subsequent egress, which results in destruction of the infected cell, are central to pathogenicity. This manuscript focuses on understanding how maternal resources, specifically cellular organelles, are shared between daughter parasites during cell division. Many organelles are present as a single copy, making their division and inheritance essential for successful replication. In T. gondii, our understanding of how organelles are divided during cell division remains limited, and this study helps address this important knowledge gap.

Strengths:

The major strength of this study is the use of a Halo-based pulse-chase assay to characterize patterns of organelle inheritance and to monitor protein synthesis, turnover, and movement. This approach will be of considerable interest to the field. Using this method, the authors identify three major modes of organelle inheritance:

(1) Organelles present in multiple copies (such as micronemes and rhoptries) are partitioned between daughter parasites, with additional contributions from newly formed vesicles. Newly synthesized and pre-existing material remain as distinct populations within the cell.

(2) Single-copy organelles, such as the Golgi and apicoplast, are expanded through the incorporation of newly synthesized material before division.

(3) Cytoskeletal structures are synthesized de novo during each round of cell division.

These findings provide a more refined understanding of organelle inheritance and demonstrate that secretory organelles are not generated entirely de novo during each round of division, as was previously thought.

The paper places particular emphasis on the fate of maternal micronemes and rhoptries during division. The data show that (1) during division in wild-type cells, maternal micronemes and rhoptries are detectable in the residual body (RB); however, the majority of these organelles are localized within the parasite body, either at the apical or basal ends of the daughter parasites (Fig. 6). (2) In the absence of the myosin motor MyoF, micronemes and rhoptries accumulate in the residual body and are not properly trafficked to the daughter cells. Upon restoration of MyoF protein levels, these organelles redistribute to the daughter cells, although in an uneven manner.

Weaknesses:

The second half of the paper focuses on a more detailed characterization of microneme and rhoptry recycling. While the authors propose that the RB acts as a central hub for recycling both organelles, the current data do not fully support this conclusion.<br /> The model that microneme and rhoptry recycling is RB-dependent relies largely on the MyoF depletion phenotype and the limited detection of maternal organelles in the RB of wild-type parasites. Alternative models remain plausible, including direct trafficking to daughter cells, with RB accumulation upon MyoF depletion reflecting impaired trafficking rather than an obligatory RB-dependent recycling pathway, as now discussed by the authors.

Reviewer #2 (Public review):

Summary:

The manuscript shows that nutrient resorption efficiency in Phragmites australis is largely genetically canalized rather than plastic under effective salt stress, with variation mainly associated with phylogeographic lineage, ecotype, and latitude.

Strengths:

The common-garden experiment with 110 genotypes, paired control and salt treatments, multi-level stress validation, and element-specific analyses provides compelling support for the central conclusions.

Weaknesses:

The generality is limited by the focus on a single species and a single growing season, and some mechanisms are inferred indirectly.

Reviewer #2 (Public review):

Summary:

The study uses transient optogenetic silencing of the dorsal hippocampus or prefrontal cortex in mice using a delayed response task in a T-maze, with a temporal delay of 1s after a visual cue, followed by a central stem run period before choice execution. Silencing of either the dorsal hippocampus or the dorsal prefrontal cortex is executed for varying time periods during the stem/ central arm running epoch or the 1s temporal delay epoch by targeting either PV+ or Dlx interneurons in a block-wise or random-trial design. The main result reported is that silencing of either region during long periods of stem running impaired choice behavior, and silencing during the temporal delay period did not have an effect.

Strengths:

The major strength of the study is using the optogenetic silencing strategy to target different temporal periods of the task.

Weaknesses:

(1) A major weakness of the study is the lack of a balanced design with equal time periods of silencing during the stem running period and temporal delay period in many of the animals, which precludes any conclusion about distinct functional roles of the regions during these two phases of the task. The central question of this study is not new, with many previous studies investigating distinct and overlapping roles of hippocampus and prefrontal cortex in spatial working memory tasks and memory-guided navigation, using inactivation of one or both regions, crossed inactivation approaches, as well as targeting direct and indirect connections between the regions (PMIDs: 20074655, 9030646, 17045348, 30179661, 27511010, 10491611, 26017312, etc.), in addition to several physiology studies. A key extension for the current study would have been to show a distinction between roles in the temporal delay period after the cue and the stem-running working memory period. However, the inactivation period during stem running shows effects only for long inactivation periods, 2s initial periods and later ~1.6s periods (run after 0.8s, >2/3 total running periods), whereas the temporal delay period inactivation is 1s for the large majority of animals, which is a clear mismatch in inactivation periods, obviating this conclusion of distinction.

(2) It is not clarified why such short delay periods were used compared to long periods of ~10s in T-maze spatial alternation tasks with delay, and whether the temporal delay period of 1 sec is strictly distinct from the spatiotemporal delay period during stem running in terms of short-term memory function. The choice of time windows for inactivation needs to be better justified, which currently appears to be rather random (2s initial running period, run after 0.8s, run after 1.6s; for an average reported running period of ~2.4-2.5s). The ideal design clearly would have been to use a 2s temporal delay period so that the inactivation time in this epoch matched the initial 2s run period. Only a subset of 4 animals were run with a longer temporal delay period, and too with only with hippocampal inactivation (Figure 4d). The main conclusion of distinction between temporal delay and stem running delay periods is therefore not adequately tested for the prefrontal cortex, and the statistics in terms of number of animals for this important control for hippocampal inactivation are also not comparable to the main experiment.

(3) The mixture of PV-Cre animals (5 animals), Dlx targeting (2 animals), and one WT animal is also suggestive of a fragmented approach, and inactivation efficacy cannot be assumed to be similar for different animals. Importantly, there is no physiological evidence for confirmation of suppression in the optogenetic experiments, even in exemplar animals.

Reviewer #2 (Public review):

Summary:

In this short paper, a clever single-cell Strand-seq method was used to study the number and location of sister chromatid exchange events (SCEs) in cells after CRISPR/Cas9-induced DNA double-strand breaks (DSBs). Unique as well as multiple genomic loci were targeted. Cas9-induced cuts at unique genomic locations led to statistical enrichment of SCEs at the target site, whereas Cas9 targeted at repetitive targets revealed only mild enrichment of on-target SCEs unless analysis was restricted to a subset of cells with >8 SCEs per cell. Interestingly, reciprocal daughter-cell pair analysis revealed large-scale structural alterations on some chromosomes. Whereas disruption of DNA repair genes, including LIG3, LIG4, XRCC1, and XRCC4, did not measurably alter SCE frequency per cell within 24 hrs, consistent with delayed functional loss following editing and selection against essential genes. Together, these findings demonstrate that Cas9-induced DSBs are potent local triggers of SCE at unique loci and can be associated with structural alterations, highlighting the influence of lesion type and genomic context on recombination outcomes during genome editing.

Strengths:

The data in this paper represent a very rich resource of how parental DNA template strands are distributed in paired daughter cells after various treatments. Abnormalities observed in only one of such paired daughter cells provide a novel and exciting approach to study mechanisms of DNA instability and DNA repair at a genome-wide level in general and following Cas9-induced DSB in particular.

Weaknesses:

The effect of Cas9-induced DSBs in the cells that are used will depend on the cell cycle stage of the cells that are targeted, as well as the number of times cuts are made. The latter could happen before, during, and after DNA repair reactions on one or both alleles in a diploid cell. As a result, it is very difficult to extrapolate the mechanisms of DNA instability and DNA repair from the observed genomic rearrangements. Novel approaches are needed to limit the number and timing of Cas9-induced breaks to overcome some of these limitations.

Reviewer #2 (Public review):

Summary:

The study confronts a major obstacle in repertoire analysis. Given that individual TCR sequences are diverse and sparsely detected across repertoires, identifying sample-specific enrichment of TCRs based on sample-to-sample comparison of exact clonotype sequences can be intractable. This paper attempts to build on insights that sequence-similar TCRs can share antigen recognition, such that aggregating similar sequences derived from multi-sample joint-graph communities (i.e. clusters of tightly connected nodes) could reduce sparsity and boost signal.

Strengths:

The study is a well-motivated effort to address a need in the field. The paper takes a unique approach. The core method is modeling community occupancy with a hierarchical Dirichlet-Multinomial model that attempts to account for the high level of overdispersion present in repertoire sampling, a technique that has been previously applied to compositional microbiome data.

The authors apply this framework to both single-cell paired-chain and bulk single-chain TCR data. They develop a set of examples from public and synthetic data, with the most promising real-world application shown in reanalysis of longitudinal data during treatment of cancer patients with checkpoint inhibitors.

The manuscript is well structured and cogent. The authors are to be commended for contributing a well-documented, open-source R/Bioconductor package and for providing the underlying analysis datasets in a well-organized repository. In the joint graph construction step, the authors opt to use an existing implementation of the BLAST algorithm on CDR3 sequences, which is a slightly odd choice since it ignores potential contributions of other V-gene germline-encoded CDRs, but the authors also envision that their statistical package could be extended to include community graphs developed with other established TCR clustering tools. This will allow others to potentially explore the utility of Bayesian hierarchical Dirichlet-Multinomial models for differential occupancy analysis of immune receptors under varied clustering criteria.

The methods described here were demonstrated on relatively small datasets from 2-5 samples, and future work is likely needed to extend the joint graph differential occupancy concept to larger datasets. The authors are transparent about this and some of the other limitations in their current tool, most notably the computational cost of graph construction based on an all-versus-all sequence alignment to construct a joint sequence graph and the challenge of Bayesian parameter estimation as the number of subgraph entities scales with input data size. Since efficient approximate methods exist to find edges between similar text strings, the underlying idea of applying uncertainty-aware statistical inference to subgraph communities is promising, and the paper advances its primary goal.

Weaknesses:

The paper proposes the utility of the joint-graph community occupancy framework through three examples. I discuss potential weaknesses apparent in each separate example in turn.

(1) Weaknesses in Example 1

A broad weakness of the first results section ("Detecting EBV- and MART1-antigen reactive T cell communities from single cell datasets") is its reliance on a single vendor-generated dataset generated by the company ParseBio with no published experimental protocols and limited, if any, prior peer review. For reasons I will explore in greater detail below, the EBV-sample data may be particularly prone to chimeric pairings that confound the authors' primary analysis goals, and, at the very least, may not reflect physiologically realistic conditions for identifying antigen-reactive TCRs in other contexts.

Let us first consider Dataset 1 in more detail. The authors compare 2 antigen-stimulated repertoires with 3 unstimulated controls. To improve on single clonotype-level comparisons, the authors propose comparing the cell count aggregated across cells within joint graph communities constructed from paired CDR3 sequences across all the samples. Thus, one of the most relevant questions one hopes the authors answer in this section is whether the resolved graph communities are made up of many distinct clonotypes (i.e., are they polyclonal), allowing the method to function as intended by aggregating across multiple clones with putative shared antigen-reactivity.

Supplementary Figure 1B shows the size of all the communities with callouts for the putative EBV-expanded communities. The authors listed communities strongly enriched in the EBV-stimulated sample as e1, e2, e3, and e5. Each contains {greater than or equal to}100 clonotypes, and the authors note they contain at least one clonotype with a CDR3 sequence matching an EBV-annotated clone in VDJdb - a database of TCRs with some experimental evidence of epitope-reactivity. Community "e2" is notable for its remarkable size, including 1,339 unique clonotypes. At first glance, this seems promising for a method attempting to boost signal through community detection. However, it is worth re-investigating the individual clone sizes and sequences within this extraordinary community.

In the EBV-associated community "e2", a look at the data provided by the authors on the paper's GitHub repository suggests a single clonotype (clonotype_9; TRAV12-3 CATQGSNDYKLSF / TRBV9 CASSTGQVATNEKLFF) comprises 26,917 cells. As such, it makes up 29% of the sample with a total of 90,588 cells. If one clone supplies most of a graph community's cell counts, the community-level posterior estimate of β (Figure 1B) effectively tracks a single-clone estimate, and the premise of borrowing statistical power across a polyclonal expansion in this example is hard to assess.

There is also considerable evidence to believe that the apparent mega-polyclonality of cluster e2 may be partially an illusion, stemming from an artifact of this hyper-expanded clone's massive size and the experimental method used to assign TCRα-TCRβ chain pairings. In fact, the same α-chain CDR3 (CATQGSNDYKLSF) appears in ~1,219 clonotypes paired to distinct β chains, generating much of e2's remarkable 1,339-clonotype count. I believe two features warrant caution here. First, a single clonotype making up ~29% of total T cells in the sample is highly unexpected in ex vivo repertoires, suggesting intense non-physiological expansion conditions unlikely to generalize to other settings. That is, one would almost never expect to see a signal this strong.

Second, one TCR-α chain paired to ~1,220 distinct and diverse β chains in one sample is also biologically unexpected given what we know of the best-characterized epitope-specific responses for EBV, including to the well-known HLA-A*02 EBV BMLF-1 epitope, which recruits a tetramer-stained repertoire with conserved CDR3 motifs in both chains and at least some constraint on favored V-gene/α-β pairing (See Extended Data Figure 5 in Dash et al., Nature 2017). This raises the concerning possibility of barcode collision and mispairing against a hyperexpanded clone in the ParseBio split-pool method, unlikely to be robust to a clone occupying a third of the sample. Most of the ~1,219 β chains paired to the dominant α have a cell count of 1, further raising the concern of artifactual pairing versus genuine convergence. The second largest community "e1" also seems to suffer from the same issue, with a TCRβ sequence from one super clone making up 7% of the sample potentially being artifactually over-paired to >500 rare single-cell-count TCR α chains.

Taken together, these observations suggest that the authors' first positive control example passes but probably for the wrong reason since at least some of the "antigen-specific communities" are strongly anchored by a single hyper-clone. This could be remedied by repeating the same type of analysis on an ex vivo single-cell repertoire following more modest stimulation or natural infection (e.g., yellow-fever vaccine, influenza, or SARS-CoV-2 single-cell TCR datasets). I would advise future work using an alternative data source with better-documented experimental protocols, given the concerns above.

(2) Weaknesses in Example 2

Example 2 explores the application of Bayesian methods for identifying sample-enriched joint graph communities found in longitudinal data from many participants at two time points and longitudinal data from 1 person (Pt4) at 5 time points. A potential weakness of Case Study 2 is that it yields limited additional biological insight compared to what was previously shown by the authors of the underlying input data. Previously, Formenti et al. 2018 showed that the number of expanded clones after treatment strongly reflects responder status in this cohort, greater in patients with CR/PR versus SD or PD (See Figure 2b of the study). This 2018 primary analysis showed that tracking individual clonotypes was sufficient to reveal biological insight without the need for the computational demands of constructing a massive sequence similarity graph, finding communities on joint graphs, or Bayesian statistical inference. Thus, the impact of Case Study 2 in proving the unique utility of ClustIRR is somewhat diminished.

It is not clear how this study's result is more "robust" than the original analysis. Perhaps the authors could further clarify what is learned from the uncertainty-aware approach that could not be learned from exact clone tracking in time series.

Thus, example 2 shows that a complex method recapitulated the finding of a much simpler method for analyzing longitudinal TCR data where a strong signal of expansion was already present at the single-clonotype level. Since the Dirichlet method is sensitive to absolute counts, the large expanding clone in each community at 22 days may alone have carried most of the signal, which is not fully explored.

In this section, the authors make an interesting observation that CDR3β detected in both PBMC and patient-matched tumor samples were enriched in contracting communities (7/14) versus expanded communities (1/41). The authors do not indicate whether a similar enrichment of tumor-infiltrating lymphocytes (TILs) matched Day 0-22 contracting communities in the other 4 patients with tumor-matched samples, which, if consistent, would strengthen their finding.

(3) Weaknesses in Example 3

The final example explores "convergent repertoire differences between species." This is intriguing in principle but less informative due to the use of synthetic data with somewhat predictable properties that some may reasonably consider baked in by the data-generating process. That is, some of the results might be guaranteed by the way the data is constructed using the OLGA/IGoR generative model. For instance, the authors observe a positive correlation between CJ community size and Pgen of constituent clonotypes, stating: "This indicates that CDR3 sequences with high Pgen are statistically more likely to be generated, leading to convergence of similar sequences into public CJs." I may be mistaken, but this conclusion is almost guaranteed by the way the data-generating OLGA model outputs more similar high-Pgen sequences and fewer lower-Pgen sequences.

An interesting finding in this section is shown in Figure 4B, where community-level aggregation allowed for discrete clustering of human samples away from mouse samples that was not possible by comparing cosine similarity of a sparse clonotype occurrence matrix, a result that would be higher impact if it could be shown to separate real repertoire samples from humans with differential serology, vaccination status, or HLA backgrounds.

(4) Weaknesses in General

More generally, one aspect of the method that seems under-emphasized is the fact that many of the nodes in a multi-sample joint sequence similarity graph may have no edges. These zero-degree nodes would probably frequently occur in only one sample but be absent in other samples. It is not strongly emphasized in the paper how the model would infer whether such a singleton found in only one sample in the graph could be reliably inferred to be sample-specific enriched (see, for example, the large single node in Figure 3D, the orange node labeled "GQYF" in the far-right position of the lowest row in panel D). Presumably the number of cell counts represented in this single-sequence node is so great at sampled timepoint Day 22 as to yield a statistically strong signal in the multinomial model; however, the authors may wish to comment on how, for such singleton sequences, the power to detect sample-specific enrichment differs from prior single-clonotype-based methods.

With any large effort to find statistically significant features from a large candidate set examined all at once, a reader might be concerned with the potential for false discovery. Throughout, the authors seem to assign statistical significance when the 95% high-density interval (HDI) of the posterior estimate excludes zero or when the 95% HDIs of two features do not overlap. There is little discussion of how this implicitly handles multiplicity adjustment via shrinkage, which the authors could address more directly and explain more clearly to a broad audience, including many non-statisticians, who will read this paper.

Reviewer #2 (Public review):

Summary:

This manuscript by Pletenev et al. provides a meta-analysis of 59 published studies on decision-related activity in the macaque visual cortex. The work does not contain original research material, but by conducting extensive analyses and compilations of previously published data, it provides several new insights not already conveyed in recent reviews on this topic.

Strengths:

The work is scholarly and helps organize and integrate a broad set of findings. A difficulty in such an undertaking is making sure that the original studies are accurately characterized and tabulated. The lead authors should be commended for the rigorous approach they've taken; the inclusion of many of the authors of the original studies as co-authors provides additional reassurance that trends visible across studies reflect an accurate quantification of what each study has shown.

Weaknesses:

My sole scientific concern is on the 'duration' section (lines 487 to 536). Specifically, the authors relate the dependence of choice probability (CP) on time to two competing (but not mutually exclusive) views of how CPs arise-the 'feedforward' vs 'feedback' views. The basis for the predictions in this section was not clear to me. In particular, it was not obvious that the predictions fully considered all the relevant factors. For instance, did the feedforward predictions consider how the response covariance depends on duration and how this would affect CP values (Equation 1)? (See for example Figure 4 of Kang and Maunsell, 2012, JNP.)

I would suggest either explaining the basis of the predictions much more carefully or moving the predictions to a supplementary section where they can be presented in more detail (i.e., more convincingly). Alternatively, since the section is inconclusive in the end (there is not strong evidence in favor of FF or FB), the authors could mention the theoretical predictions in passing only, i.e., much more briefly, just to make the reader aware that the different theories can provide predictions about the duration dependence.

Reviewer #2 (Public review):

The authors developed a reinforcement learning framework to identify stimulation patterns that produce a predefined activity pattern in neuronal networks cultured on a microelectrode array. The main contribution of this study lies in the development and validation of the experimental framework, rather than in providing new insight into neuronal response properties or the biological mechanisms underlying network activity.

A major strength of the work is that the framework is implemented using open-source software. Electrical stimulation through microelectrode arrays is already widely used, and the proposed approach is therefore likely to be of interest to researchers seeking to automate the exploration and optimization of stimulation parameters. The authors also provide the software, hardware configuration, and experimental data, which substantially improves transparency and should facilitate reproduction and further development of the system by other laboratories.

The experiments provide a useful proof of concept showing that the framework can search for stimulation patterns associated with the predefined task in the tested neuronal network. The evidence is solid for demonstrating the feasibility of the approach within this specific experimental configuration. In particular, the closed-loop integration of stimulation, recording, evaluation of the neuronal response, and subsequent selection of stimulation patterns is clearly implemented and experimentally tested.

An important limitation is that the framework was evaluated using a relatively small and highly structured network. Four stimulation electrodes were positioned around a single network, and stimulation was delivered to microchannels in which axons were concentrated. This configuration is well suited to the initial demonstration, but it remains uncertain whether the same approach will perform similarly in conventional monolayer dissociated cultures, larger networks, or systems with different numbers and spatial arrangements of electrodes. Additional validation across a broader range of network structures and experimental configurations would therefore be needed to establish the general applicability of the framework.

Overall, this study presents a valuable and reproducible methodological framework for the closed-loop optimization of electrical stimulation in cultured neuronal networks. Its likely impact lies primarily in providing an accessible experimental and computational platform that can be adapted for studies requiring systematic exploration of stimulation patterns, although the extent to which the current findings generalize beyond the tested network configuration remains to be determined.

Reviewer #2 (Public review):

Summary:

This study addresses a question that has been essentially inaccessible in humans: whether the early somatosensory relay nuclei are engaged by anything other than peripheral drive. Using functional and quantitative MRI in individuals with chronic cervical spinal cord injury, the authors show that the cuneate nucleus and VPL are robustly engaged during overt or attempted hand movement, and that this engagement persists in a participant with complete hand paralysis and no detectable muscle activity. They further report structural degeneration of the cuneate nucleus that is unrelated to the preserved task-evoked activity, and interpret the residual activity as reflecting top-down corticocuneate signalling.

Strengths:

The work is well conceived and clearly written, and the demonstration that the cuneate nucleus and VPL are robustly engaged during (attempted) hand movement after chronic cervical spinal cord injury is, to my knowledge, novel at this level of anatomical resolution. The finding is convincing. The dissociation between preserved task-evoked activity and marked structural degeneration of the cuneate nucleus is a highly interesting result with clear implications for rehabilitation. The manuscript is straightforward to follow, and the imaging protocol is carefully executed.

Weaknesses:

My main reservation concerns the inferential step from "not peripheral" to "corticocuneate". The data establish the former convincingly; the latter may not.

(1) Attribution of the observed activity to corticocuneate projections.

The central claim rests on an argument by elimination: because bottom-up drive is excluded in PT01, the residual activity must be top-down and, by extension, corticocuneate. Two distinct gaps should be addressed. First, "top-down" is not equivalent to "direct corticocuneate". Descending influence could reach the cuneate nucleus through multiple indirect pathways. Second, the activity observed at the three levels (cuneate, VPL, S1) need not be serially propagated, since layer 6 corticothalamic projections, for example, could drive VPL independently of any cuneate contribution. I would ask the authors to either provide evidence bearing on the routing, or to consistently use a route-neutral term (e.g. "descending" or "top-down") and reserve "corticocuneate" for the discussion of candidate mechanisms.

(2) Afferent input arising above the lesion level.

The EMG control in PT01 was restricted to hand and forearm muscles. Musculature innervated above C4 (cervical paraspinals, trapezius, and to a variable extent the shoulder girdle) remained available to this participant, and attempted hand movement is frequently accompanied by increased proximal co-contraction, postural stabilization, and altered respiratory effort. Afferent to the upper cervical cord is known to project to the ipsilateral cuneate nucleus, and its activity would produce lateralized, ipsilaterally dominant cuneate input - that is, precisely the pattern reported. This alternative is not excluded by the present control and should be addressed directly, ideally with proximal EMG in PT01 (and, if possible, in the other participants), or at minimum with an explicit discussion. Relatedly, the authors recorded respiratory and cardiac signals: please report whether respiratory volume or heart rate differed between movement and rest blocks, and between groups, since the dorsal medulla lies adjacent to cardiorespiratory nuclei.

(3) Functional significance of the preserved top-down signal.

The discussion establishes that top-down input persists but says relatively little about why it should. If the principal role of descending input to the cuneate nucleus is the gating of incoming afferent traffic, then in the absence of afferents there is nothing left to gate, and one might have expected the signal to be lost. Its persistence is the most interesting aspect of the finding and deserves fuller discussion. Candidate accounts the authors may wish to consider include: an efference copy or predictive signal delivered to a comparator that no longer receives its input, in the framework the authors already invoke (references 27, 28); engagement of the non-lemniscal outputs of the dorsal column nuclei (e.g., cuneocerebellar, cuneo-olivary projections); attempted movement engages motor imagery and attention, in which case the relevant question becomes what distinguishes these from movement-related gating. A related interpretational point: in behaving primates, movement-related modulation of cuneate transmission is bidirectional and includes prominent suppression (refs. 7/12). Note also that BOLD increases are compatible with increased inhibition, so they do not indicate facilitated throughput.

Reviewer #2 (Public review):

The manuscript by Thompson and Gold reported that, although baseline and evoked LC activity were positively correlated with baseline pupil size and evoked pupil dilation, respectively, there were no reliable cross-epoch relationships between LC activity and pupil size - that is, between baseline LC activity and evoked pupil dilation or between baseline pupil size and evoked LC activity. A major strength of the study is its large dataset, comprising recordings from more than 100 LC single units collected across 83 recording sessions in two monkeys. The authors further strengthened their conclusions by performing several control analyses to rule out potential confounds, including whether the findings were influenced by (1) the use of residuals in the main analyses, (2) the possibility that the auditory stimulus failed to evoke the full dynamic range of LC activity and pupil responses, and (3) nonlinear cross-epoch relationships between LC activity and pupil size.

These findings are important because they remind researchers to exercise caution when assuming that non-illuminance-mediated fluctuations in pupil size can reliably serve as a proxy for LC activity under all experimental conditions

With that being said, I have two suggestions that I believe would further strengthen the manuscript.

(1) Previous studies have suggested that the LC is organized into functionally distinct subpopulations (e.g., PMID 28920933 and PMID 40770025). It is therefore possible that a subset of the LC neurons recorded in this study does not participate in controlling pupil size. I encourage the authors to discuss this possibility in the Discussion. In addition, this possibility could be tested by repeating the cross-epoch relationship analyses using only LC units that exhibit a strong correlation with pupil size (the darker points in the last column of the top two rows of Figure 1?).

(2) The manuscript would benefit from a clearer visualization of the analyses addressing the possibility that the auditory stimulus failed to evoke the full dynamic range of LC activity and pupil responses. A supplemental figure illustrating the distributions or ranges of LC activity and pupil responses, together with the corresponding control analyses, would help readers better understand this important point.

Reviewer #2 (Public review):

Summary

This manuscript investigates whether neurofeedback based on the N1 component of the temporal response function can be used to modulate neural responses during selective attention to continuous speech. Participants listened to two competing audiobooks and were instructed to attend to one of them. In the neurofeedback group, trial-by-trial N1 responses were converted into visual feedback, whereas the sham-feedback group received replayed feedback from other participants. The authors found a significant interaction between group and block for the N1 response to target speech over a small fronto-central cluster, with larger N1 responses during feedback blocks in the genuine neurofeedback group but not in the sham group. No neurofeedback effect was found for the distractor response. The authors also reported exploratory post-training effects and an association between changes in N1 and speech-comprehension performance at right fronto-central electrodes.

Overall, the study is conceptually interesting and novel. The online, trial-by-trial estimation of neural responses from continuous speech is an attractive development for auditory neurofeedback, and the inclusion of a randomised sham-feedback group is an important strength. However, the manuscript provides stronger evidence for modulation of a neural response during feedback than for learning or training of selective attention. Some aspects of the analysis and interpretation also require further consideration/clarification, particularly the use of group-specific N1 time windows, the spatial confound between target and distractor streams, the absence of artefact correction in the signal used for feedback, and the relatively weak behavioural evidence.

Strengths

The main strength of this study is its novel use of neurofeedback during continuous competing speech. Rather than providing feedback based on a general measure of brain activity, the authors targeted a specific neural response associated with selective auditory attention. The online implementation is technically impressive, allowing neural responses to be estimated from 22-second speech segments and converted rapidly into feedback. The inclusion of a sham-feedback group is another important strength, as it helps distinguish effects of genuine neurofeedback from nonspecific effects such as task engagement or motivation. The relatively large sample for a neurofeedback study and the use of natural continuous speech also increase the robustness and ecological relevance of the work.

Weaknesses

(1) The effect was present during the feedback blocks but did not increase across training blocks, and the post-training effect was not found at the same electrodes used for feedback. The evidence therefore supports online modulation more strongly than learning or lasting self-regulation, and claims about successful training or persistent learning should be interpreted cautiously.

(2) The offline analysis used different N1 time windows for the neurofeedback and sham groups. This introduces a potential bias in the group comparison because the dependent measure was defined differently between groups. Confirmation of the main result using a common, independently defined N1 window would strengthen the evidence.

(3) The target speech was always presented from the front and the distractor from behind. Differences between target and distractor responses therefore cannot be attributed entirely to attention because spatial location is also different. This limits the interpretation of target-versus-distractor differences and may also contribute to the weaker reliability of the distractor response.

(4) Feedback blocks always contained two speakers, whereas half of the baseline trials contained only one speaker. Since the presence of a distractor altered the neural response, it is important that the neurofeedback comparison is based on acoustically matched multi-speaker baseline trials. If this were not the case, differences between baseline and feedback could partly reflect differences in the acoustic condition rather than neurofeedback.

(5) No artefact correction was applied to the signal used for online feedback. Because feedback was derived from fronto-central electrodes, eye or muscle activity could potentially contribute to the measured signal. An offline demonstration that the main neural effect remains after appropriate artefact control would strengthen the interpretation that the effect reflects neural modulation rather than systematic changes in non-neural activity.

(6) The behavioural evidence is weaker than the neural evidence. There was no significant overall improvement in speech comprehension in the neurofeedback group. The reported behavioural effect is instead based mainly on associations between changes in the neural response and changes in comprehension, and some of these effects were weak before the whole-scalp analysis. Therefore, these findings are better interpreted as exploratory associations rather than evidence that neural modulation directly caused improved comprehension.

(7) Adding the distractor did not significantly reduce comprehension performance. The absence of a significant distractor effect on comprehension suggests that the listening condition may not have produced a strong behavioural cocktail-party difficulty in this sample. The large spatial separation between speakers and the nature of the behavioural task may have reduced sensitivity to distraction, which could limit the strength of the conclusions regarding improvement of speech understanding in challenging listening conditions.

(8) The study is described as double-blind, but the manuscript provides limited detail on how blinding was maintained, particularly when the experimenter manually checked the N1 estimate. In addition, participants' belief in or perceived control over the feedback was not formally assessed. These factors make it difficult to determine how effectively expectancy or motivation-related effects were controlled.

Overall assessment:

This study provides a valuable methodological and conceptual advance by showing that online, trial-by-trial neurofeedback can modulate the neural response to attended speech during competing speech. The evidence is solid for an immediate neural effect during feedback, and it was supported by comparison with a sham-feedback group. However, it remains incomplete for broader claims about learned self-regulation, persistent effects, distractor suppression, and improved speech understanding.

Reviewer #2 (Public review):

Summary:

The study by presents a sophisticated molecular dissection of ribosome-associated complexes (RCs) in two well-defined cortical projection neuron subtypes (ScPN and CPN) during early postnatal development. The authors develop and optimize an rRNA immunoprecipitation-mass spectrometry (rRNA IP-MS) workflow to recover RCs from FACS-purified, retrogradely labeled neurons, achieving remarkable subtype specificity and biochemical resolution. Through proteomic profiling, they reveal both shared and distinct ribosome-associated proteins between ScPN and CPN, with a focus on non-core RC components and their potential functional relevance. The work advances our understanding of cell-type-specific translation regulation, moving beyond the transcriptome to explore the proteome-level complexity in neuronal subtypes.

Strengths:

This work stands out for its technical sophistication and innovation. The authors combine retrograde labeling, FACS purification, and an optimized rRNA IP-MS approach (low input) to isolate ribosome-associated complexes from highly specific neuronal subtypes in vivo, a challenging issue that they execute with impressive rigor. The methodological pipeline is both elegant and well controlled, yielding high-quality, reproducible data. The depth of proteomic coverage is remarkable, with nearly all known cytoplasmic ribosomal proteins identified, along with hundreds of ribosome-associated proteins (RAPs), including translation factors, chaperones, and RNA-binding proteins. The analysis not only reveals shared components between ScPN and CPN RCs but also uncovers subtype-specific differences in associated proteins.

Particularly notable is the integration of this new proteomic dataset with previously published transcriptomic and ribosome footprinting data, which helps to validate the specificity and relevance of the findings. Overall, the clarity of the writing, the robustness of the data, and the transparency of the methods make this a strong and compelling contribution.

Weaknesses:

Despite the depth and high quality of the dataset, the study remains descriptive. While the identification of subtype-specific RC components is intriguing, the current version of the manuscript does not explore their functional roles or biological consequences of their alterations. There is no perturbation, causal testing, in vitro or in vivo manipulation to demonstrate whether these proteins are necessary for ScPN or CPN identity, specific axonal targeting, metabolism or synaptic function.

One important point that is also highlighted by the authors in their discussion and that is critical to establish the subtype specificity of the identified protein. One important point highlighted by the authors in the discussion - and critical for establishing the subtype specificity of the identified proteins-is that some ribosomal complexes may be specialized for specific developmental stages, rather than exclusively for the subtype-specific needs of projection neuron development. The work presented here provides a valuable starting point for further investigation into such RC specialization. However, it will be essential to determine to what extent these RCs exhibit true subtype specificity, independently of their temporal maturation context.

As a result, key mechanistic insights remain a bit speculative. Although several of the identified proteins have known roles in processes like synaptogenesis or metabolism, their relevance to the specific neuronal subtypes under study is not experimentally addressed. That said, given its rich content and the comprehensive early postnatal dataset, the manuscript represents an extremely valuable resource for the community. While primarily exploratory, it lays a strong foundation for future functional studies aimed at uncovering the biological impact of the identified ribosomal complexes.

Reviewer #4 (Public review):

The authors use a d' defined for 2-alternative forced choice experiments, but their data are 4-alternative (for color) and 8-alternative (for number) forced-choice. So, the d' is not computed correctly. For mAFC experiments, the authors should use the Hacker-Ratcliff (1979) method, also defined in chapter 10 of the Macmillan & Creelman textbook.

Normalization of the stimuli was arbitrary, and consequently the unexpected improvement in performance in reverberation compared to anechoic condition is still not explained. A natural normalization across different environments is to take the direct portion of the BRIR (and HRTF for the anechoic condition) and make sure that that is scaled identically across the different simulated rooms (with the reverberant tails scaled naturally). That corresponds to the situation when the sources are emitting the sound at the same level in each environment. The current study scaled the overall levels. As a minimum, it should be reported how this scaling boosted/attenuated the targets and maskers in each environment.

The potential that the listeners are tuning to individual voices, as opposed to rooms, has not been eliminated. The authors suggest that a lack of interaction with different voices is evidence that that is not the case. This is not correct: lack of this interaction just means that there are no differences in tuning between the voices. But that does not mean that the same amount of tuning is happening for each voice, as observed in previous studies. Unless the authors provide a follow-up data with randomly varying voices within each trial, these claims should be tuned down.

Reviewer #2 (Public review):

Summary:

The manuscript by Luden et al. investigates the molecular function and DNA-binding modes of AHL15, a transcription factor with pleiotropic effects on plant development. The results contribute to our understanding of AHL15 function in development specifically and transcriptional regulation in plants more broadly.

Strengths:

The authors developed a set of genetic tools for high-resolution profiling of AHL15 DNA binding and provide exploratory analyses of chromatin accessibility changes upon AHL15 overexpression. The generated data (CHiP-Seq, ATAC-Seq and RNA-Seq is a valuable resource for further studies. The data suggest that AHL15 does not operate as a pioneer TF, but is likely involved in gene looping.

Weaknesses:

The authors have extended the motif analysis to the top 1,000 shared peaks, addressing part of my previous concern. It did not erase my worries about overclaiming completely, but I think it can be considered as a terminological issue, rather than technical one. Therefore, it could be addressed through minor revision, without further analyses.

Specifically, the absence of a predominant enriched motif does not establish that AHL15 binds non-specifically or lacks sequence preferences. Low motif prevalence limits the proportion of peaks it could explain but does not exclude a genuine binding preference; a binding motif need not be rare in the genomic background (binding can be supported by other factors). Please revise the interpretation in lines 188-194, and the conclusion in lines 204-206 to state that the present analysis did not identify a strongly enriched motif accounting for a substantial proportion of AHL15-associated regions. Any equivalent claims of non-specific binding elsewhere in the manuscript should be toned down.

Additional minor points:

(1) Please provide the exact HOMER background-selection procedure, and definition of the 50-bp search windows.

(2) Figures 2B-C and the related supplementary figures, add the x axis label.

Reviewer #2 (Public review):

Summary

Wu and Turrigiano investigate how Shank3 loss affects experience-dependent changes in sensory representations during conditioned taste aversion learning and extinction. Using longitudinal two-photon calcium imaging in the gustatory cortex, the authors show that Shank3 knockout mice acquire taste aversion more slowly but, after additional conditioning, reach an aversion comparable to wild-type mice; this learned aversion then extinguishes more rapidly. At the neural level, knockout mice exhibit reduced stimulus-evoked suppression and increased correlated variability; while learning and extinction are accompanied by changes in the reliability, selectivity and population-level discriminability of taste representations. The revised manuscript more clearly distinguishes baseline genotype-dependent differences in cortical activity from learning-associated changes and appropriately frames the relationship between neural activity and behaviour as associative rather than causal.

Strengths

A major strength of the study is the combination of longitudinal cellular-resolution imaging with a behavioural paradigm that allows cortical population activity to be followed across acquisition, retrieval and extinction. This provides a rich description of how sensory representations evolve as learned value changes, and how these dynamics differ following Shank3 deletion. The observation that knockout mice eventually acquire a robust aversion but subsequently extinguish it more rapidly is particularly useful because it separates impaired acquisition from subsequent instability of the learned association.

The revised manuscript has substantially addressed several concerns raised in the original review. Importantly, the authors now show that reduced stimulus-evoked suppression is already evident during pre-learning habituation and that increased coactivity therefore appears to reflect, at least in part, a pre-existing network property rather than a consequence of learning. They additionally report that the level of coactivity at the beginning of conditioning correlates with subsequent behavioural acquisition, providing a useful link between individual variation in cortical activity and learning performance. This analysis strengthens the association between cortical network state and behaviour without establishing causality.

Another useful addition concerns the potential contribution of licking behaviour to taste decoding. Because sampling behaviour necessarily differs as animals acquire an aversion, separating sensory representations from movement-related activity is difficult in this paradigm. The authors now perform decoding during the ten-second post-sampling period and find above-chance decoding after licking has ceased, making it less likely that differences in licking alone explain the principal population-decoding results.

The manuscript is also clearer in its anatomical and conceptual terminology. The recordings are now appropriately described as being from gustatory cortex rather than implying coverage of the broader anterior insular cortex, and the conclusions have been restricted primarily to conditioned taste aversion rather than generalised to cognitive flexibility more broadly. The latter is particularly important because whether these findings generalise to reversal learning, probabilistic learning, or other forms of adaptive behaviour remains unknown.

Weaknesses

The principal remaining limitation is mechanistic. The experiments establish a robust association between Shank3 deletion, altered cortical activity and altered learning dynamics, but they do not establish the causal relationships among these observations. The new correlation between early coactivity and subsequent learning is informative, but manipulating the relevant network property would ultimately be required to determine whether increased correlated variability contributes directly to slower acquisition. The authors now acknowledge this limitation and have appropriately removed language implying causality.

Similarly, the cellular or circuit origin of the altered correlated variability remains unresolved. Reduced inhibition is an interesting potential explanation, but the authors do not directly measure interneuron function or inhibitory transmission here. Consequently, the proposed relationship between Shank3 loss, altered inhibition, increased correlated activity, and impaired sensory encoding should remain a hypothesis emerging from the results rather than a demonstrated mechanism.

A further limitation is the absence of a full conditioned-stimulus-only Shank3 knockout control group. The newly analysed habituation recordings partly address this issue by demonstrating reduced suppression and a tendency toward increased coactivity before learning, and the cross-session decoding analysis suggests that naïve knockout cortical populations can nevertheless distinguish water from saccharin. These analyses considerably improve interpretation of the existing experiment, although they are not equivalent to longitudinal comparison with a knockout control group undergoing the complete protocol without aversive conditioning.

Finally, the interpretation of population activity in terms of taste identity, learned value and their interaction remains necessarily limited by the task design. The results clearly demonstrate experience-dependent changes in population discriminability, but because taste identity, learned value, and sampling behaviour covary during conditioned taste aversion and extinction, the present experiments cannot fully separate the precise information represented by these population changes.

Overall assessment

The revision has addressed the major interpretational concerns raised in the previous review and has strengthened the manuscript through several useful additional analyses. In particular, distinguishing pre-existing cortical abnormalities from learning-associated changes, relating early coactivity to subsequent behaviour, controlling more carefully for licking-related activity, and removing causal language better align the conclusions with the evidence. The study therefore provides solid evidence for altered experience-dependent sensory coding and learning dynamics following Shank3 loss, while the mechanisms connecting these phenomena remain an important question for future work.

Reviewer #2 (Public review):

Summary:

Haast et al. investigated the organization of the zona incerta (ZI) in the human brain based on its structural connectivity to the neocortex. They found that the ZI is organized according to a primary rostro-caudal gradient, where the rostral ZI is more strongly connected to the prefrontal cortex and the caudal ZI to sensorimotor cortex. They also found that the central region of the ZI is differently connected to neocortex compared with the rostral and caudal regions and could be important as a deep brain stimulation target for the treatment of essential tremor.

Strengths:

I think the overall quality of this work is great, and the results are presented in a very clear and organized manner. I particularly appreciate the effort that the authors put into validating the results using 7T and 3T data, as well as test-retest data.

Weaknesses:

The initial version of the manuscript left me with a couple of minor concerns that the authors addressed in the revised version. These were related to the clinical relevance of the work (now addressed with the lower-resolution data), and to the initial emphasis on a dorso-ventral gradient that the data did not strongly support.

Reviewer #2 (Public review):

Summary:

In this work, Lohse and colleagues develop a system for doing targeted photostimulation in mouse cortex. The system uses a camera image to target laser stimulation to stereotactically defined locations in mouse dorsal cortex.

Strengths:

The hardware is well designed, and the software is well documented and supported. The build guide and well-documented software package should allow for simple implementation of the technology. Without a doubt, this is a valuable community resource for the circuit neuroscience field.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #2 (Public review):

Summary:

It is demonstrated that sponge larvae prepare for receiving the environmental cue (sunset) by extensively modifying their chromatin accessibility in the absence of large gene expression changes - "anticipatory" chromatin remodeling. This program can be offset by modifying the cue (making light constant), leading to a novel molecular state. These are the most important novel findings. Then, around metamorphosis there is dramatic regulation of transcription factors and their targets. While this result is hardly surprising, it is nice to see it demonstrated thoroughly in a phylogenetically fundamental animal study system.

Strengths:

This is a top-notch study of a key life cycle transition in an organism of great phylogenetic importance, involving concurrent gene expression and chromatic accessibility profiling (to the best of my knowledge, this has never been done in non-bilaterians and likely anywhere outside Vertebrata). The result is highly non-trivial. There is also an additional experiment modifying the key environmental cue (constant light), adding additional insight.

Weaknesses:

The paper presents a great amount of material, which somewhat obscures results related to the major novel finding ("anticipatory" chromatin remodeling). Among them is the fact that there is no significant statistical association between regions opened during remodeling and genes subsequently regulated during metamorphosis. This lack of direct association suggests that there may be more to the story than just priming the genome for metamorphosis.

Reviewer #2 (Public review):

Summary:

The manuscript "Thermodynamic principles of enzymatic regulation in biomolecular condensates from reaction-coupled molecular modeling" reports results of a very coarse-grained molecular dynamics (MD) simulation of three types of particles that undergo phase separation and chemical reactions. The study focuses on a molecule that can exist in two states (phosphorylated vs. unphosphorylated), whereas the third species is an enzyme that catalyzes the reaction in one direction of the transition. This topic of chemically active multicomponent mixtures is timely, and its connection to biomolecular condensates is well motivated in the introduction. Using their minimal setup, the authors find that driven reactions affect the composition of the dilute and dense region, and can even suppress phase separation completely. Moreover, the reaction fluxes are heterogeneous and exhibit a pronounced peak at the interface. The authors then interpret this acceleration of reactions in light of the role of condensates as reaction centers and conclude that the effect they report is relevant in cells.

Strengths:

A strength of the manuscript is the setup of a minimal system to understand the complex roles of enzymatically controlled, active reactions in condensates. This is a subtle topic since the physics of phase separation, implying non-ideal systems, affects the reactions, which can then no longer be described in a dilute approximation. The authors tried to take special care to ensure thermodynamic consistency, which is key to describing the interplay of the two effects accurately. The authors also perform relevant numerical tests, e.g., by determining the effect of reactions on the phase diagram, and measuring densities and reaction fluxes carefully. Moreover, they attempt to interpret the obtained quantities with intuitive pictures, although this is not always convincing.

Weaknesses:

The main weakness of the work is its presentation: I could not follow the detailed setup of the model since important details (such as the concrete interaction potentials and particularly the implementation of the reactions) are not described thoroughly. In particular, the implementation of the actively driven reaction is mysterious, and a proper negative control without activity is missing. Such a control is crucial since it would allow testing whether the implementation of the code ensures local detailed balance and thus thermodynamic consistency. Furthermore, such a passive null model would surely help in establishing the effects of activity, which is currently unclear. Since I don't understand the detailed setup, I cannot judge whether the major result, namely that reactions are accelerated at interfaces, is correct. It might very well be true, but I'm unable to judge this based on the current presentation. I detail my criticism in separate points below, and I hope that addressing these points helps the authors to improve their manuscript:

(1) I find the model setup unclear, which makes it difficult for me to gauge the correctness of the results. I think the details of the model need to be explained in more detail, both in the main text and the SI. There are three different aspects that I find lacking:

1a) The setup of the interactions in the model is unclear. First, only single interaction parameters \epsilon_i are specified, but the simulation likely needs to specify pairwise interactions. Table S2 is not particularly helpful since it uses a different notation (Is this switch of notation necessary?). Second, the statement that k_BT = 0.75 is confusing since k_BT should have units of energy. Third, the authors mention "a truncated and shifted LJ potential", but it is unclear whether this is the potential they use or not. Given this lack of details, I would not be able to immediately repeat the simulation, even without reactions.

1b) In the main text (page 6), it is unclear whether an active or passive system is studied. The subsequent results suggest that the system is active (and thus has sustained energy fluxes), but this needs to be explained in detail. In any case, I would like to see an explicit activity parameter, so that a passive control can be added. For instance, I would expect that a passive system remains the same when reaction rates are changed, but the energetics are kept constant. In any case, since it is not explained how activity enters the system, the subsequent results are unclear to me.

1c) It is unclear how thermodynamic consistency (i.e., local detailed balance) is ensured. For instance, how is the formation of a scaffold-enzyme complex performed (page 6)? The SI is also light on details. For instance, it is unclear whether the transitions in Equations (1-8) refer to individual particles (in this case, how are bimolecular reactions implemented?) or refer to densities or even overall particle counts. It is also suspicious that the reactions are given for the "dilute limit", whereas the main text clearly discusses condensed regions. It is also unclear how ΔU is calculated and what it means. Generally, I would expect a detailed discussion of detailed balance conditions in the SI, if not even in the main text. Here, it might help to clearly separate thermodynamic aspects (involving detailed balance) from kinetic considerations.

(2) If the authors study an active system, reporting averaged concentrations and partition coefficients might be misleading. Generally, active systems develop gradients in the dilute and dense regions (Figure 1), so any averaged measurement (such as a density) will depend on the size of the region. It is thus necessary to clearly define the measurements in the main text (so readers know how to interpret the plots), and the discussion needs to be much more careful, particularly when comparing to phase diagrams, which typically discuss thermodynamically large systems.

(3) The strongly increased fluxes at the interface are a bit suspicious, particularly since they are hardly visible in passive systems (Figure S6). The enhancement might originate from large reaction rates, leading to a short reaction-diffusion length scale (although I would then expect balanced reactions in the phases). Alternatively, they might originate from the microscopic details, such as the cut-off length of the kinase-interaction range. In any case, it would be important to establish a negative control using a passive setup and (based on this) explain the observed flux increase clearly.

(4) I am not convinced by the statement about the bias of reactions in the dense and dilute phase, e.g., on page 12. I would find it extremely helpful to start with a passive system (without energy input), where I would expect balanced chemical potentials, so that all reactions are balanced at all points in the system. Already in this case, there might be accelerated reactions at the interface (due to kinetic details), but the two reactions need to be balanced to have overall homogeneous chemical potentials. Starting from such a base state, one could then discuss how activity biases reactions in a certain direction. While the current explanation correctly mentions that a transition can be hindered by energetics, it fails to account for the different abundances. In a passive system, these two aspects are perfectly balanced, leading to a linking of reaction rate constants and partition coefficients (e.g., see https://arxiv.org/abs/2202.13646). These aspects need to be discussed much more cleanly (and they are intimately linked to the setup of the model, which is currently unclear; see point 1).

(5) I think the title is misleading. The authors do not establish new "Thermodynamic principles". I am also not sure what they mean by "reaction-coupled molecular modeling". Finally, the 
"enzymatic regulation" is hardly discussed in the main text, which instead seems to emphasize the accelerated reactions at the interface.

Reviewer #3 (Public review):

In this revised version of the manuscript, the authors have addressed several of the Reviewer's prior questions and comments. However, the main elements of the critique remain largely intact in the absence of additional experiments.

Major:

(1) The authors and Reviewer disagree as to the extent to which the main findings replicate prior work vs. represent true novelty. In their response, the authors state, "This appears incorrect. It was known that direct cortical stimulation (as was done in the Rosenthal paper) can drive a calcium event that resembles a CSD. It was also known that CSD can drive Fos expression. What was not known is that the Fos expression driven by ECS is fully explained by the calcium event (putative CSD)." First, what appears incorrect is the author's statement that Rosenthal et al. use direct cortical stimulation. So far as the Reviewer can discern, Rosenthal et al. in fact did not use intracranial or direct cortical stimulation, as stated repeatedly in the rebuttal. Instead, Rosenthal et al. use stimulating electrodes that are attached to the skull (i.e., non-penetrating), as clearly shown in Figure 1 of that paper. Describing this as "intracranial stimulation" and equating it with direct cortical stimulation used to induce CSD in Leão (1944), is incorrect and misleading.

(2) The authors ask for guidance in improving the clarity of their messaging around this and cite the Abstract as clearly stating what they view as the novelty here. Yet, the key section of the Abstract reads: "We show that CSD drives increased expression of the immediate early gene Fos, a key marker of neuronal plasticity, and is associated with factors that predict positive ECT therapeutic outcome. Our results suggest that the therapeutic efficacy of ECT may be mediated by CSD. This challenges the seizure-centric model and implies that CSD, a currently unmonitored neurophysiological event, may serve as a more relevant biomarker for predicting and optimizing therapeutic outcomes of ECT." The Reviewer finds this quite misleading, as it suggests (at least to this Reviewer) that the authors view themselves as showing, for the first time, that ECT generates CSD and that this may be the therapeutic mechanism of ECT, while not clearly defining what is new. Instead, one could perhaps more accurately write: "Prior work has shown that ECT generates CSD and that CSD is associated with increases in the immediate early gene Fos, a key marker of neuronal plasticity. We provide data suggesting that ECT-associated CSD in fact drives this increased Fos expression. Our results imply the hypothesis that the therapeutic effect of ECT may be mediated by CSD-induced Fos expression and could serve as a biomarker of ECT efficacy."

(3) In claiming novelty, the authors further assert that Rosenthal et al. provided "no confirmation beyond the observation of the calcium event that this is indeed a CSD." This also appears to be incorrect or misleading. Rosenthal et al. show simultaneous calcium and intrinsic optical imaging of hemoglobin dynamics associated with the propagating event. Rosenthal et al. also show DC-coupled electrophysiology demonstrating the slow potential shift characteristic of CSD using auricular ECS (the very stimulation model that the authors argue distinguishes their work from Rosenthal et al.). Hence, speculation that the "cortical seizure Rosenthal and colleagues find is a methodological artifact" is unsupported and should be removed.

(4) In the rebuttal, the authors clarify the novelty of their manuscript as "that ECS drives CSD dependent immediately early gene expression (Fos)." The Reviewer agrees with this, yet as summarized in the bullet point, finds this novelty to be relatively limited and not particularly surprising, given that Fos is robustly induced in association with CSD in various contexts. This novelty could be markedly enhanced by linking increased Fos expression mechanistically to the therapeutic efficacy of ECT. Instead, any relevance of the CSD-dependent Fos induction is unclear, and statements to that effect, while interesting, are hypothetical, yet follow logically from Rosenthal et al. Any association between CSD occurrence and clinical outcome in human patients could establish predictive value but would also not prove causality, as suggested. Also unclear is how Fos might be used as a biomarker in humans, and how this might be valuable as a biomarker beyond CSD itself (which can actually be recorded), is unclear. The authors should state these limitations.

Minor:

(1) Regarding the microprism preparation, the added three-week postoperative interval is helpful toward alleviating this concern. Still the absence of spontaneous CSD does not address the fact that cortical incision and the CSD likely elicited during implantation could alter subsequent susceptibility to experimentally evoked CSD. This should simply be acknowledged as a limitation.

(2) The Reviewer recognizes that the authors have added Tables S3 and S4 detailing stimulation parameters. However, the response that properties of the traveling calcium event do not depend on stimulation charge does not resolve the concern regarding analyses in which stimulation parameters are used to predict whether CSD occurs. Manually selecting stimulation parameters rather than selecting them randomly or systematically may confound these analyses, an issue which could simply be acknowledged.

Reviewer #2 (Public review):

Summary:

Mione et al. aim to resolve a long-standing question in comparative neuroscience: whether the macaque brain contains a functional analogue to the distributed human multiple-demand (MD) network. To address this, the authors employ a direct cross-species fMRI comparison using a multi-step saccadic maze task in humans and a simplified two-step version in macaques. By contrasting goal-directed navigation against a control condition that requires similar motor responses but no strategic planning, the study isolates the neural signatures of cognitive control across species.

Strengths:

The most compelling aspect of this work is its methodological alignment. Previous attempts to compare these systems often relied on comparisons of human BOLD signals and macaque single-unit recordings. By running parallel fMRI protocols, the authors establish a shared measurement basis that allows for a more direct comparison. The resulting activation maps demonstrate conserved network topology in dorsolateral and dorsomedial frontal cortex and insula.

Weaknesses:

However, there is concerning inter-individual variability in the macaque data, along with a notable design asymmetry between the human and monkey tasks. In the human experiment, 2-, 4-, and 6-step trials were mixed within the same blocks, whereas macaques performed only 2-step problems. This design difference likely places human participants in a state of sustained proactive cognitive control (Braver, 2012), as they must remain prepared for more demanding trials at any moment. This elevated baseline arousal may potentially inflate MD network activation even during the simpler 2-step trials in humans, making direct comparisons with the macaque data difficult.

The authors acknowledge this concern and note that similarities between species survive despite this procedural difference. However, their own individual-level data raise doubts about how robust these "similarities" really are. A defining feature of the MD system is consistent recruitment across individuals performing the same task. In this study, however, the two monkeys show markedly different activation maps in parietal cortex. The conjunction map (Supplementary Figure 6) does not support the claim that lateral and medial parietal regions are commonly activated across both animals at any meaningful statistical threshold, even when using a permissive threshold of |t| > 1.5, corresponding to approximately p ≈ 0.13 - 0.14 (two-tailed).

This suggests that the group-level parietal activations are largely driven by a single animal, raising questions about whether these regions can reliably be considered as part of the monkey MD network. Notably, in Premereur et al. (2018), the only other direct macaque fMRI study of a cognitively demanding task, their conjunction map between the two animals also failed to show common parietal activation during task switching. Taken together, these findings suggest that either the monkey MD network is not as similar to its human counterpart as claimed, or the monkey version of the maze task was simply not sufficiently challenging to fully engage parietal MD nodes.

Because of the design asymmetry described above, it remains difficult to determine which of these possibilities is correct (a genuine species difference versus insufficient task demands in macaques). The authors' current claims treat the group-level activations, which appear largely driven by one animal, as reliable evidence for homology, but without consistent individual-level support, such conclusions remain tentative.

In summary, the authors demonstrate functional correspondence between human and macaque cognitive control networks in dorsolateral and dorsomedial frontal regions. However, assertions that this extends to lateral and medial parietal cortex are not consistently supported at the individual level. While the overall conclusions are plausible, they would be significantly strengthened by demonstrating consistent activation across both animals in all claimed MD regions, ideally with a properly thresholded conjunction map.

Reviewer #2 (Public review):

Summary:

The authors provide the first thorough profiling of neurons in Tribolium characterized by the expression of the transcription factor foxQ2, which will be useful for developmental neurobiology. They use state-of-the-art methods convincingly to not only identify the neurons, but also to further characterize them anatomically and neurochemically.

Strengths:

Thorough and meticulous application of state-of-the-art anatomical methods in a non-standard laboratory organism.

Reviewer #3 (Public review):

Summary:

This manuscript by Choucri and Treiber responds to a recent paper by Azad et al., which responds to a paper by Treiber and Wadell (Genome Research, 2020). The controversy relates to the detection of transcripts with transposable elements (TEs) spliced into them in the Drosophila brain.

Strengths:

The authors now argue convincingly that these transcripts exist using an improved, updated version of their pipeline. They also validate some of their findings using RT-PCR and explain why Azad et al. failed to detect these transcripts due to methodological errors. Overall, I am convinced that these transcripts exist and that the TE-derived transcripts described by Choucri and Treiber are real.

Weaknesses:

The authors should mention that combining PCR-amplified cDNA generation with short-read sequencing is suboptimal for detecting TE-fusion transcripts. Recently, direct long-read ONT RNA sequencing, which does not require amplification and spans the entire transcript, has been used to detect similar transcripts in human stem cells and the human brain (PMID: 40848716 & Garza et al, BioRxiv) . Had the authors used this technology to validate their findings, there would be no question about these transcripts. If not doing such experiments, then they should at least discuss the possibility and the advantage of the approach.

Reviewer #2 (Public review):

The revised manuscript by Boni et al. is substantially improved, and in my view the authors have responded constructively to the concerns raised during the first review. Most importantly, they now explicitly distinguish the bistable green/blue states generated by the toggle switch from the reversible red and yellow quorum-sensing outputs and acknowledge that the latter do not constitute irreversible differentiated states. This clarification improves the conceptual accuracy of the work.

The other revisions are also well justified. The authors clarify that Fig. 2d is derived quantitatively from flow-cytometry data and explain how the cross-sections in Fig. 2e were obtained; they introduce the nullcline interpretation of the toggle-switch landscape and distinguish stochastic single-cell fate from predictable population-level proportions. They also provide more information on pLux engineering, report the functional forms and parameters of fitted curves, quantify the different HSL induction ranges, explain the heuristic treatment of entry into stationary phase, and clarify image selection and sender-receiver distance calculations.

The principal strength remains the systematic engineering of a sequential multi-circuit programme in a single bacterial genetic circuitry (spread over two plasmids). The work combines bistable symmetry breaking, LuxI/LuxR-mediated communication and an orthogonal CinI/CinR layer to generate spatially organised colony-level behaviours. The DBTL engineering cycle is also convincing: substantial cross-talk in the initially tested Lux/Las quorum-sensing combination was experimentally identified and addressed by switching to the more orthogonal Lux/Cin architecture, while subsequent promoter leakiness was identified and mitigated by replacing pLux with pLuxLac.

Overall, I consider the revisions sufficient to address all the important concerns.

Reviewer #2 (Public review):

Summary:

The authors of this study developed a closed-loop optogenetic stimulation system with high temporal precision in rats to examine the effect of medial septum (MS) stimulation on the disruption of hippocampal activity at both behavioral and compressed time scales. They found that this manipulation preserved hippocampus single-cell-level spatial coding but affected theta sequences and performance during a spatial alternation task. The performance deficits were observed during the more cognitively demanding component of the task and even persisted after the stimulation was turned off. However, the effects of this disruption were confined to locomotor periods and did not impact waking rest replay, even during the early phase of stimulation-on. Their conclusion is consistent with previous findings from the Pastalkova lab, where MS disruption (using different methods) affected theta sequences and task performance but spared replay (Wang et al., 2015; Wang et al., 2016). However, it differs from a recent study in which optogenetic disruption of EC inputs during running affected both theta sequences and replay (Liu et al., 2023).

Strengths:

The experiments were well designed and controlled, and the results were generally well presented.

Comments on revised version.

The authors of this study addressed all my concerns, some of them successfully. The stimulation disrupted theta oscillations, making quantification of theta sequences problematic. Within the constraints of their experimental design, the authors tried their best to address my concerns. Therefore, I am satisfied with the current version and express no further comments.

Reviewer #2 (Public review):

Summary:

The manuscript presents an interesting and potentially important single-molecule study of Cdc13 assembly on telomeric ssDNA. The experimental observations are intriguing, particularly the identification of distinct FRET states associated with different Cdc13 occupancies. However, I have substantial concerns about whether the current data support the central mechanistic conclusion as strongly as the authors claim. In particular, the manuscript does not yet clearly distinguish between the observation that two Cdc13 molecules are associated with DNA and the stronger mechanistic claim that Cdc13 loads sequentially as monomers and subsequently dimerizes on DNA.

Major concerns

(1) The central mechanistic conclusion is stronger than the evidence

The manuscript's principal model is that Cdc13 proceeds through the pathway monomer → DNA-bound monomer → recruitment of a second monomer → stable DNA-bound dimer. However, the experiments establish this sequence only indirectly.

The authors show that FRET state II is associated with one Cdc13 molecule. FRET state III is associated with two Cdc13 molecules. Cdc13-DM produces state II but not state III. WT Cdc13 can undergo I→II→III transitions. Direct I→III transitions also occur.

These observations are consistent with sequential binding, but they do not uniquely demonstrate that the second Cdc13 molecule first binds as a monomer and subsequently undergoes dimerization on DNA. In particular, the direct I→III events indicate that a preassembled dimer or another cooperative pathway can also contribute.

I therefore recommend substantially tempering statements such as "Cdc13 initially loads onto telomeres as a monomer." A more defensible formulation would be: "The data support a kinetically favored sequential pathway involving an initial Cdc13 binding event followed by recruitment of a second Cdc13 molecule." The authors could still present sequential loading as the preferred model, but the language should clearly distinguish a kinetically supported pathway from a uniquely established molecular mechanism.

(2) The assignment of FRET states II and III to exactly one and two Cdc13 molecules requires stronger validation

The interpretation of states II and III as one- and two-Cdc13 states is central to the entire mechanistic model. Because labeling efficiency, incomplete labeling, photophysics, and heterogeneous molecular populations can all affect the observed distributions, the authors should provide a quantitative probabilistic model. Specifically, the authors should calculate the expected distribution of one- and two-labeled-Cdc13 species given the experimentally determined labeling efficiency and compare these expectations with the observed FRET-state populations. This analysis would provide an important independent validation of the proposed stoichiometric assignments.

(3) The TG12 substrate raises an important stoichiometric and conformational question

The manuscript argues that a single Cdc13 molecule binds approximately 11 nt, while two Cdc13 molecules can bind a 12-nt ssDNA substrate. This raises an immediate mechanistic question: if one Cdc13 occupies approximately 11 nt, how can two Cdc13 molecules simultaneously associate with only 12 nt of ssDNA? This issue should be addressed experimentally rather than only structurally or schematically. A particularly informative experiment would be to systematically vary ssDNA length. The probability and kinetics of state III formation could then be quantified as a function of substrate length. Such an experiment would determine whether formation of the two-Cdc13 state genuinely requires additional DNA and whether the two proteins occupy overlapping or distinct regions of the substrate.

(4) The "salt-resistant" interpretation is overstated

The manuscript repeatedly describes state III as "salt-resistant" and uses this observation to support the existence of a highly stable physiological complex. However, the reported experiment examines only a relatively modest range of NaCl concentrations (50, 75, and 100 mM).

I recommend either expanding the salt-dependence analysis substantially or using more quantitative language. For example, the authors could report the fraction and lifetime of state III as a function of salt concentration and define explicitly what they mean by "salt-resistant." The current data support persistence under the tested conditions, but they do not by themselves establish exceptional physiological stability.

(5) The mass-photometry experiment does not establish the physiological solution equilibrium

The mass-photometry data are useful for demonstrating that Cdc13 can exist in monomeric and dimeric forms, but the current experiment does not establish the equilibrium between these species under physiologically relevant conditions.

A concentration series would be valuable. The observed monomer/dimer populations should be fit to an explicit equilibrium model to obtain an apparent dimerization constant and assess how strongly the equilibrium depends on Cdc13 concentration. This would also help connect the solution behavior to the single-molecule observations and determine whether the observed DNA-bound dimer could plausibly arise from a pre-existing solution dimer.

(6) The WT + R635C mixing experiment is overinterpreted

The interpretation of the WT + R635C experiment is currently complicated and somewhat speculative. The experiment is potentially informative, but the conclusions appear stronger than what can be directly inferred from the data. The authors should clearly distinguish between the observations directly supported by the mixing experiment and the mechanistic interpretation proposed from them. In particular, the experiment does not necessarily establish the precise sequence of DNA binding and dimerization events.

(7) The requirement for DNA-binding activity in both Cdc13 molecules is not fully established

The manuscript concludes that both Cdc13 molecules must possess DNA-binding activity. However, the R635C experiment does not clearly distinguish between:<br /> two independently DNA-bound Cdc13 molecules; and one Cdc13 molecule directly bound to DNA plus a second molecule whose DNA-binding surface is required for allosteric stabilization of the dimer.

This distinction is mechanistically important. Additional experiments using DNA-binding-defective mutants in defined heterodimeric configurations would help determine whether both molecules directly contact DNA or whether DNA binding by one molecule promotes recruitment/stabilization of the second through protein-protein interactions.

(8) Stronger controls are needed for FRET-state assignment

The FRET states are treated as discrete molecular states, but alternative explanations for heterogeneous FRET populations should be considered more explicitly.

Important controls would include: concentration-dependent FRET measurements in the absence of DNA binding; fluorescence controls to determine whether the observed states could arise from dye-protein interactions; Cdc13 mutants with altered DNA-binding specificity; alternative dye positions; demonstration that the major FRET states are reproduced with independent labeling configurations. The duplex-positioned Cy3 controls, which show little FRET change, are useful. However, they do not completely exclude the possibility that protein-induced changes in DNA conformation contribute to the observed FRET states. Independent labeling geometries would substantially strengthen the assignment.

(9) The physiological relevance of the 12-nt substrate requires better justification

The authors use TG12 as their primary substrate and state that telomeres contain approximately 12-14 nt of ssDNA during most of the cell cycle. This rationale requires greater biological context. Telomere length and the extent of the exposed G-rich strand are dynamic and heterogeneous, and Cdc13 has established functions throughout telomere replication. The authors should explain more carefully why TG12 is biologically representative and how the proposed mechanism is expected to behave on substantially longer telomeric substrates. The TG25 experiment is useful, but at present it functions primarily as a stoichiometric observation. A systematic substrate-length analysis, as suggested above, would turn this observation into a mechanistic test.

(10) The relationship to existing structural studies requires deeper discussion

The manuscript presents sequential loading as a novel mechanism, but existing structural and biochemical studies of Cdc13 dimerization and Cdc13-DNA architecture are essential for interpreting these observations. The authors should explicitly reconcile their proposed model with the existing structural literature. In particular, they should address:

Does the known Cdc13 dimerization interface permit simultaneous DNA binding by both subunits?

Is the dimerization interface compatible with the proposed DNA-bound state II?

Could DNA binding alter the dimerization interface?

Are the two Cdc13 molecules predicted to bind overlapping or distinct portions of the telomeric sequence?

Can the structural models accommodate the apparent stoichiometry on a TG12 substrate?

Without this reconciliation, the proposed sequential-loading mechanism remains somewhat disconnected from the established structural framework.

Other important issues:

(11) The Kd comparisons are confusing

The manuscript should include a table summarizing the different Kd values and explicitly explaining why they differ. In particular, describing 3.7 nM as the Kd for the first monomeric binding step while reporting an apparent Kd of 1.2 nM requires careful kinetic and statistical justification. The authors should distinguish clearly among microscopic Kd values, apparent Kd values, and parameters inferred from kinetic models.

(12) The direct-dimer pathway deserves greater attention

The occurrence of direct I→III transitions is mechanistically important and should not be treated primarily as an exception to the sequential pathway. A more balanced conclusion would be:

"Both pathways contribute to formation of the final Cdc13-DNA complex, with the sequential pathway being kinetically favored under the experimental conditions." This interpretation appears better aligned with the data and would still constitute a strong mechanistic conclusion.

(13) The "kinetic proofreading" interpretation is currently speculative

The Discussion proposes that the first Cdc13 monomer provides a kinetic proofreading step. This is an interesting hypothesis, but it is not directly demonstrated by the current experiments.

I recommend changing this to language such as "a kinetic proofreading-like mechanism may be possible" unless the authors can provide direct evidence that the first binding event selectively promotes productive complex formation or rejects nonproductive substrates.

(14) Biological-function claims should be clearly separated from the in vitro findings

The manuscript frequently connects the stable state III complex with telomere protection, telomere length regulation, CST formation, Est1 recruitment, Pol α recruitment, and prevention of DNA degradation. None of these functions are directly tested in the present study.

The experiments establish a biochemical/single-molecule mechanism in vitro. They do not establish that state III is the functional protective species in vivo.

This distinction should therefore be maintained throughout the Abstract, Discussion, Key Points, and concluding statements. The authors can appropriately discuss these possibilities as<br /> implications or hypotheses, but should avoid presenting them as demonstrated functions of state III.

Reviewer #2 (Public review):

Summary:

The main aim of the presented manuscript was to test and validate proton observed proton edited 13C MRS and track the 13C label from uniformly labeled U-13C-glucose into glutamine, glutamate, GABA and lactate in rodent and human brain in vivo.

Strengths:

In contrast to already established methods of 13C and 2H MRSI this method applies only proton RF and thus can potentially be implemented on a standard clinical scanner.

Weaknesses:

The validation of the method in a human setting is rudimentary. First, even at ultra-high field strength of 7T, the authors did not reach sufficient SNR to detect and quantify GABA with good CV, and further, the experiment needs optimisation to reach metabolic and fractional enrichment steady state and/or for additional conditions to show the possibility of lactate detection.

Reviewer #2 (Public review):

This study by Wang et al. longitudinally tracks hippocampal CA1 population dynamics in Alzheimer model rats during repeated exposure to different environments. The authors dissociate two levels of neural coding. "Explicit" spatial coding, assessed by rate maps and population vector correlations, is severely impaired in AD rats and does not improve with experience. In contrast, "implicit" temporal cofiring structure, quantified by pairwise Kendall's tau and population cofiring correlations, becomes progressively more context-specific over days, mirroring behavioral learning. This preserved temporal coding is not merely a byproduct of spatial overlap, as position-independent rate analysis confirms that learning-dependent discrimination arises from intrinsic temporal dynamics rather than from residual spatial tuning. Moreover, offline sharp-wave ripple reactivation shows increasing consistency across days specifically in AD rats. These findings reveal a dissociation in the AD hippocampus and propose that temporally structured population dynamics, rather than spatially selective firing, may support residual cognitive function.

Overall, the findings are novel and thought-provoking; the analyses are comprehensive and well-controlled, and the proposed re-registration framework offers a compelling new lens for understanding cognitive resilience in Alzheimer's disease, with clear potential to guide future neuromodulation interventions.

I have only a few minor comments.

(1) Justification of terminology ("explicit" vs. "implicit").

The manuscript should clearly define the two terms early in the Introduction, ideally in a dedicated paragraph. In my opinion, the current use of "explicit" and "implicit" is not intuitive and may even be misleading.

(2) Does the dissociation reflect differential impairment between rest and running states in AD?

The authors could elaborate on this.

(3) Effect size in Figure 1D - the difference does not appear very large.

The statistical significance in Figure 1D is accompanied by relatively modest effect sizes. The authors may tone down the claim about "failure to distinguish" in the manuscript ("failed to distinguish different contexts during the second transition" on Page 8).

(4) Definition of "confused cells" - why not a fixed correlation threshold?

The authors define "confused cells" as those whose B2‑A2 spatial correlation exceeds the 95th percentile of the A1‑B1 baseline distribution within the same animal. This seems to be unnecessary. A fixed correlation threshold (e.g., r > 0.5 or r > 0.6) would have a direct biological interpretation: it would identify cells that maintain similar firing fields across two putatively distinct environments, i.e., cells that truly fail to remap. In contrast, a relative percentile threshold defines "confusion" not against an absolute standard of similarity, but against the degree of remapping observed during the first A‑B transition.

(5) SVM decoding on spike‑train temporal structure - missing justification.

The authors state that "the temporal structure of spike trains contained distinct contextual information" (P9) and then directly apply an SVM decoder to the data, but the logical bridge is missing. Why is a decoder necessary here, and is it useful for such a task?

(6) Figure 6 - learning occurs during reactivation but does not transfer to the online state (theta state), even after days of training.

What does this mean in terms of different phases of memory? Is the consolidation phase affected more? The authors may provide more discussion along these lines.

Reviewer #2 (Public review):

This manuscript reports on reinforcement learning in participants with current depression, remitted depression (without current depression), in people without depression but with first-degree relatives with depression, as well as healthy controls. Participants completed two common tasks measuring reinforcement learning and risk aversion, and their behavior was fit to computational models assessing processes on these tasks.

Participants with remitted depression showed a lower punishment learning rate and more value-concordant decisions on the reinforcement learning task. Relatives of depressed participants, as well as people who are currently depressed, had higher inverse temperature, indicating more value-driven choices. Within the non-depressed participants, the punishment learning rate was negatively associated with symptoms of anhedonia and apathy.

I have reviewed this paper at a previous journal. This revised version is responsive to most of my concerns, particularly in terms of placing the manuscript more in the context of other related literature and providing more details on methods. There are some remaining concerns about sample size and the appropriateness of some of the methods (e.g., interpreting participant-level parameter estimates from hierarchical models estimated using BMA), but the latter has been adequately addressed with sensitivity analyses.

Reviewer #2 (Public review):

Summary:

Dietary restriction (DR) increases lifespan, an effect that has been consistently observed in several organisms, but we still lack a clear mechanism to explain this phenomenon. In this work, Hwangbo et al. revisited the role of the circadian clock in DR-mediated lifespan effects. They found that the increase in lifespan produced by DR is missing on a clock mutant, a clock dependency that is also observed at the level of nutrient-dependent egg laying. By conducting RNA-seq with an impressive temporal resolution, they showed that DR triggers an increment in the number of cycling genes expressed in the fat body, the fly functional analog of the mammalian liver. Interestingly, from these genes, a group of them are de novo daily expressed genes, meaning that their expression was not rhythmic under the control diet but appear rhythmically expressed under DR. Among those, genes encoding proteasome subunits are enriched. The authors finally showed that adult-specific knockdown of these genes in the fat body prevents the increase in lifespan under DR, further supporting a role of the proteasome in this process. Overall, the conclusions are mostly supported by the evidence presented, and the authors' discussion nicely frame their results with other research in the field.

Strengths:

- Many studies have limited their observations of DR on lifespan to a few dietary conditions which makes the reach of some previous conclusions somewhat limited. The dilution strategy that the authors used in this work provides a strong indication that the effect of DR on lifespan relies on clock expression regardless of the conditions used. Furthermore, the inclusion of the egg-laying assay is a good addition to support this hypothesis.

- Because the strength of the rhythmicity statistics relies heavily on the number of data points collected, the temporal resolution used for the RNA-seq experiments (every 2 hrs per 48hrs) is remarkable. This allows exquisite dissection of the phase of rhythmic genes in different conditions. The dataset produced in this work might be of use to other groups interested in weighting the role of other represented gene clusters in DR.

Weaknesses:

I see only minor flaws in this work, that if addressed, might strengthen the authors' conclusions, particularly:

- The results of the lifespan assays are quite variable and in some instances contradictory (Fig. S8) across trials, possibly because there are other unaccounted variables we still do not understand. The fecundity assay, in contrast, seems to be a better readout (Fig. 2). Confirming at least the two genes picked for the study (Fig. 5) would be good support for the claim that the proteasome mediates the effects of DR.

- According to the model, the acute effect of DR on gene expression is related to CLOCK protein function. However, I am not sure how this link was established. It is tempting to assume that CLOCK upstream is the reason for having an increase in rhythmic genes under DR, but the experiments did not test this. The tests conducted either assessed the role of clk or the effect of an impaired proteasome on DR-dependent extension of lifespan. Thus, it is difficult to assert the authors' claims on the link between CLK and the changes in cycling genes and to the proteasome upon DR.

Comments on revised version:

In this new version, Hwangbo and colleagues add new data to support a role of the clock in the effects of DR on lifespan. While adding this new data helps to alleviate some of the concerns previously raised, I think there are still some gaps. Below are my main concerns:

ClkJrk transcriptomic data: Adding this data supports a role of the clock in daily rhythmicity of genes in the fat body, likely due to a circadian role. However, it does not show that de novo rhythmicity of proteasomal genes is clock-related since there is no ClkJrk DR dataset. Thus, there is still a possibility this is a pleiotropic effect. While redoing an entire RNA-seq dataset might not be feasible, a possible way to support the circadian claim would be to use proxy genes observed in the Ctrl vs DR conditions and compare them by qPCR in ClkJrk Ctrl vs DR.

Feeding data: Are the flies reared in DR conditions, or do they just start the DR at the beginning of the experiment? If so, is it possible the flies will show a different feeding pattern after consecutive days of DR affecting overall (5-10 days) food consumption?

Clk expression is important for non-circadian roles in the ovaries (Wang et al., Cell Mol Life Sci, 2025). Therefore, it is possible that the fecundity effect is at the low level of the ovary/egg development instead of integration and processing of DR. This might be a confounding effect when interpreting data in Fig 2 in ClkJrk as solely the effect of DR.

Line 215: Considering the discussion above, I'd rather change "circadian-dependent change" to "daily"<br /> Considering that the ClkJrk RNA-seq transcriptomic was generated, presumably, at a different date/time than the original transcriptomic data from Control vs DR, comparative metrics (seq depth, mapping rate, etc.) between these are needed.

How is the feeding analysis conducted? I believe the method information was not updated.

Reviewer #2 (Public review):

Summary:

The aim of this work was to characterise in detail the cellular organisation of the growth plate in the growing mouse limb, the effect of mechanical loading due to physical activity, and the role of primary cilia in mediating this effect as putative mechanosensors. Primary cilia are generally thought to function as mechanosensors in a range of different organs and tissues, e.g. in the kidney. Exposure to mechanical loading is a normal part of post-natal limb growth, and the central hypothesis underlying this work was that primary cilia will play an important role in transducing the effects of mechanical loading into cellular responses during this process. To investigate this, the authors used a mouse model encoding fluorescent markers for primary cilia, and also allowing conditional knockout of IFT88, a protein that plays a key role in primary cilium biogenesis. The authors compared the effects of mechanical loading by comparing tissue from mice with normal or surgically immobilised limbs. To investigate the role of primary cilia, the authors performed the same experiments with mice treated with tamoxifen to induce IFT88 knockout.

Strengths:

A major strength of this work is that it studied primary cilia in a fully in vivo system. The authors employed cutting-edge imaging approaches and image analysis pipelines to rigorously study cellular organisation, centriole positioning, cilium length, and orientation across thousands of cells in limbs from 18 animals. The imaging results presented are of an exceptionally high-quality. The analysis of imaging data is extremely quantitative and robust and utilised appropriate statistical analyses, which were clearly stated throughout. The imaging data were complemented with transcriptomic data and analyses, which provided an orthogonal dimension for understanding cellular responses. An interesting and unexpected outcome from this is that the primary cilia are oriented in the same direction, and inclined at a roughly 45{degree sign} angle to the mediolateral and proximal-distal axes of the limb. The authors speculate as to how this might arise and the role it may play in sensing.

Weaknesses:

Overall, the work reported in this study was of a very high quality, and I could not find any significant shortcomings. However, I did feel that the paper was not very well written in many places, which made it difficult to read.

Overall, I think that the authors did achieve their aims in this study. It will be interesting to unpick the cellular mechanisms that lead to alignment of the cilia, the role of this alignment in mechanosensing, and the molecular mechanisms by which the cilia sense mechanical strain. Thus, this work provides a fertile ground for future studies, which will have important consequences for the study of primary cilia in vivo.

Reviewer #2 (Public review):

Summary:

In this review, the authors set out to synthesize how cryo-EM is reshaping RNA structural biology, moving the field from the determination of static, individual conformations toward the reconstruction of dynamic conformational ensembles and energy landscapes. Through eight case studies spanning ribozymes, riboswitches, viral RNAs, and synthetic RNA assemblies, they aim to show how cryo-EM has revealed mechanisms of RNA motion, including folding, ligand-dependent switching, and cooperative assembly, and to provide a practical account of the experimental and computational challenges (construct design, sample preparation, vitrification, data analysis) that are specific to dynamic RNA targets.

Strengths:

The manuscript succeeds in bringing together a wide and genuinely current range of case studies illustrating the field's shift toward dynamics-focused cryo-EM, several published within the last one to two years. The dedicated "Challenges" section, which walks through construct engineering, buffer and vitrification optimization, grid screening, and heterogeneity-resolving computational approaches, is a particularly useful and practical contribution; it goes beyond simply cataloguing structures and gives readers new to the area a genuine methodological roadmap. The figures are detailed and well matched to the quantitative claims made in the text (helix rotations, distances, RMSDs), which strengthens the paper's value as a reference resource.

Weaknesses:

The coverage of two areas in particular, the SL5 viral RNA element and RNA quaternary/multimeric assemblies, would benefit from incorporating additional recent primary literature that is directly relevant but currently omitted. This does not undermine the manuscript's core narrative, but it means the review is presently less complete than it could be as a field synthesis, particularly for readers using it to identify the full body of recent work on these specific RNA classes.

More concerning is that one specific structural claim, describing conformer heterogeneity in the cobalamin riboswitch (Case study 3, holo dimer 4), appears to invert the finding reported in its own source paper (Ding, Deme et al., 2023). As written, the manuscript states that P2 and the distal half of P6 are structured in dimer 4, whereas the source paper reports that these are the regions that could not be modeled. This is worth flagging prominently because it is a factual claim about a specific structure, not an interpretive point, and readers relying on this review as a secondary source could come away with an inverted understanding of that structure's flexibility.

The manuscript also contains several minor internal inconsistencies. None of these individually threatens the paper's core arguments, but together they suggest the manuscript would benefit from a careful proofreading and reference-list audit pass.

Overall, the authors largely achieve their stated aim. The case studies convincingly illustrate that cryo-EM can now resolve discrete and continuous conformational states of RNA at near-atomic resolution, and the Challenges section substantiates the claim that construct design, sample preparation, and computational innovations have been jointly responsible for this progress. The gaps in coverage of SL5 and multimeric RNA literature, and the inverted claim in Case study 3, are the main respects in which the manuscript falls short of being a fully comprehensive and accurate synthesis at this stage, but these are correctable issues rather than flaws in the overall argument or framework.

This review is likely to be a useful entry point and practical reference for researchers moving into RNA cryo-EM, particularly given the level of methodological detail in the Challenges section. Its impact would be strengthened by two additions. First, a short discussion of recent cryo-EM advances in tRNA would round out the manuscript's coverage of classical RNA structural targets alongside the ribozyme, riboswitch, and viral RNA case studies already included. Second, the raiA non-coding RNA, currently mentioned only briefly, has in the last two years become a genuine model system for cryo-EM-based ncRNA structure determination, progressing from a single novel-fold discovery to a comparative structural framework spanning multiple raiA subtypes and candidate protein partners. Expanding this into a full case study would let the manuscript showcase, in a single worked example, exactly the kind of field-level progression (from novel fold to scaffold-based strategy and further to comparative structural framework) that the review's own framing describes as the trajectory of the field as a whole.

Reviewer #2 (Public review):

Summary:

The manuscript by Nilssen et al. presents a comprehensive study of the circuitry linking the medial and lateral entorhinal cortices (MEC and LEC). Using a combination of anatomical tracing, optogenetics, and in vitro electrophysiology, the authors convincingly demonstrate that the MEC sends both glutamatergic and long-range inhibitory SST+ GABAergic projections to the LEC, with distinct laminar and cell-type-specific targeting. Notably, they reveal that SST+ inhibitory projections selectively suppress the activity of layer IIa neurons, whereas excitatory inputs preferentially engage neurons in layers IIb and III, thereby differentially modulating hippocampal-projecting populations.

Strengths:

The experiments are carefully executed, the results are compelling, and the conclusions are well supported by the data. This work will be of broad interest to researchers studying memory circuits, cortical inhibition, and the organization of long-range connectivity.

Weaknesses:

Although the in vivo relevance of these connections remains to be determined, this is an important and timely contribution to our understanding of entorhinal-hippocampal interactions.

Comments on revised version.

The authors have addressed my comments satisfactorily, and I am satisfied with the changes made in the revised version.

Reviewer #2 (Public review):

Summary:

This manuscript presents a thoughtful and well-executed study of critical period plasticity in the Drosophila larval motor circuit. The authors examined how transient heat, 32C, during embryonic stage, altered network properties, showing that premotor interneurons A27h increase excitatory drive onto motoneurons, which respond with a reduction in excitability. At the NMJ, synaptic terminals expand and GluRIIA distribution shifts, yet synaptic transmission remains largely unaffected. Despite these local compensations, the treated larvae display slower crawling and prolonged recovery from seizures, indicating that the network is functionally compromised.

Strengths:

(1) One of the major strengths of this study is the elegant dissection of a defined circuit, tracking changes from premotor interneurons through motoneurons to the NMJ. The multimodal approach provides a comprehensive view of how connected elements respond to CP perturbations.

(2) An interesting finding is that NMJ morphology changes dramatically without corresponding deficits in synaptic transmission, challenging the common assumption that larger boutons necessarily indicate stronger synapses.

(3) Another intriguing result is that even with two layers of homeostatic compensation, locomotor behavior is still impaired, highlighting the limits of compensation and underscoring the critical role of CP timing.

(4) Beyond these scientific insights, the study benefits from a well-defined, tractable system and simple experimental manipulations, which together make the results highly interpretable and reproducible.

Comments on revised version.

The authors have carefully considered my comments and recommendations and have made substantial efforts to improve the clarity and validity of the study. Although additional electrophysiology experiments using shorter heat stress windows were not feasible, the authors performed additional analyses of postsynaptic GluRs and provided a clearer discussion of the study's limitations. Overall, this is a strong and well-written paper that establishes a valuable foundational framework for addressing interesting and important questions about adaptive responses in developing neural circuits.

Reviewer #2 (Public review):

Using electrophysiological recordings in freely moving rats during a spatial navigation task, this study investigated the role of beta oscillations in the hippocampal-prefrontal network. Through a set of appropriate analyses-including oscillation-oscillation coupling, oscillation-spike modulation, and behavioral dependant measurements-the study presents convincing evidence for uncoupled beta activity between the two regions. These findings offer important insights into the network mechanisms of spatial navigation and may have significant implications for related neurological disorders.

Comments on revised version.

The authors have carried out additional analyses and made corresponding revisions to the manuscript in response to the earlier review comments, which have made the conclusions more convincing. However, please note that the last question about coexistence of beta and SWR has not been fully answered. Please supplement your response to address this point completely.

Reviewer #2 (Public review):

Summary:

The goal of this study was to find evidence for local adaptation in survival and fecundity of the model plant Arabidopsis thaliana. The authors grew a large set of Swedish Arabidopsis accessions at four common garden sites in northern and southern Sweden. Accessions were grown from seed in trays, which were laid on the ground at each site in late summer, screened for survival in fall and the following spring, and fecundity was determined from rosette size and seed production in spring. Experiments were complemented by 'selection experiments', in which seeds of the same accessions were sown in plots, and after two years of growth, plants were sampled to determine fitness from genotype frequencies, providing a more comprehensive evaluation of lifetime fitness than can be gleaned from fecundity alone.

As the main result, southern accessions had higher mortality in northern sites in one of two years, but also suffered more slug damage in southern sites in one year, indicating a potential link between frost tolerance and herbivore resistance. Fecundity of accession was highest when growing close to the 'home' environment, but while accessions from one sand dune population in southern Sweden had among the lowest fecundities overall, they consistently had the highest fitness in the selection experiment. Accessions from this population had large seed size and rapid root growth, which might be related to establishment success when arriving in a new, partially occupied habitat. However, neither trait could fully explain the very high fitness of this population, suggesting the presence of other, unmeasured traits.

Overall, the authors could provide clear evidence of local adaptation in different traits for some of their experiments, but they also highlight high temporal and spatial variability that makes prediction of microevolutionary change so challenging.

Strengths:

A major strength of this study is the highly comprehensive evaluation of different fitness-related traits of Arabidopsis under natural conditions. The evaluation of survival and fecundity in common garden experiments across four sites and two years provides an estimate of variability and consistency of results. The addition of the 'selection experiment' provides an extended view on plant fitness that is both original and interesting, in particular highlighting potential limitations of 'fitness-proxies' such as seed production that don't take into account seedling establishment and competitive exclusion.

Throughout the study, the authors have gone to impressive depths in exploring their data, and particularly the discovery of 'native volunteers' in selection experiment plots and their statistical treatment is very elegant and has resulted in compelling conclusions. Also, while the authors are careful in the interpretation of their GWAS results, they nonetheless highlight a few interesting gene candidates that may be underlying the observed plant adaptations, and which likely will stimulate further research.

Overall, this study will likely make an important contribution to the field of evolutionary biology, and it is another very strong example of how the extensive molecular tools in Arabidopsis can be leveraged to address fundamental questions in evolution and ecology, to an extent that is not (yet) possible in other plant systems.

Reviewer #2 (Public review):

Summary:

The manuscript by Chang et al. presents the discovery and deorphanization of two bona fide PRXamide GPCRs, and their peptide agonists, in Aplysia californica (sea hare, a type of sea slug). Focusing on MMG2-DPb, the most potent peptide agonist, the authors demonstrate that pyroglutamination on the extracellularly facing N-terminus of the peptide reduces its agonist potency towards ApPRXa-R1 and increases potency towards ApPRXa-R2. Model-guided pocket and peptide mutagenesis demonstrate multiple differential dependencies and sensitivities of ApPRXa-R1 and ApPRXa-R2, supporting distinct mechanisms of peptide binding. The authors then show that a pyroglutaminated version of the dog Neuromedin U peptide has an increased potency towards human NMUR1, relative to the free-Gln version or the non-Gln-containing human peptide, whereas the potency towards NMUR1 is slightly reduced. These examples delineate N-terminal pyroglutamination of a peptide agonist as a receptor subtype selectivity controlling mechanism even when the peptide binds the receptor C-terminus.

Strengths:

(1) Identification of a class of GPCR-signaling peptides in a poorly characterized organism (Aplysia californica, a type of a sea slug).

(2) Deorphanization of two Aplysia californica GPCRs and the establishment of an Aplysia analog of the human Neuromedin U signaling system.

(3) A rigorous structure-function study of the Aplysia receptors and peptides.

(4) Identification of pyroglutamination as a mechanism controlling subtype selectivity in Aplysia PRXamide receptor system.

(5) Very clear and compelling graphics.

(6) Overall, the biochemical and pharmacological part of the study is strong, exciting, and well-presented.

Weaknesses:

(1) My biggest problem is with the emphasis on the 'non-contacting role' of the N-terminal pGlu: "purely on positional grounds, N-terminal pQ/Q is unlikely to form direct contacts with receptor residues", "Despite the absence of direct pQ/Q-receptor interactions...", "indirect PTM distal steering", etc. The absence of direct contacts between pGlu and the receptor residues is likely an artifact of limited precision in modeling and reflects insufficiently advanced modeling tools. The authors used Swiss-Model, Robetta, and HPEPDOCK: why not go for the state-of-the-art AI modeling software like Boltz, Chai, or AlphaFold? A quick AlphaFold3 generates a model where Gln and pGlu form perfect contacts with the receptor N-term and ECL2 (in ApPRXa-R2) or N-term and ECL3 (in ApPRXa-R1). The N-termini of both receptors are quite long, form well-defined tertiary structures, and fold onto the receptor extracellular loops; in the case of ApPRXa-R1, the N-terminal domain is stabilized by an intra-domain disulfide bond C15(NT)-C133(NT) and stapled to ECL2 via another disulfide bond C16(NT)-C322(ECL2), suggesting that these interactions are real. The 3D models generated by the authors are not available for review but based on figures, I don't see these N-terminal domains modeled at all, which would obviously affect the docked position of the peptide N-terminus and its contacts with the receptor. For example, in an AlphaFold model, ApPRXa-R1 F161, E317, R340 are in direct vicinity of the peptide's Gln / pGlu and are quite distinct in ApPRXa-R2 - direct contacts with these or nearby residues may easily explain the differential preferences for free Gln vs pGlu but none of them were tested via mutagenesis.

(2) Some conclusions and interpretations in the manuscript are overstated and lack precision. For example: "...a single N-terminal lactam modification converts a minimal chemical difference into bidirectional GPCR subtype outputs" makes the reader think about something as dramatic as an inversion of efficacy, an agonist becoming an antagonist, or at least the introduction of signaling bias, whereas in reality, the authors established that pyroglutamination reduces the potency of the peptide at one receptor but increases it at the other receptor. The use of PTMs and proteolytic processing is a broadly utilized mechanism for controlling GPCR peptide selectivity. The effects of pyroglutamination on the potency of MMG2-DPb towards its two Aplysia receptors very well align with this mechanism and should be described as such. Other examples of sentences that are similarly misleading: "... the same ligand, ..., produces opposite functional outcomes: pQ suppresses ApPRXa-R1 activation while enhancing ApPRXa-R2 activation" (in reality, both receptors are activated and the functional outcomes are the same, just achieved with different potency), "A single N-terminal lactam enables bidirectional GPCR subtype tuning...", "This bidirectional, subtype-specific effect of pQ is unprecedented in neuropeptide signaling..." (not bidirectional and not as unprecedented as the authors make it sound), etc.

(3) Overall, what the authors present as a new "indirect PTM distal steering" mechanism is really not supported by the data. The described systems fit the classical "lock-key" and "induced fit" paradigms rather than challenging them. I recommend the authors revisit their modeling approaches and model-guided interpretations of the biochemical/pharmacological data. Importantly, I do not think such revision would undermine the key strengths of the paper (listed above in Strengths). Based on experimental data alone, this is a very strong and interesting study and with more rigorous modeling and adequate interpretations, it can be made exceptional.

Reviewer #2 (Public review):

This manuscript addresses an important public health question in adolescents using a large longitudinal ABCD cohort. It is generally well organized and presents a compelling story linking central adiposity, brain development, and cognition. The authors use baseline and 4-year follow-up data from the ABCD Study (N = 8,519 baseline; N = 1,873 longitudinal, the use of 4-year follow-up only should be justified) to examine how adiposity relates to cognitive performance and cortical structure in early adolescence. Major findings include: (1) central adiposity indices (BRI, WHtR) show stronger cross-sectional associations with cognition than BMI, partially mediated by frontotemporal cortical morphology; (2) the rate of adiposity accrual, rather than static adiposity at either timepoint, predicts follow-up cognition and altered (attenuated) cortical thinning; and (3) among overweight/obese adolescents, central fat reduction is accompanied by accelerated cortical thinning and catch-up in inhibitory control.

The use of large longitudinal data from ABCD is considered a strength as most prior neuroimaging work is cross-sectional. The focus on adiposity change velocity within adolescence is considered novel over single-timepoint designs, and the comparison of central-fat indices (BRI/WHtR) against BMI in a neurodevelopmental context is important given growing interest in these markers in cardiometabolic research. The subgroup observation that fat reduction may be accompanied by normalization of both cortical trajectories and inhibitory control is potentially clinically meaningful, suggesting reversibility rather than fixed deficit, and would be of broad interest if it withstands more rigorous analysis.

However, several major issues were identified. For example, the strength of the causal language is not supported by the design, the headline comparison between adiposity indices rests on coefficients that are described as standardized but evidently are not, and several analytic and reporting issues (attrition and selection, family clustering, regression to the mean in the subgroup analysis, internal inconsistencies in reported p values) must be resolved before the conclusions can be considered established.

Major Concerns

(1) ABCD provides more follow-ups and should be included. Also, there is a severe longitudinal attrition (8519 vs 1873) that needs to be addressed. No comparison of completers versus non-completers is provided. The cohort description of having 78.5% White seems to be wrong, suggesting either a coding error in the race variable or strong selection introduced by the exclusions. The exclusion of 483 participants for "extreme adiposity indicator values" is especially concerning in a study of obesity and should be justified with explicit criteria in the main text, with sensitivity analyses retaining these participants where possible.

(2) Adiposity is heavily influenced by socio-economic status, which itself contributes heavily to cognition and mental health as well. Many ABCD studies have reported this. Another important factor contributing to adiposity is sleep, which itself can contribute significantly to cognition and mental health. Many ABCD-based sleep studies have been published and can be reviewed.

(3) The manuscript repeatedly uses causal language that the observational design cannot support (e.g., "longitudinal fat accumulation ... drives cortical alteration," "fat reduction activated adaptive neural change," "neuroprotective management"). The cited literature (Likhitweerawong et al., 2022) is explicitly bidirectional: poor inhibitory control may promote weight gain rather than the reverse. The baseline mediation analysis is particularly problematic because exposure, mediator, and outcome were measured concurrently, so the temporal ordering required for mediation is assumed rather than established. Causal claims should be tempered throughout. Similarly, mediation analyses should be interpreted cautiously as statistical rather than causal.

(4) zBMI, WC, BRI, and WHtR are highly intercorrelated, as are follow-up adiposity and delta-adiposity (Table 3). Entering them simultaneously invites unstable estimates and sign flips. Please report pairwise correlations and variance inflation factors, and demonstrate that the key conclusions are robust.

(5) Subgroup analysis: regression to the mean, group labels, and internal inconsistencies. (a) Stratifying on delta-BRI >= 0 enriches the "decreasing" group for high baseline values, so the observed cognitive "catch-up" and accelerated thinning may partly reflect regression to the mean; the percentile-based sensitivity analysis mitigates but does not resolve this. Group comparisons should adjust for baseline BRI and baseline cognition. (b) zBMI >= 1 corresponds to overweight by WHO criteria, not obesity; labelling this group "obese adolescents" throughout (including the Abstract) is inaccurate. (c) The Abstract claims a Flanker slope difference of p < 0.05, but the Results report p = 0.058 versus controls (described, incorrectly, as significant) and p = 0.021 only versus the increasing-BRI group; these statements must be reconciled. (d) It is unclear whether subgroup p values were FDR-corrected and how t-tests/Wilcoxon tests produced "adjusted" p values; if covariate adjustment was intended, regression models are required.

Tables 2-4 mention that beta values are standardized, yet the magnitudes are clearly scale-dependent (e.g., WHtR beta = -7.5 vs. zBMI beta = -0.42 in Table 2; WHtR delta-beta = -780 in Table 3). This undermines the central claim that BRI/WHtR "outperform" BMI, since predictors cannot be compared on unstandardized coefficients. Please either truly standardize all predictors and outcomes, or compare indices formally (e.g., delta-R2, AIC/BIC, or non-nested model comparison tests), and report 95% confidence intervals for all estimates.

(6) Underspecified mixed models and family clustering. The random-effects structure of the LME models is never stated. ABCD includes twins and siblings, so family nesting must be modelled (and the treatment of site - fixed covariate versus random effect - clarified). As written, the analyses may be anti-conservative. Please provide full model specifications and confidence intervals.

(7) Interpretation of accelerated cortical thinning is overly strong. The manuscript equates faster thinning with better maturation and slower thinning with delay. This reading is contested: apparent cortical thinning also reflects myelination-related signal changes, and thickness-cognition associations are age- and region-dependent. Moreover, the present null finding for baseline adiposity contradicts Kaltenhauser et al. (2023), who reported that baseline adiposity attenuates thinning in ABCD; the discrepancy is cited but never reconciled and deserves direct discussion (differences in sample, covariates, or modelling) Unclear units and implausible mediation estimates. Table 1 gives delta-WC = 0.264, interpretable only if delta is per month (age in months), yet the text describes "annualized" change. Units for all delta variables must be stated explicitly and used consistently. Mediation effects reported as wide ranges (e.g., ACME = -29.95 to -0.18; "ACME = -0.1.17" is a typographical error) are uninterpretable and, at face value, implausibly large relative to the cognitive score scale; please report per-indicator ACMEs with confidence intervals and proportion mediated. Not sure how the ranges such as "beta = -0.29 to -0.00" cited as significant effects are informative.

(8) Pubertal confounding. Only baseline pubertal category is adjusted for, but pubertal tempo over the follow-up window confounds both adiposity change and cortical development. This should be adjusted for change in pubertal stage or acknowledged as a substantive limitation. Puberty interactions and sex-specific analysis should be performed too.

(9) Effect sizes should be discussed.

(10) Inclusion of both baseline adiposity and adiposity change in longitudinal models should be justified.

(11) Would there be nonlinear age/puberty effects?

Reviewer #2 (Public review):

Summary:

This study from Cavallini-Speisser et al. cleverly leverages a tissue layer-specific mutant, single cell and bulk RNA-sequencing, and ChIP-sequencing to decipher tissue layer-specific regulation of petal development in petunia by the PhDEF transcription factor. The authors find common and unique targets of PhDEF between the epidermis and mesophyll and conclude that the activity of transcription factors like PhDEF are influenced by the pre-existing environment in the cell they are expressed in. Understanding when and how a given transcription factor drives expression of unique target genes in various contexts is an important aspect of developmental biology that can be elusive outside of highly tractable model systems. As such, I think this study is of high value and has strong potential to expand our understanding of how developmental specificity is mediated by commonly employed transcriptional regulators. However, I think there are some issues with possible over-interpretation and some places where documentation of experimental design and data quality control are lacking. I elaborate on these concerns below.

Major Comments From Review at Review Commons:

(1) Line 155: Assigning mesophyll clusters by default without any positive marker genes strikes me as problematic, especially as much of the analysis rests on comparing the transcriptomes of epidermis and mesophyll cells. Can the authors perhaps leverage published scRNA-seq datasets to find potential mesophyll markers, even homologs from other species, to improve confidence in the cluster assignment?

(3) Line 228: Through the description and interpretation of the ChIP-seq dataset, the authors use the fact that peaks are more abundant and bigger in the epidermis to conclude that binding of PhDEF is "stronger" in the epidermis. This implies a difference in physical interaction between the TF and the DNA that I don't think can be concluded from the data presented. This could be confounded by biology; if expression of PhDEF is more heterogeneous in the mesophyll than in the epidermis, the peaks from that tissue will be averaged out and appear smaller when in fact the binding is the same strength. This could also be a technical artifact if the ChIP was less efficient in one sample versus another. This conclusion requires reinterpretation. The authors have the power to address this at least partially with the scRNA-seq by measuring PhDEF heterogeneity. I believe assessing ChIP efficiency would have required a spike in control, but perhaps there is a computational way to address this. It is important to discuss these confounding factors in the text.

(3) Line 376: The authors risk overinterpreting a lack of differential gene expression detection in their analysis of PhDEF binding profiles. This can be affected by how deeply a library was sequenced or how many cells were analyzed per cell type. A gene might not be found to be DE if low depth or few cells resulted in noise or dropout. Lack of detection does not mean lack of differential regulation so the biological relevance of this portion of the analysis should be interpreted with caution.

(4) Line 476: The authors state there is a mismatch in developmental timing between the RNAseq and ChIP datasets. Why is this? This is mentioned briefly in the Discussion, but has the potential to be majorly confounding to the joint interpretation of the ChIP and RNAseq datasets. This experimental design choice should be justified more thoroughly and a consideration of the limitations it brings to data interpretation should be more prominent in the text.

Significance:

Strengths: The authors employ a unique and powerful mutant system to explore a fundamental developmental biology question. In addition, the datasets generated will likely be useful to other researchers working in petunia or flower development.

Limitations: While the mutant system employed here is a creative way to get at tissue-layer specific transcription factor activity, the ChIP samples still include heterogeneous cell types, which may impact the findings presented here.

Advance: This study uses a unique system to test the function of a transcription factor in distinct cell types. As stated above, understanding when and how a given transcription factor drives expression of unique target genes in various contexts is an important aspect of developmental biology.

Audience: I believe this work will be of primary interest to the plant development, single cell, and chromatin biology communities. These are specialized, basic research communities.

Reviewer Expertise: I am a plant developmental biologist who works with multiple modes of cell-type-specific NGS datasets including bulk and single cell RNAseq and ChIPseq among others.

Comments on latest version:

I find the authors' response to my review to be comprehensive and I think the revision looks solid. The eLife assessment works for me.

Reviewer #3 (Public review):

Summary:

The manuscript evaluated behavioral phenotypes in the Cntnap2 knockout mouse using two behavioral paradigms: trace fear conditioning and a radial maze task. The trace fear conditioning training is normal, but memory generalization is impaired. The inflexibility is suggested to be related to low activity in dCA1 neurons, which can be rescued by ChR2. The radial maze task data suggested a similar conclusion. Brain-wide cFos mapping indicated impairments in the Cntnap2 knockout mouse. The brain-wide cFos mapping does not show direct correlations with Cntnap2, limiting the interpretation of these data in the context of this paper.

Strengths:

The behavior data are solid.

Comment on revised version.

The authors have addressed all my concerns.

Reviewer #2 (Public review):

The authors sought to determine the mechanistic relationship between the mitochondrial intermembrane space proteins CLPB and HAX1, whose genetic, proteomic, and disease associations have long suggested a functional partnership. Using biochemical reconstitution with purified proteins, they provide evidence that HAX1, an intrinsically disordered protein, acts as a direct activating cofactor of CLPB, stimulating its oligomerisation, ATPase activity, disaggregase activity, and refoldase activity. They further identify a region of HAX1 required for CLPB interaction and propose a model in which HAX1 promotes formation of a distinct active CLPB assembly.

A major strength of the work is the comprehensive biochemical approach. The authors combine activity assays, mutational analysis, interaction studies, and oligomerisation measurements to address the central question from multiple angles. The data provide convincing evidence that HAX1 is not simply a client of CLPB but instead functions as a positive regulator.

The principal weakness concerns the mechanistic model of oligomer remodelling. While the SEC and stoichiometric analyses are consistent with the formation of a smaller HAX1-bound CLPB complex, the proposed transition from a dodecameric to a hexameric assembly is inferred rather than directly demonstrated. Additional structural or biophysical evidence would strengthen this aspect of the study.

Overall, the authors largely achieve their aims. The evidence strongly supports the conclusion that HAX1 is a direct stimulatory cofactor of CLPB and provides an important mechanistic framework linking two proteins associated with overlapping mitochondrial and hematological disease phenotypes. Some aspects of the proposed oligomeric remodeling mechanism should be interpreted more cautiously, as they remain supported primarily by indirect evidence.

Case #: Male, 60 years old

DiseaseAssertion: He was diagnosed with late-onset Stargardts disease.

FamilyInfo: Has a family history with his father possibly having macular degeneration.

CasePresentingHPOs: HP:0007401, HP:0025010, HP:0012045 (sub-retinal/yellow), HP:0030602

CaseHPOFreeText: Proband presents with blurry vision (right eye) from over the past two years, with the right eye having 20/40 vision, while the left was 20/20. In addition to this, through dilation scattered subretinal yellow flecks and macular atrophy bilaterally, were revealed. The flecks were hyperautofluorescent. Obscuration of background choroidal fluorescence and window defects were also found. As well as foveal atrophy and hyperreflective deposits (RPE).

CaseNotHPOs: None found

CaseNotHPOFreeText: No abnormalities in the anterior segment examination. Through a full-field electroretinogram, normal rod and cone responses were found.

Genotyping Method: The genotyping was done through the use of target enrichment and next-generation sequencing.

PreviouslyPublished: N/a

Variant: 1) NM_000350.3(ABCA4):c.5461-10T>C 2) NM_000350.3(ABCA4):c.5603A>T (p.Asn1868Ile)

ClinVar: 1) 92870 2) 99390

CAID: N/a

gnomAD: 1) Highest minor allele frequency was 0.00031 (https://www.ncbi.nlm.nih.gov/clinvar/variation/92870/) 2) Highest minor allele frequency was 0.05787 (https://www.ncbi.nlm.nih.gov/clinvar/variation/99390/)

SupplementalData: I was able to find a variant (NM_000350.3(ABCA4):c.[5461-10T>C;5603A>T]) that addresses both of the variants. Also, Fig.1 shows the phenotype of the proband through tests.

Reviewer #2 (Public review):

The remarkable evolvability of the olfactory system enables animals to rapidly adapt to dynamic and chemically complex environments. Over the past two decades, substantial effort has been devoted to uncovering the evolutionary principles that drive the diversification of odorant receptors (ORs), yielding key insights into the forces shaping their striking variability in both vertebrates and insects. In this manuscript, Zhang and colleagues analyze the OR repertoires of over 100 insect species, leveraging sequence and structural similarity to infer patterns of gene family evolution within this diverse and ecologically important clade. By integrating sequence-based and structure-based comparisons, their study builds on a compelling and recently emerging line of research made possible by the advent of AlphaFold, which has previously clarified the phylogenetic relationship between insect Ors and the gustatory receptor gene family and revealed the unexpectedly deep evolutionary origins of this ancient structural fold.

Applying this approach to a large set of ORs derived from species throughout the insect phylogeny, the authors confirm many previously reported patterns of OR evolution. More importantly, with their large-scale approach the authors found new structural features of different Or clades, resulting in intriguing testable hypotheses about Or evolution that have the potential to support future directions in studying this important gene family. For example, the authors identify a structural feature mostly unique to the OR co-receptor ORco, a beta-sheet in EL2, which they functionally show reduces odorant binding affinity - a key aspect of ORco, which does not bind ligands in the ancestral ligand-biding site. This is a particularly strong part of the manuscript, since the authors support their in silico-derived hypothesis with functional data.

In an attempt to assess the relationship between sequence identity and structure on one hand and function on the other, the authors perform an in-silico structure prediction and chemical docking analysis. While not suitable to assess the actual function of individual receptors without functional testing, this strategy revealed potential receptor clades with differences in the number and type of docked chemicals. As a response to the previous reviews, the authors now changed their writing to refer to these results as 'docking-derived function' instead of just 'function'. While I appreciate that the authors changed their wording, this semantic change still can be read as referring to docking-derived predictions as conclusions of actual biological function. However, this is not the case. Any functional claims based solely on the docking approach need to be interpreted with caution, as in silico-only approaches are currently not well enough predictors for actual receptor function. Accordingly, I strongly recommend to remove the parts that connect receptor function to structure or ecology.

Assessing their docking analyses with respect to species ecology, the authors identify correlations suggesting that Or repertoires might follow predictable evolutionary trajectories broadly aligned with species ecology. These are interesting hypotheses. However, due to the inability to predict actual receptor function the author's claims about ecology-function relationships are not well supported. Accordingly, the authors should change the respective parts of the manuscript in way that it is clear these are currently untested hypotheses. This includes adjusting the title, since the work does not find any experimental support for ecological tuning.

I believe that future studies combining functional testing with an explicit evo-eco framework could benefit from this work and the explicit hypotheses it generates. But the authors should be careful to not overstate their in-silico derived results.

Reviewer #2 (Public review):

Summary:

The work titled "Geomagnetic and visual cues guide seasonal migratory orientation in the nocturnal fall armyworm, the world's most invasive insect" provided experimental evidence on how geomagnetic and visual cues are integrated, and visual cues are indispensable for magnetic orientation in the nocturnal fall armyworm.

Strengths:

It has been demonstrated that the Australian Bogon moth could integrate global stellar cues with the geomagnetic field for long distance navigation. However, data are lacking for other insects. This study suggested that the integration of geomagnetic and visual cues may represent a conserved navigational mechanism broadly employed across migratory insects.

Weaknesses:

The visual cues used in the indoor experimental system designed by the authors may have some limitations in ecological relevance. The author may need more explanations on this experimental system.

In the revised manuscript, the authors have added explanations in the discussion section. I am fine with the revision.

Reviewer #2 (Public review):

Summary:

In this study, Davis et al., embarked on the quest of the molecular elements responsible for the regulation of lymphatic phasic contractile activity in response to variation of transmural pressure, a mechanism (termed pressure-induced lymphatic chronotropy by the authors). This contractile activity is critical for drainage of interstitial fluid from the tissue and transport of lymph and its cellular and molecular constituents back to the blood circulation. The authors' aim was to investigate the mechanism(s) involved in the pressure-induced regulation of lymphatic pumping, and test whether activation of cation channels, shown in other systems to play mechanosensitive roles are directly at play, and/or whether mechano-activation of Gq/11-coupled GPCRs is necessary to generate second messengers to activate those channels, as it has been suggested for the regulation of myogenic tone in arteries. To achieve their goal, the authors used their well-described, highly reliable protocols of mouse lymphatic vessel isolation, pressure myography, and data acquisition to obtain frequency-pressure relationships and other contractile parameters from transgenic mice where specific channels or molecular elements of interest have been ablated. They combined these data with scRNAseq analysis of these gene targets to determine their respective role and level of expression in lymphatic muscle cells. Their findings demonstrated that, with the exception of ANO1 Cl channels, part of the contractile pacemaker mechanism, none of the exhaustive list of tested ion channels was critical to pressure-induced lymphatic chronotropy, but only ablation of Gq/11 genes or IP3R1 gene in smooth muscle cells led to chronotropy inhibition. To strengthen these findings, they then repeated the myography experiments in the presence of a Gq/11 inhibitor and screened frequency-pressure relationships in vessels from transgenic mice lacking each of the 7 most expressed GPCR according to their scRNAseq dataset. While none of the GPCR ablation showed significant inhibition, they could conclude that transmural pressure activates GPCRs coupled to Gq/11 which generate IP3 to induce SR Ca2+ release through IP3R1 and activate ANO1-mediated depolarization and increase contraction frequency.

Strengths:

The manuscript's strengths reside primarily on very robust, clean and unequivocal pressure myography data and analysis. The research team is mastering these techniques they developed more than a decade ago and have implemented to mouse lymphatics to study their contractile properties, with consistent and convincing outcomes. They also provide data from an impressive list of transgenic mice in order to determine the role of the targeted genes in pressure-induced lymphatic chronotropy, relaying on pharmacological small molecule inhibitors to confirm their findings. Finally, the use of scRNAseq analysis they gathered from previously published dataset brings novelty with respect to expression of the genes of interest in all populations of cells comprising the lymphatic vessels, but more critically to validate or contrast the potential impact of genetic alteration of the given gene in the ability of lymphatic muscles to respond to change in pressure.

Comments on revised version.

The new experiments, additional data, and the now extensive discussion better temper the authors' initial conclusion, the new title as well. The authors should be commended for such an impressive amount of work.

Reviewer #2 (Public review):

This work provides a detailed metabolic reconstruction of sediment microbiomes along a depth profile in a Spartina patens salt marsh in Massachusetts, USA. Using a combination of genome reconstruction, co-occurrence network analysis, and metabolic profiling, the authors describe the metabolic potential of co-occurring microbial consortia in understudied deep sediments.

Major strengths of this study include the detailed metagenomic and metatranscriptomic characterization of the understudied deep marsh sediments. The authors recovered genomes representing a substantial portion of the deep sediment microbiome (up to ~60%) and provided an initial explanation of pathways related to the potential for organic carbon decomposition in this environment. Of particular interest is the genomic capability of the deep sediment microbiome to process complex organic compounds, highlighting the need for a collaborative consortium to carry out their decomposition. Improved understanding of the microbial transformation of deep sediment organic carbon in blue carbon ecosystems is vital to better understand the fate of this large carbon pool in the face of climate change.

After assessing a revised version of the manuscript, I only have one methodological comment that readers may find useful as going through the manuscript:

The authors normalize the relative abundance of MAGs by dividing the reads mapping to a MAG by the reads mapping to their whole set of recovered MAGs. They provide a rationale for following this approach in the Methods section. However, I still argue that these values are misleading due to differences in read recruitment levels across samples (and importantly, across depths). In particular, the relative abundance of MAGs in the surface layers (0-40 cm) is overestimated, since the higher diversity in these samples recruited fewer reads due to poor assembly in samples with high sequence-space diversity (shown in Supp. Fig 2). A better normalization approach would be to divide MAG abundance by genome equivalents (e.g., using MicrobeCensus) or any other approach leveraging the abundance of universal single-copy genes in prokaryotes. These approaches are assembly-independent and leverage existing databases of universal single-copy genes. That being said, these methodological limitations do not seem to greatly impact the biological findings of this manuscript.

All other comments have been addressed.

Reviewer #2 (Public review):

Summary:

Elley and colleagues induced a synthetic torpor-like state in rats (a non-hibernating species) by chemogenetically activating neurons in the medial preoptic area of the hypothalamus. They show that this state substantially reduced cardiac infarct size in an ex vivo ischemia-reperfusion model. They further report that protection persisted when ambient temperature was raised to prevent hypothermia and used exploratory phosphoproteomics to identify candidate cardioprotective signaling pathways.

Strengths:

This is the first demonstration that a torpor-like state is cardioprotective in a species that does not naturally enter torpor, which meaningfully advances the potential clinical utility of synthetic torpor. The experimental design is logical, and the controls are generally appropriate. The characterisation of the responsible neuronal population using ISH against QPLOT markers adds mechanistic depth and supports the cross-species conservation argument. The phosphoproteomic analysis, though exploratory, generates plausible and biologically coherent hypotheses grounded in the hibernation literature.

Reviewer #2 (Public review):

Summary:

This manuscript presents an elegant and innovative imaging approach to visualize DNase activity at the interface between macrophages and extracellular substrates. The platform is technically strong and enables the study of localized DNA degradation with high spatial resolution. The work is of clear interest and provides a useful framework to investigate how immune cells process extracellular DNA. However, several aspects of the mechanistic interpretation and conceptual framing would benefit from clarification.

Strengths:

(1) The study introduces a creative and well-designed imaging platform that allows visualization of localized DNase activity at cell-substrate interfaces.

(2) The approach is technically robust and represents a valuable tool that could be broadly useful to the field.

(3) The experiments are thoughtfully designed and address an important question regarding how immune cells interact with extracellular DNA.

(4) The work opens interesting avenues for studying DNA processing in contexts such as infection and inflammation.

Comments on revised version.

The authors have carefully addressed the points raised in my previous review, and the manuscript has been revised accordingly. I am satisfied with the revisions and have no further comments at this stage.

Reviewer #2 (Public review):

The Beauchamp and Chardon model, like any computational model, is only as informative as the physiological parameters it includes. By varying only three synaptic related dimensions, namely neuromodulatory (i.e., PIC strength from 5HT inputs, etc.), the pattern of inhibition relative to excitation, and the distribution of excitatory input across smaller versus larger motoneurons, the model necessarily holds constant many other properties that may differ substantially between individuals and between controls and patients with MS. These properties include intrinsic membrane conductance properties (with some conductances like NaV that may differ in MS not included in the Chardon model), afterhyperpolarization properties, tonic inhibition, dendritic and axonal structure, synaptic kinetics, glial changes, etc., etc. The present paper then moves one step further away from direct physiological estimation because it does not fit these three model parameters to each participant's data, but instead combines control-normalized firing pattern features using weights derived from the original simulations. As a result, the resulting "excitation," "inhibition," and "neuromodulation" scores should be regarded as indirect similarity scores within a restricted model space, with a substantial risk that changes caused by unmodeled physiology are misattributed to one of the three modeled components. An additional limitation is that the underlying motoneuron models were originally tuned to intracellular recordings from medial gastrocnemius motoneurons in decerebrate cats and then manually modified to generate more human-like firing rates and hysteresis, rather than being formally fitted to human motor unit recordings.

The authors should justify their conclusions using the Chardon reverse engineering method. In addition, more of the raw firing rate profiles should be presented to give a better sense of the data and the quality of the motor unit identification.

Reviewer #2 (Public review):

The authors presented evidence from various in vivo and in vitro experiments demonstrating the mutual interaction between CCL5 and astrocytic miR-342-5p in the ipsilateral core of cerebral ischemia. However, miR-342-5p was downregulated only late after MCAO (D3-7). Additionally, this downregulation was observed not only in the ipsilateral core but also in the ipsilateral penumbra and contralateral sides. Therefore, it is not convincing that the upregulation of CCL5 in the ipsilateral core at later time points (D3 and D7) is attributable to the decreased expression of miR-342-5p. In particular, infarct injury was already evident within a short time period (say 24 h) following MCAO.

(1) The temporal and spatial expression patterns of miR-324-5p do not match those of CCL-5, especially at D1 and D3 (see Figure 1C, 1D). Despite the inverse relationship between miR-324-5p and CCL-5 expression at D7 after MCAO, what was the purpose of administering miR-324-5p agomir (or antagomir) at D1 post-MCAO? If the connection cannot be clearly established, the conclusion reached at the end will be difficult to accept.

(2) Would administering miR-342-5p or anti-CCL5 at later time points (e.g., after D3) reduce infarct size or improve functional recovery? If this is not the case, the effect of CCL5 on neuronal cell damage (infarct size formation) must occur within a very short time after MCAO. Additionally, if the increased CCL5 expression is due to the downregulation of miR-342-5p, its impact would likely be less significant.

(3) While the study offers valuable insights into the roles of CCL5 and its connection with the regulation of miR-342-5p (though this connection is somewhat weak), it is recommended that the authors explore potential translational applications of these findings.

Overall, given the experimental designs and results, it is difficult to support the conclusions drawn in the manuscript.

Reviewer #2 (Public review):

Summary:

This study examines how genes involved in cellular recycling (autophagy) influence lifespan under different experimental conditions. The findings help clarify why previous studies have reported conflicting results about whether blocking autophagy shortens or extends lifespan. The work will be of interest to researchers studying aging and cellular stress responses, particularly those using model organisms.

Strengths:

The findings are valuable, as they help resolve inconsistencies within a specific subfield of aging research. The evidence presented is solid, as the data broadly support the primary claims of the study. In addition, the discussion is thorough and thoughtfully integrates the findings within the broader context of the field.

Weaknesses:

Additional functional validation would further strengthen the conclusions.

Comments on revised version.

I have reviewed the revised manuscript. Overall, the authors have addressed most of my concerns, and the revised manuscript has been improved. The study provides a comprehensive examination of the context-dependent effects of autophagy gene knockdown on lifespan in C. elegans. The data are solid and provide valuable information for understanding the conflicting results currently reported in the literature regarding the role of autophagy in longevity.

Reviewer #2 (Public review):

Summary:

Kirk et al. use RNA-Seq and CRISPRi to provide evidence that KLF family transcription factors regulate postnatal neuronal maturation of pyramidal neurons. The genetic programs regulating postnatal neuronal maturation are not well understood. The authors first analyzed chromatin accessibility and gene expression data from layer 4 and 6 pyramidal neurons and found that KLF TFs are predicted regulators of postnatal neuronal maturation. They then use CRISPRi knockdown and find that KLF activators first activate genes and then this is followed by KLF repressors repressing genes. Interestingly, some genes, such as those with cytoskeletal functions, are shared targets of KLF activators and repressors.

Strengths:

The study is well-executed and the paper is well-written. A major strength of this study is the application of state-of-the-art transgenic approaches. The CRISPRi approach used to knock down multiple KLFs is compelling. The genomic data generated appears to be high quality and is carefully analyzed. The presented findings provide important insights into the genetic programs that regulate postnatal maturation in cortical pyramidal neurons. The discovery that KLF family activators/repressors regulate gene expression changes during this critical step of neuronal development fills an important gap in the field.

Weaknesses:

While beyond the scope of the current study, future studies should investigate the contributions of KLFs on postnatal morphological and physiological changes.

Reviewer #2 (Public review):

Summary:

This is an elegant, rigorous, and thought-provoking study that examines how different neural circuits are altered in response to loss of a common sensory input. To study this question, the authors use the mouse retina as a model system to investigate how downstream retinal circuits undergo modifications following a well-controlled partial loss of cone photoreceptors.

Strengths:

The experiments were conducted with a high degree of rigor, and the authors carefully considered and implemented appropriate controls throughout the study. Multiple parameters were tested, including pharmacological approaches to assess responses from different ganglion cell types. In addition, the authors complemented their functional data with confocal imaging to further support their findings. Overall, this is a well-written paper that provides a thorough analysis demonstrating how two similar ganglion cell types undergo distinct adaptations (i.e., compensation versus remodeling) in response to the loss of the same sensory input.

Weaknesses:

No additional experiments are needed. However, the authors may wish to consider the following points:

(1) Do the differences in compensation versus remodeling observed in ganglion cells reflect changes in the OPL? Different bipolar types may remodel their dendrites and form aberrant contacts with rods in the absence of cones. However, this would be challenging to test because there are currently no good markers for different bipolar types.

(2) It would be interesting to determine whether these functional changes can be detected at the transcriptomic level or whether they are mediated primarily through post-translational modifications.

Reviewer #2 (Public review):

Summary:

This paper describes the employment of a Delphi process, a common consensus-reaching practice based on consecutive rounds of discussion and voting. In this study, the authors engaged in the Delphi process with stakeholders to reach consensus on recommended guidelines for use in tenure and promotion, specific to reproducible and transparent research practices.

Strengths:

The paper provides very practical guidelines for research institutions willing to push for reform regarding reproducible and transparent practices. Guidelines like these are important for institutional leadership that wishes to drive change at their institutions but does not necessarily hold topic expertise on research and reproducibility. The study's authors represent longstanding expertise on the topic, and the use of the Delphi process ensures that stakeholders were engaged in the recommendations, something that helps ensure uptake and buy-in.

Weaknesses:

The biggest weakness of the paper is a lack of engagement with the literature of tenure and promotion reform. This is a field that has been heavily studied with regard to incorporating change in practice to promote equity and inclusion. Other specific subfields that have dealt in this space are those that have pushed for reform to increase the importance of teaching, service, and (inclusion of) mentorship in tenure and promotion packages. Because this paper deals heavily with implementing change in this specific space, and provides guidelines with very practical implications for change, some literature review in the introduction and discussion on where past efforts have succeeded or faced roadblocks would be welcome. This would add to the study's value in helping people implement these practices in a very real and tangible way. The other weakness is a lack of further expansion on the recommendations that did not reach consensus, many of which were added in Round 3. Are more rounds warranted for future studies? Changing landscapes require changing recommendations, which is probably why things like the use of LLMs emerged later and did not reach consensus. How should the readers engage with these results, and what future steps are needed to implement guidelines (if any) around these topics?

Reviewer #2 (Public review):

This manuscript introduces the tuxedo sea urchin, Mespilia globulus, as a new experimental model for developmental, reproductive and genomic biology. The authors establish culture methods that permit completion of the life cycle in a landlocked aquarium facility, demonstrate the applicability of developmental biology tools including HCR-FISH and CRISPR/Cas9-mediated gene disruption, generate chromosome-scale genome assemblies from two color morphs and both sexes, investigate potential sex determination mechanisms, and compare genome organization and gene family evolution with other sea urchin models. The authors conclude that M. globulus combines a relatively rapid life cycle with genomic tractability and therefore represents a valuable addition to the growing repertoire of genetically accessible sea urchin model systems.

Overall, I found this to be a strong and timely contribution that is well suited for the Tools and Resources category of eLife. The authors provide a comprehensive suite of resources, including husbandry protocols, genomic resources, developmental staging information, and proof-of-principle functional manipulations. The manuscript appropriately places M. globulus in the context of established sea urchin models, particularly Lytechinus pictus, and clearly argues that the new species is complementary rather than a replacement for existing systems. Given the increasing importance of genetically tractable echinoderm models, the development of an additional species that can be maintained and bred in closed aquarium systems is of considerable value to the field.

The evidence supporting the establishment of this model system is convincing, with multiple complementary datasets including life-cycle culture methods, genome assemblies, gene expression analyses, and gene-editing experiments.

My concerns primarily relate to two broader issues that should be addressed, followed by several specific comments.

(1) Genetic Background and Aquarium Trade Populations: A central argument of the manuscript is that M. globulus is attractive as a laboratory model because it is widely cultured in the aquarium trade and may exhibit reduced genetic variability due to captive propagation.

The manuscript states:

"M. globulus is a popular species in the aquarium industry and has excellent properties in tropical aquariums where it has been bred for many years in farming operations that reduce genomic heterogeneity..." (lines 96-98) and later:

"Captive breeding may also lower genetic variability compared to wild-caught individuals..." (lines 389-390).

However, the manuscript does not provide sufficient information to evaluate these claims. Several important questions remain unresolved:

(1) How genetically representative are the sequenced individuals relative to natural populations?

(2) What is known about the provenance and breeding history of the aquarium trade stocks used in this study?

(3) Are these animals derived from a small number of founder populations?

(4) Is there evidence for substantial inbreeding or genetic bottlenecks within commercial brood stocks?

(5) How similar are commercially available animals from different vendors and geographic sources?

These questions are important for both practical and biological reasons. From a practical perspective, researchers wishing to adopt M. globulus need to know whether animals purchased from aquarium suppliers are expected to resemble those analyzed here. From a biological perspective, reduced diversity or founder effects could influence genome assembly characteristics, heterozygosity estimates, gene family analyses, developmental traits, or responses to experimental manipulation.

Even if little information is currently available, the manuscript should explicitly discuss these uncertainties and provide available information regarding stock origin, aquaculture practices, and potential differences between captive and natural populations. A clear discussion of potential limitations would significantly strengthen the manuscript.

(2) Presentation and Interpretation of HCR and Phalloidin Data: Although the HCR and phalloidin experiments are not central to the major conclusions of the paper, they serve as important demonstrations of experimental tractability. At present, however, the presentation of these data is not fully convincing.

The HCR images show detectable signal, but the expression domains are only minimally documented. The manuscript states that expression patterns are consistent with known functions of Nodal and Notch signaling, yet the figures do not sufficiently guide readers to these conclusions (especially readers not familiar with sea urchin development). The signal is relatively diffuse and weak in some panels, and there is little annotation explaining exactly which embryonic territories are expressing the genes of interest. For readers without extensive sea urchin developmental biology expertise, it can be difficult to assess the validity and biological significance of the observed expression patterns.

Similarly, the phalloidin-labeled images provide limited anatomical information because the larvae are largely not labeled. I recommend:

(1) Adding labels identifying relevant embryonic regions and structures.

(2) Including arrows or overlays indicating key expression domains.

(3) Providing higher-magnification insets of relevant regions.

(4) Including selected optical sections rather than relying exclusively on 3D projections.

(5) Identifying known larval muscle groups in the phalloidin images.

(6) Improving image contrast and figure annotation where possible.

These changes would substantially strengthen the claim that established developmental biology methods are readily transferable to M. globulus.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigated the relationship between the Tat system and MPIase, a glycolipid that facilitates protein integration into the bacterial cell membrane. The TAT (twin-arginine translocation) system is a unique membrane transport machinery that exports fully folded proteins containing a twin-arginine signal peptide. Using both in vivo and in vitro approaches, the authors demonstrated that a sufficient amount of MPIase is required for Tat-dependent protein translocation. Furthermore, the authors successfully reconstituted the Tat transport system by combining recombinant TatA, TatB, TatC, MPIase, and FoF1-ATP synthase.

Strengths:

The reconstituted system clearly demonstrated the requirement for each component, as substrate translocation occurred only when all components were present. Based on these findings, the authors proposed a mechanistic role for MPIase in facilitating Tat-mediated membrane translocation. Previous studies have shown that MPIase is involved in Sec-dependent protein translocation and membrane protein integration, as well as YidC-dependent membrane insertion. The present study further demonstrated that MPIase also plays an essential role in the Tat translocation pathway. Overall, this work highlights the central importance of MPIase in bacterial membrane protein biogenesis and provides new insights into the molecular mechanism of Tat-dependent protein transport.

Comments on revised version from Reviewer 2 and Reviewing Editor:

(Reviewer 2) It is nice to find the mistake in Fig. 4B (RR, EK413, (not +MPIase)) and corrected it in the revised version. However, the data of Fig. 4B and the related modified text on P.9 L256-269 are not very easy to understand what the authors are trying to argue. This reviewer believes that the magnitudes of MPIase expression level are Ek413 > +MPIase > delta-MPIase. Therefore, the means of supernatant in the delta-MPIase and EK413 represents mostly cytosol and periplasm, respectively. On the other hand, the +MPIase shows less supernatant that means a large part of TorA-GFP are trapped nearby membrane but not aggressively transported. Similar results are found also in (KK) case, whereas the sup level in EK413 is lower than that of (RR) case due to the lack of twin-arginine motif in the substrate.

Although these results are convincing in explaining the model in Figure 5, authors might be able to attempt to refine them more understandable. Especially, the revised text on P.9 L256-269 is not enough for leading readers to the author's claim. So that, the authors should revise the text about Fig. 4B again and the related description in the manuscript. Another option is it would be possible to delete whole Fig. 4B, because Fig. 4A, C already well support the model in Fig. 5.

(Reviewing Editor) I agree with Reviewer 2. The first paragraph in the section "MPIase serves as the TAT signal receptor" runs very long and the clarity of the descriptions on Fig. 4B could be further improved. Fig. 4A, C would sufficiently support author's arguments. I recommend that authors consider these options for the final version.

Reviewer #3 (Public review):

Summary:

Jiang et al. described findings aimed at interrogating the interactions of the antibiotic polymyxin B with human kidney proteins that mediate nephrotoxicity. Their findings using both computational molecular dynamics simulations and experimental approaches illustrate the importance of aspartic acid residues (D215) in mediating the antibiotic uptake into the cells, and upon mutagenesis with Alanine, the effects are less pronounced. Further, they could modify the antibiotic units interacting with proteins into less toxic peptides with retained antibacterial properties.

Strengths:

I was impressed by this text, which advances the knowledge of how the antibiotic causes human nephrotoxicity and how this could be exploited into less problematic antibiotic peptides.

Comments on revised version.

Majority of the raised issues were addressed satisfactorily.

Reviewer #2 (Public review):

This manuscript by Hisler, Rees, and colleagues examines the cardiac regenerative ability of two livebearer species, the platyfish and swordtail. Unlike zebrafish, these species lack cortical myocardium and coronary vasculature. Cryoinjury to their hearts caused persistent scarring at 60 and 90 days post-injury and prevented most of the myocardium from regenerating. Although the wound size progressively shrinks and fibronectin content decreases, the myocardial wall does not recover. Transcriptomic profiling at 7 dpi revealed significant differences between zebrafish and platyfish, including alterations in ECM deposition, immune regulation, and signaling pathways involved in regeneration, such as TGFβ, mTOR, and Erbb2. Platyfish exhibit a delayed but chronic immune response, and although some cardiomyocyte proliferation is observed, it does not appear to contribute to myocardial recovery significantly.

Overall, this is an excellent manuscript that tackles a crucial question: do different fish lineages have the ability to regenerate hearts, or is this capability limited to a few groups? Therefore, this work is relevant to the fields of cardiac regeneration and comparative regenerative biology for a broad audience. I am very enthusiastic about expanding the list of species tested for their heart regeneration abilities, and this study is detailed and rigorous, providing a solid foundation for future comparative research. However, there are several aspects where additional work could significantly strengthen the manuscript.

Comments on revised version:

The authors have done a fantastic job addressing all my comments, covering both the experimental and the more conceptual aspects of my critique. I am fully satisfied with their response.

Reviewer #2 (Public review):

Summary:

The authors' data reveal that the spectraplakin Shot, which can interact with both actin and microtubules, is essential for the proper pruning of dendrites in a Drosophila model. A molecular basis for the coordination of these two cytoskeletons during neuronal developmental has been elusive. The authors' data support a role for Shot in regulating microtubule polarity in young neurons, and the authors link this early function to a later role in dendrite pruning. The story has many interesting components, but lacks experimental depth to solidly (and sufficiently) support the authors' claims.

Strengths:

(1) A strength of the manuscript is the authors' data supporting the idea that Shot is needed for proper dendritic microtubule polarity in young neurons and that Shot regulates dendrite pruning.

(2) Another strength of the manuscript is the data in support of Rab11 functioning as a MTOC in young larvae but not older larvae; this is an important finding that may resolve some debates in the literature. The finding that Rab11 and Msps coimmunoprecipitate is nice evidence in support of the idea that Rab11(+) endosomes serve as MTOCs.

Weaknesses:

(1) A major concern is that many of the authors' main conclusions are weakly supported. The idea that Shot exerts an effect on microtubules via an interaction with actin relies predominantly on the findings that deleting Shot's actin-interacting domains (CH domains) does not rescue the dendritic microtubule polarity defect and also alters the dendrite-tip enrichment of Shot (the change in dendrite-tip enrichment, however, appears fairly modest as Shot appears to still be localized quite well near the tip of the dendrite, if not at the actual tip). The over-expression of Mical, which was intended the disrupt actin, does not affect actin in distal dendrites in 1st instar neurons; 1st instar distal dendrite tips is where (and when) the authors propose Shot functions. It does not seem possible to draw conclusions about the actin cytoskeleton from the overexpression of Mical since this manipulation only (mildly) affects actin in the proximal dendrites of 1st instar neurons.

Given the reliance on the Shot transgenes, it is also important to test, or at least comment on, whether the Shot transgenes are expressed at equivalent levels (the transgenes appear to be integrated at different positions in the genome). A difference in transgene expression should be ruled out as a potential contributing factor.

(2) The authors propose that Shot acts by (locally) stabilizing microtubules. This conclusion is not supported by the data. For example, the fluorescently tagged tubulin images show signal that is quite diffuse, and the images are not convincing (fluorescent tubulin reports both the free tubulin pool as well as microtubules). A high-resolution image in the supplementary materials is also not convincing. These images are of primary dendrites, but the authors' model proposes a local role for Shot in dendrite tips.

(3) A general weakness is the use of data derived from different parts of the dendritic arbor, and at varying developmental stages, to generate a model that proposes a spatially and temporally specific role for Shot.

Reviewer #2 (Public review):

Summary:

The authors analyzed genetic population structure of two clingfish species in the eastern Mediterranean Sea. They used a genome-wide DNA sequence dataset representing several populations of both species and found general patterns of isolation that agree with likely patterns of dispersal and described parallel signatures suggesting genomic adaptation. The results support that similar species follow the same evolutionary trajectories when they evolve in the same ecological and geographical context and when meta -populations are subject to the same constraints with respect to dispersal.

Strengths:

The findings as such include a population genetic analysis of taxa for which data are lacking. Conclusions on population subdivision as well as genomic divergence suggestive of genomic adaptation are well supported. The analysis of the genetic data is according to established standards and useful.

Weaknesses:

The idea to apply drift simulations to study dispersal in pelagic fish larvae makes sense; I do however, wonder to what extent the neutral drift scenario applies to the species under study, as an earlier paper by the authors ( J Fish Biol 2020 Nov 3; 98(1): 64-88) emphasizes a strong near-coastal retention of larvae. Accordingly, the drift simulations are in agreement with the observed population structure, but I note that a simpler isolation-by-distance model and dispersal primarily along coastlines would equally agree with the data. A demonstration that drift simulations contribute to a refined model of population subdivision would require a denser sampling of populations and measures of drift between these. Ideally, this should include a demonstration that areas where drift is reduced coincide with genetic discontinuity in the absence of deeper areas of the sea. This is not to say that the approach as such is not interesting, but the conclusions towards this goal are not very well supported by data.

One aspect in the study should be clarified: between-species gene flow is common between closely related species of fish. If this occurs, this would dramatically affect the interpretation of parallelism and convergent evolution at the genomic level. Hybridization should be ruled out or discussed. The two focal taxa are not sister taxa according to a phylogenetic tree (Figure 6) based on only a few genetic loci. Even if a more complete dataset is missing for most relevant taxa to recalculate this phylogeny, I would like to see an assessment of the degree of separation between the two taxa at a genome-wide level. The authors mention that the genetic variants that are subject to parallel evolution are not identical. This is promising and should be highlighted more in case hybridization is likely.

Reviewer #2 (Public review):

Summary:

The authors combine a value-based choice task with a gaze-contingent covert attention paradigm to investigate how covert attention relates to value-guided decisions. This is an important and timely question, as many models of decision-making implicitly equate gaze with attention despite extensive evidence that covert attention can be dissociated from eye position. I particularly appreciated the relatively naturalistic task design, which allows participants to freely explore the options while covert attention is sampled during the decision process. Overall, the manuscript is well written, the experiments are carefully executed, and the analyses are generally appropriate and clearly presented.

My main reservation concerns the strength of some conceptual claims. The data convincingly demonstrate that covert attention can be decoupled from gaze and that probe report is modulated by option value. However, I found the evidence less directly supportive of the stronger conclusions that covert attention dynamically competes with overt attention throughout the decision process and that covert attention itself causally influences subsequent choice. Many of the reported analyses are consistent with these interpretations but, in my view, do not uniquely support them.

Strengths:

The manuscript addresses an important and timely question at the intersection of visual attention and value-based decision making. The experimental paradigm is elegant and relatively naturalistic, allowing covert attention to be sampled during ongoing decision making while participants freely explore the choice display. The experiments are carefully executed, and the analyses clearly demonstrate that covert attention can be dissociated from gaze and is systematically modulated by option value. The control analyses addressing the contribution of presaccadic attention are an important addition that substantially strengthens the manuscript, even though I had questions regarding their interpretation.

Weaknesses:

(1) Interpreting the causal role of covert attention in guiding choice

The manuscript puts forward two central conclusions: first, that covert attention is shaped by decision-relevant factors such as option value; and second, that covert attention, in turn, influences subsequent choice behavior. I found the evidence for the first conclusion compelling. My reservations concern the second conclusion, namely whether the current data uniquely establish a causal influence of covert attention on choice. Throughout the manuscript, the authors conclude that covert attention exerts "downstream consequences for choice behavior" and that an estimate of covert attention "influences the final choice." However, I am not sure that the presented analyses fully disentangle a causal influence of covert attention from a common influence of option value on both covert attention and the eventual decision.

One aspect that illustrates this concern is the interpretation of the last-fixation bias. While the reported association between the final fixation and the chosen option is clear, it is less obvious that the final fixation itself biases the subsequent choice. An equally plausible interpretation is that participants have largely committed to a decision and subsequently direct one final gaze shift toward the option they have already selected before executing the button press. In other words, the relationship may reflect choice influencing gaze rather than gaze influencing choice. Unless the temporal dynamics allow these alternatives to be distinguished, I would encourage the authors to use more neutral language (e.g., an association between last fixation and subsequent choice) or explicitly discuss this alternative interpretation.

More generally, I found myself looking for a more direct demonstration that covert attention biases choice toward the attended option. Most analyses ask whether successful peripheral probe report attenuates established gaze-related choice biases, such as the last-fixation or time-advantage bias. While these are interesting findings, they remain relatively indirect. A particularly compelling demonstration would be to examine situations in which option value cannot explain the relationship between covert attention and choice.

(2) Dynamic competition between covert attention and overt attention

I think one of the key strengths of this manuscript is its explicit attempt to dissociate covert attention from gaze-a distinction that is often overlooked in both empirical studies and computational models of value-based decision making. My reservation concerns the stronger claim that covert attention is dynamically reallocated during decision making and competes with overt attention. The present results clearly show that covert attention can be dissociated from gaze, but I found less direct evidence for the proposed dynamic competition. Throughout the experiments, probe report accuracy remains highest at the currently fixated option, whereas peripheral benefits, although reliable, are comparatively modest (e.g., Figures 2 and 3). This raises the question of how often, and under which circumstances, covert attention is actually reallocated away from the current fixation.

Because the authors possess precise eye-movement timing, I think the manuscript would be substantially strengthened by an analysis of attentional dynamics within individual fixations, to provide more direct support for the proposed dynamic interplay between covert and overt attention.

More generally, I found several interpretations somewhat stronger than the presented evidence. For example, the statement that increased covert attention to higher-valued peripheral options occurs "at the expense of" overt attention at the fixated location seems to imply a direct trade-off that is not explicitly demonstrated. Unless such temporal dynamics can be shown, I would encourage the authors to distinguish more clearly between demonstrating that covert attention can be dissociated from gaze and demonstrating that it dynamically competes with overt attention throughout the decision process.

(3) Dissociating covert from presaccadic attention

The control analyses aimed at dissociating covert from presaccadic attention are an important strength of the manuscript. However, I found the rationale underlying these analyses difficult to follow. The key conclusion-that value modulates probe report even when covert attention is not presaccadic-rests on the distinction illustrated in Figure 6, yet it remained unclear to me exactly how this distinction isolates presaccadic from non-presaccadic covert attention. My understanding is that the authors compare probe performance at previously fixated locations depending on whether those locations are subsequently re-fixated. If this interpretation is correct, a more explicit explanation of why this operationalization isolates presaccadic attention would help readers evaluate this conclusion. Conceptually, I found it more intuitive to think about a dissociation based on probe performance at locations that are subsequently fixated versus not subsequently fixated following the first fixation, when the currently fixated option, the upcoming saccade target, and a third unfixated option are distinct. Such situations would seem to provide the clearest opportunity to separate attentional enhancement associated with the upcoming saccade target from covert attention directed elsewhere.

I also found the interpretation of the time-to-saccade control stronger than the evidence directly supports. The authors argue that a purely presaccadic account predicts a monotonic decline in probe accuracy as a function of time-to-saccade. This assumption seems stronger than supported by the presaccadic attention literature, which primarily characterizes attentional enhancement during approximately the final 100-200 ms before saccade onset. Within this temporal window, the present data actually appear consistent with such an increase (Figure S6). By contrast, I am not aware of theoretical or empirical work predicting a monotonic presaccadic attentional shift extending hundreds of milliseconds, or even a second, before saccade onset.

The observed U-shaped relationship is nevertheless inconsistent with a simple presaccadic-only account, as probe performance remains elevated even when the upcoming saccade is relatively distant. I therefore agree that the findings suggest additional attentional processes. However, I do not think they uniquely establish that probe performance indexes covert attention independently of presaccadic planning.

Reviewer #2 (Public review):

Summary:

The present paper studies the entropy production rate (EPR) before, during, and after thalamic sonication in freely moving mice from a previously published dataset. The motivation is that EPR contains information about the dynamics of neural activity that is not present in the average calcium amplitude. It was observed that the change in EPR reaches a maximum at an intermediate stimulation dosage, in contrast to the change in overall calcium amplitude, which increases monotonically with dose in the on-target condition. The paper further observes a statistically significant relation between the baseline EPR and the change in EPR during stimulation, but not for recovery, as well as a similar relation for the calcium response. The manuscript draws an analogy between the non-monotonic dependence and stochastic resonance.

Overall, these results present an interesting analysis of how acoustic stimulation affects the dynamics of neural activity. In my view, these tools would be quite useful for quantifying changes in neural activity under different perturbations.

Strength:

The study is well motivated by the argument that quantifying neural response requires information about dynamics that is not contained in the average calcium amplitude. EPR or irreversibility has been shown to be a powerful tool in describing out-of-equilibrium biological processes. The paper successfully quantifies irreversibility using an ordinal surrogate of the entropy production rate. The non-monotonic dependence is an interesting result, which demonstrates (with caveats stated in Weakness) that EPR contains more information about neural response than the average calcium amplitude. Furthermore, the paper also clearly states limitations of the approach and performs a robustness check by varying the parameters used in the ordinal EPR estimation.

Weakness:

(1) My main concern, as the authors have touched upon in the Discussion, is that although irreversibility clearly provides a readout of the neuronal dynamics, it remains unclear what its biological significance is. While it is related to susceptibility, the mechanistic link remains weak. I suspect that this interpretability issue will limit the impact of this approach.

(2) I wonder how the measured irreversibility is affected by the asymmetric response of GCaMP, which typically rises quickly and decays slowly. It seems possible that this temporal asymmetry, combined with the average calcium response, already leads to a non-monotonic irreversibility curve. The paper should investigate this and other potential contributors to the temporal irreversibility in the readout.

(3) As shown in Fig. 2AB, the baseline EPR already varies considerably with acoustic intensity, by an amount comparable to the change in EPR after stimulation (Fig. 2E). Since the baseline window precedes stimulation, this variation cannot be caused by the sonication. It suggests either that the uncertainty in EPR quantification is larger than the error bars indicate, or that trials at different nominal intensities differ systematically in some other respect. It should be examined whether the non-monotonic trend is statistically significant in light of this baseline fluctuation.

Reviewer #2 (Public review):

This manuscript presents a broad and potentially impactful investigation of inflammation-associated ovarian cancer implantation and identifies IRAK4 as a candidate therapeutic node linking inflammatory signaling to tumor seeding. Major strengths include development of an injury-associated metastasis model, complementary genetic and pharmacological interrogation of IRAK4, extensive characterization of the novel inhibitor UR241-2, and incorporation of both xenograft and immunocompetent models. The observation that genetic or pharmacological disruption of the IL1R1/IRAK4 pathway preferentially affects tumor formation at injured sites rather than generalized omental disease is particularly interesting and potentially novel. However, several conclusions currently exceed the mechanistic evidence. Most importantly, the manuscript does not conclusively demonstrate that the antitumor effects of UR241-2 are mediated through IRAK4, particularly given the concentration differences between pathway inhibition and antiproliferative activity and the compound's measurable off-target kinase activity. In addition, host versus tumor-intrinsic IRAK4 functions are not resolved, the functional contribution of the altered macrophage/neutrophil populations remains unproven, and the needle-injury model should be described more cautiously as a model relevant to rather than fully recapitulating port-site metastasis. Addressing these issues would substantially strengthen the mechanistic foundation and translational significance of the work. The manuscript has potential, particularly if the authors sharpen the central claim and add experiments establishing that UR241-2's phenotypes are actually IRAK4-dependent.

Major concerns:

(1) UR241-2 decreases tumors at the injury site but apparently does not significantly decrease omental tumor burden in the syngeneic MiM model. It argues against simple nonspecific antitumor activity and supports a possible role for IRAK4 specifically within an inflammation/injury-dependent metastatic niche. The authors should make much more of this distinction-but also mechanistically prove it.

(2) The manuscript does not yet establish that UR241-2's antitumor effects are mediated primarily through IRAK4. The compound has measurable activity against additional kinases, including MAP4K2 and LRRK2, and activity against other kinases is reported at higher concentrations. More importantly, there appears to be a substantial concentration disconnect across assays. IRAK4 phosphorylation is inhibited at nanomolar concentrations in some experiments, whereas colony formation/viability phenotypes occur largely in the micromolar range. For example, colony effects are reported at 5-20 µM and viability experiments at 20-60 µM. Thus, are the antiproliferative effects observed at 10-60 µM actually caused by IRAK4 inhibition? The manuscript needs a stronger pharmacological/genetic causality experiment. Ideally, the authors should test UR241-2 in IRAK4-knockdown/knockout cells. If UR241-2 retains essentially identical cytotoxic activity after IRAK4 loss, the mechanistic interpretation would need substantial revision. A rescue experiment with WT versus inhibitor-resistant IRAK4 would be even stronger.

(3) The distinction between host IRAK4 and tumor-cell IRAK4 is insufficiently resolved. The Il1r1 experiments manipulate the host, whereas IRAK4 knockdown manipulates the tumor cell. UR241-2, meanwhile, presumably inhibits IRAK4 in both compartments. Consequently, the current experiments combine at least two mechanistically distinct possibilities: tumor-intrinsic IRAK4 versus host IRAK4. The manuscript would be considerably stronger if these compartments were experimentally separated. For example, IRAK4-deficient tumor cells implanted into WT versus pathway-deficient hosts, or pharmacological treatment of mice bearing IRAK4-deficient tumor cells, could determine how much of UR241-2 efficacy is tumor-intrinsic versus microenvironment-mediated.

(4) The immune conclusions are presently associative. The increase in MHC-II-positive macrophages and neutrophils is interesting, but describing these populations as demonstrating an "antitumor immune response" is stronger than the evidence warrants. MHC-II expression does not itself demonstrate antitumor function. Likewise, neutrophils in ovarian cancer can be either tumor-promoting or tumor-suppressive depending on context. The authors show that UR241-2 changes immune composition/phenotype. They do not yet demonstrate that these cells mediate the therapeutic effect. This could be addressed by macrophage or neutrophil depletion, functional assays, cytokine profiling, T-cell activation measurements, or potentially single-cell profiling.

(5) The drug-development claims are somewhat premature<br /> The ADMET package is useful, but several features deserve more cautious interpretation. The compound shows: very high plasma protein binding, rapid mouse microsomal turnover, evidence of efflux, relatively rapid IV clearance, and measurable off-target kinase activity. The authors report mouse microsomal half-life of only ~8.7 min compared with ~209 min in human microsomes and an efflux ratio of ~4.14. These are not fatal problems for a proof-of-concept molecule, but they make language implying a near-clinical candidate premature. UR241-2 currently looks more convincing as a lead/tool compound demonstrating therapeutic tractability of IRAK4 than as an advanced drug candidate.

(6) Exposure-response relationships need considerably more attention. Analysis of IRAK4 and/or NF-κB pathway in the treated tumors should be examined

(7) Some mechanistic observations need deeper validation. The connections among IRAK4, adhesion, E-cadherin, WNT4 and ECM remodeling are intriguing but currently somewhat descriptive. At present, several pieces of this pathway appear adjacent rather than causally connected.

Reviewer #2 (Public review):

Summary:

Tagoe and colleagues present a thorough analysis of the calcium (Ca2+) binding capacity of calreticulin (CRT), an endoplasmic reticulum (ER) Ca2+-buffer protein, using a mutant version (CRT del52) found in myeloproliferative neoplasms (MPNs). The authors use purified human CRT protein variants, CRT-KO cell lines, and an MPN cell line to elucidate the differing Ca2+ dynamics, both on the level of the protein and on cell-wide Ca2+-governed processes. In sum, the authors provide new insights into CRT that can be applied to both normal and malignant cell biology.

First the authors purify CRT protein and perform isothermal titration calorimetry to quantify the Ca2+ binding capacity of CRT. They use full-length human CRT, CRT del52, and two truncations of CRT (1-339 and 1-351, the former of which should lead to the entire loss of low affinity Ca2+ binding). While CRT del52 has previously been shown to lead to a decrease in Ca2+ binding affinity in other models, the ITC data shows that this is retained in CRT del52.

Next, the authors utilize a CRT-KO cell line with subsequent addition of CRT protein variants to validate these findings with flow cytometric analysis. Cells were transfected with a ratiometric ER Ca2+ probe, and fluorescence indicates that CRT del52 is unable to restore basal ER Ca2+ levels to the same extent as CRT wild-type. To translate these findings to MPNs, the authors perform CRT-KO in a megakaryocytic cell line, where reconstitution with either CRT variant did not cause a difference in cytosolic calcium levels. The authors further test store-operated calcium entry (SOCE), an important process to maintaining ER Ca2+ levels, in these cells, and find that CRT-KO cells have lower SOCE activity, and that this can be slightly recovered with CRT addition.

Finally, the authors ask whether other effects of CRT-KO/reconstitution can affect cellular Ca2+ signaling pathway and levels. RNASeq analysis revealed showed that CRT-KO lead to an increase in various chaperone protein expressions, and that reconstitution with CRT del52 is unable to reduce expression to the same extent as reconstitution with CRT wildtype.

Comments on revised version.

The authors have sufficiently addressed my concerns from the first review.

Reviewer #2 (Public review):

Summary:

The authors aim to assess the differential role of Orai isoforms for mediating SOCE and NFAT1 and/or NFAT4 nuclear translocation primarily in the context of T cells. For this purpose, they have used genetically modified cells and appropriate human genetic conditions. All isoforms were expressed individually (with deletions of other isoforms) at roughly equivalent levels so that data across isoforms could be compared unambiguously. Their experiments convincingly identify Orai1 as the main driver of SOCE and NFAT1/4 translocation in the HEK293 cell line and in primary T cells. The two Orai1 isoforms appear functionally equivalent, and loss of the longer isoform (Orai1α) is compensated in humans by the presence of the shorter isoform (Orai1β).

Strengths:

Overall, their judicious use of appropriate knockouts, mutants and expression constructs allows for unambiguous interpretation of data regarding the key role of Orai1α and Orai1β in T cell physiology. They also demonstrate a role for Orai3 in driving SOCE in a breast cancer cell line.

Weaknesses:

The main shortcoming of this manuscript is its inability to place the findings in a context that would be of interest to a broader audience. It would be helpful to provide a metanalysis from existing public databases (human and/or murine) of the known expression in various cell types and tissues of Orai1α, Orai1β, Orai2 and Orai3. This could suggest possible roles for each Orai isoform in tissues other than immune cells. Further, the physiological relevance of the two NFAT isoforms studied, NFAT1 and NFAT4, needs some elaboration, both in the context of T cells and other tissues.

Reviewer #3 (Public review):

Summary:

Core conclusions are well-supported by data: co-folding outperforms docking in known ligand pose/affinity prediction (validated by RMSD and IC₅₀ correlation), struggles with false positive discrimination in virtual screens (lower AUC values), and is complementary to docking (non-correlated errors, distinct strengths in drug discovery stages).

Strengths:

Unprecedented prospective design with 557 novel Mac1-ligand complexes ensures rigorous, independent evaluation of co-folding methods, provides an unbiased and rigorous benchmark dataset, which contains structures and compounds absent from the co-folding models training sets. Comprehensive comparison of 3 co-folding tools (AlphaFold3, Chai-1, Boltz-2) with DOCK3.7 across diverse targets and metrics enables nuanced performance assessment. The revised results clarify an intriguing finding: co-folding can predict correct ligand poses even when protein formations are mispredicted. The study clearly demonstrates complementary roles of co-folding (superior pose/affinity prediction for known ligands) and docking (better hit prioritization), and addresses deep learning memorization concerns via ligand similarity analysis.

Weaknesses:

The study identifies a major limitation of co-folding-failure to capture rare protein conformational changes, which deserve future investigation. The authors include uncalibrated Boltz-2 affinity data (addressing a prior comment) but note that large-scale free energy perturbation (FEP) comparisons are beyond their capabilities.

Appraisal of Aims Achieved:

The authors successfully achieved their primary aims and the results provide strong, well-supported evidence for their core conclusions. Key conclusions are grounded in the study's unbiased, training-set independent data, ensures the conclusions are not confounded by model memorization and are broadly applicable to the field's use of these co-folding models.

Field Impact:

This study provides a critical reality check for the field: co-folding models are powerful tools for pose prediction but are not yet standalone solutions for virtual screening, a key distinction that will prevent over-reliance on these models and guide more rational tool selection.

Reviewer #2 (Public review):

Summary:

In vivo glia-to-neuron conversion emerges as a potential regeneration-based therapeutic strategy for neural injuries and diseases. However, controversies exist in this exciting field, largely arising from the non-stringent methods use to analyze in vivo neuronal conversions. The study by Li et al. directly tackled such a controversy on Neurod1-mediated microglia-to-neuron conversion. They took advantage of transgenic mouse lines to specifically express Neurod1 in microglia of adult mouse brains. Results from immunohistology, in vivo live cell imaging, and scRNA-seq convincingly demonstrate that microglia cannot be converted in vivo to neurons by ectopic Neurod1 expression under the specified normal or injury conditions. Instead, it induces microglia death, consistent with their earlier findings. These solid results, though negative, are critical additions to the research field and further support that stringent lineage tracing methods are essential for studying in vivo cell reprogramming. Overall, the studies are rigorously designed and executed.

Reviewer #2 (Public review):

This manuscript from Zuniga-Pflucker laboratory describes that thymic macrophages are heterogeneous in flow cytometric and transcriptomic profiles, containing two major populations characterized by TIMD4 and CX3CR1 expression. These macrophage populations are both parenchymal in the thymus but are unequal in developmental ontogeny, Flt3 expression history, and CCR2 dependency. The manuscript further reports the interesting findings that the depletion of thymic macrophages impairs thymocyte development at the DN3 beta-selection checkpoint. These results provide an important advance for further understanding of thymus biology, especially in view of the contribution of heterogenous thymic macrophage subpopulations.

Reviewer #2 (Public review):

Summary:

In this study, Zhou et al investigated the expression and function of AIRE in B cells in peripheral lymphoid tissues. First, they found the expression of AIRE protein in mature B cells in the follicles in human tonsils and spleens from healthy donors. Flow cytometry analyses using human samples as well as Aire-reporter mice demonstrated AIRE expression in germinal center B cells. The expression of Aire in B cells was induced by CD40 signals. Then, to investigate the impact of AIRE deficiency on B cell function, the authors used a method of transplanting bone marrow cells from Aire-KO and WT mice into B-cell-deficient mice, comparing B cell development and function reconstituted in the recipient mice. Their results showed that Aire-deficient B cells strongly responded to immunization with antigens, exhibiting enhanced class switching and somatic hypermutation of antibodies compared with WT B cells. The same phenomena were observed in CRISPRed B cell lines lacking Aire. The authors successfully utilized the Aire-deficient B cell line to demonstrate that Aire suppresses antibody class switching and somatic hypermutation via its interaction with AID. Finally, using B cell transfer into B cell-deficient mice demonstrated that mice harboring Aire-deficient B cells produced high levels of autoantibodies against Th17 cytokines and exhibited reduced resistance to Candida infection. This mirrors characteristic symptoms in AIRE-deficient patients. The findings of this study not only reveal an unexpected function of AIRE in B cells but also have the potential to contribute to understanding the pathogenesis of APECED and offering a new direction for developing therapies.

Strengths:

The strength of this study lies in demonstrating the expression of function of AIRE in B cells in both mice and humans. It also revealed the direct interaction between AIRE and AID, along with its binding mode (requiring CARD and NLS domains of AIRE), and showed that this interaction is crucial for AIRE function in B cells. It is also significant that the study demonstrated how B cell-intrinsic dysfunction of AIRE leads to autoantibody production against cytokines.

Comments on revised version.

My previous concerns have been properly addressed.

Reviewer #2 (Public review):

Summary:

The authors apply a hydrogel nanoliter-well in situ microRNA assay to tissue sections from a mouse model of BRCA1-related triple-negative breast cancer, in which tumors had acquired resistance to a PARP inhibitor, and test two drug combinations. They develop a spatial analysis that groups wells into microRNA "topics" and relates these topics to treatment sensitivity and to immune infiltration, aiming to show that the spatial arrangement of microRNAs can report on drug efficacy and stratify tumors by their eventual sensitivity or resistance.

Strengths:

(1) The in vivo combination-therapy experiments are technically careful, and the finding that adding Poly(I:C) to olaparib improves antitumor activity is new.

(2) The data and the analysis code are openly deposited on Zenodo.

(3) Whether the spatial organization of microRNAs carries treatment-relevant information remains a worthwhile question.

Weaknesses:

(1) It is not clear what the spatial measurement adds. The discrimination of sensitive vs resistant tumors was already reported in the authors' prior work, and in this dataset the separation reduces to the relative amount of two microRNAs, let-7a and miR-21, that the bulk analysis had already nominated.

(2) The platform is on the well-level rather than single-cell, and the effective spatial resolution (well size and the number of cells per well) is not stated, so the meaning of "spatial" is unclear, and the sensitivity and specificity of the assay are not established in this work.

(3) The sensitive vs resistant separation is an in-sample description of labels that were fixed in advance, on roughly 21 tumors with about three per treatment arm and nothing held out; the model is refit for each analysis rather than frozen, so it cannot be evaluated as a classifier.

(4) The headline association of a let-7a topic with resistance is correlative, unvalidated, and runs opposite to the canonical roles of let-7 as a tumor suppressor and miR-21 as an oncomiR, and it may reflect the abundance ratio of the two dominant probes.

(5) The topic model is simplistic, collapses to two informative topics, and does not use the existing morphology or H&E information already available on the same sections; the co-localization analysis relies on an image-quality metric that is not appropriate for this purpose and lacks a null.

(6) The conclusions are dependent on a single mouse model at a single timepoint with no human data, but the framing attempts to extend to patients.

Case#: Female, age 44 years old

DiseaseAssertion: Discordant STGD phenotype

FamilyInfo: Information revolving the sister of this patient is given as well as they both have a discordant STGD phenotype. Additionally, it mentions that the parents each harboured a mutation but were asymptomatic/had normal examination results.

CasePresentingHPOs: HP:0001141, HP:0007401, HP:0030602

CaseHPOFreeText: At 23 central vision was deteriorating and patient had a VA of 6/36 in the right eye and 6/60 in the left. Through fundus photography, bilateral yellow/white flecks were found at the posterior pole. 12 years later, her VA was retested and it was 6/60 in both eyes. After autofluorescnece (AF) imaging was done, there was localized low signal at the macula found with abnromal foci. In 2008 her macular atrophy had enlarged and the flecks were less apparent.

CaseNotHPOs: N/a

CaseNotHPOFreeText: In this article there was not a phenotype presented that was normal.

CasePreviousTesting: It mentioned that there were two previously reported variants on the same allele detected in the siblings and one unique novel variant on the second allele for this patient. However, the testing they used was not listed, it just stated that the variants were found through sequencing. For this patient the variants were p.L541P/p.A1038V and p.R881C.

GenotypingMethod: Just mentioned sequencing and ABCA4 screening to look for two variants p.L541V and p.A1038V and a third novel variant p.R881C.

PreviouslyPublished: N/a

Variant: 1) NM_000350.3(ABCA4):c.1622T>C (p.Leu541Pro) 2) NM_000350.3(ABCA4):c.3113C>T (p.Ala1038Val) 3) N/a

ClinVar ID: 1) 99067 2) 7894 3) N/a

**CAID: ** 3) Because there was not a reference or alternate allele provided in this article I was unable to find a CAID for p.R881C.

gnomAD: 1) Highest minor allele frequency was 0.00017 (https://www.ncbi.nlm.nih.gov/clinvar/variation/99067/) 2) Highest minor allele frequency was 0.00188 (https://www.ncbi.nlm.nih.gov/clinvar/variation/7894/) 3) N/a

SupplementalData: Figure 1 had information regarding imaging and other testing done on the patient that is vital for phenotypic characterization. Also, it mentions a variant known as p.R881C, but was unable to find anything on ClinVar or gnomAD.

Reviewer #2 (Public review):

This is a very interesting study from Vandendoren and colleagues examining the role of PVN oxytocin neurons during thermoregulatory behaviors, in particular during thermoregulatory huddling. The findings are important and have implications for the thermoregulation field as well as the social/naturalistic behavior field. The findings are compelling and use a combination of state-of-the-art tools (photometry, optogenetics, automated behavior tracking, thermal imaging, and core body temperature measurement), often in combination with each other, to produce a rigorous and high-dimensional dataset.

Comments on revised version.

I appreciate the effort the authors have put into addressing all of my questions, and I have no remaining concerns.

Reviewer #2 (Public review):

The manuscript by Zhu et al. describes MAIT cell activation by riboflavin metabolites presented by MR1. The authors provide solid evidence for this activation and anti-cancer functional consequence using an array of selected cell lines, primary ex vivo and engineered xenograft models. Broadly, the results are thorough and well controlled, and provide a highly informative insight into the metabolite-MAIT-cancer cell interactions. However, the majority of this work is undertaken using models that preferentially express key targets, and whilst still useful, the (current) broader implications of this research are overstated. Additionally, the suggested MAIT modulation of the tumor microenvironment requires clarification.

Major Comments:

(1) In Figures 2b-d, the authors suggest microbial metabolite stimulation of PBMC cultures increased MAIT cell frequency up to 60%. Whilst their flow data is compelling, the frequency of one population can be influenced by changes in other populations. A form of absolute or relative-to-total count should be used.

(2) The statements regarding cytokine induction in Figure 4e are too strong; many of those inflammatory cytokines are not automatically and consistently tumour-suppressive. The line 299 '...were not induced' may just reflect death of tumor cells. It would be useful to include tumour cell-only controls in Figure 4.

(3) Figure 7 is interesting, but the authors' conclusion that MAIT+5-OP-RU controls the tumor microenvironment is not robustly supported by their evidence.

a) It is not clear how CD14+ cells established a sustained suppressive environment.

b) It is not clear how the peritoneal addition of microbial metabolites 'significantly enhanced MAIT-mediated tumor control'. The authors show that the addition of 5-OP-RU reduced the number of GFP-expressing tumour cells present in peritoneal lavage fluid. There is limited evidence to suggest this occurs through MAIT cells or MR1 in this figure.

c) It is difficult to draw conclusions from peritoneal lavage flow when some experimental groups received cells IP, but then all groups were equally assessed for key populations, and all data are presented as frequencies. The authors should use absolute counts (or similar) to appropriately show changes in cell populations to account for varying total/live/cd45+ cell compartments.

d) It would be necessary at a minimum to include 5-OP-RU-only controls, and ideally include MR1 blocking or the cancer line with MR1 removed. Alongside this, the authors should substantially reduce the strength of their statements on microbial metabolite-MAIT suppression of the tumor microenvironment.

Reviewer #2 (Public review):

Summary:

In this review article, the authors discuss the whole brain activity changes induced by brain stimulation. They review the literature on how these activity changes depend on the cognitive state of the brain and divide the results by the scale of the change being induced, from microscale changes across small groups of neurons, up to macroscale changes across the entire brain. Finally, they describe attempts to model these changes using computational models.

Strengths:

The review provides an overview of the results within this sub-field of neuroscience, and the authors are able to discuss a lot of prior results. The framing of the changes in neuronal activity in terms of computational changes is also a helpful approach.

We thank the authors for the updates that they have made in response to our original comments. Their attempts to address many of the comments that we raised have greatly improved the paper. We believe that there are two major points that still require some additional changes:

(1) We raised the concern that the results within each of the three spatial scales did not join together into a cohesive single framework. The authors responded by updating the conclusion section to provide a more conceptual picture linking the different spatial scales. This is much appreciated. However, by placing this framework at the end of the paper, it prevents the reader from using this understanding to building a conceptual model as they progress through the paper. We would ask that the authors intersperse this conceptual picture within the main text, and to then re-emphasize it in the conclusions. This would frame each section in terms of the findings that led directly to it and, therefore, allow the reader to build a conceptual understanding within each section. As one example, we note that the authors have made no changes to the mesoscale processing section. Therefore, when reading that section, it is completely unclear how any of the results seen in the microscale may relate to the changes observed at the mesoscale.

(2) The authors have greatly improved their explanation of the complexity metrics. However, the paper still lacks a conceptual understanding for why "perturbation-based complexity metrics" are a reasonable way to study the state-dependent dynamics? What does studying perturbations provide that studying the spontaneous activity in different states alone, would not provide? Why is this the preferred way to study such dynamical systems? Such a justification would strongly support the analyses reviewed in the paper and would increase the reader's understanding of the methodology.

Reviewer #2 (Public review):

Summary:

Abbasi et al. examine how signaling through the major G-protein pathways (Gs, Gq, and Gi) influences tumor necrosis factor expression in astrocytes and microglia. Using a combination of pharmacological receptor activation, chemogenetic manipulation, primary rodent glial cultures, human induced pluripotent stem cell-derived astrocytes, and an in vivo astrocyte-targeted Gi manipulation, the authors report a broadly consistent pattern in which Gs- and Gq-associated signaling reduces tumor necrosis factor expression, whereas Gi signaling increases it. The study's cross-species and cross-preparation design, spanning astrocytes and microglia as well as in vitro and in vivo systems, provides a potentially valuable framework for understanding how neuromodulatory pathways may regulate glial inflammatory signaling.

Strengths:

A major strength of the study is the breadth of experimental systems used, which includes primary rat glia, human induced pluripotent stem cell-derived astrocytes, and an in vivo manipulation, allowing for comparison across species and levels of biological complexity. The use of chemogenetic receptors in astrocytes provides relatively direct control over Gq and Gi signaling, and these experiments yield consistent effects on both tumor necrosis factor messenger RNA and protein, strengthening the internal validity of the astrocyte findings. The observation that similar directional effects are seen in human-derived astrocytes and in microglial cultures further supports the idea that aspects of this regulatory relationship may be conserved across glial cell types. More broadly, the study addresses an important and timely question about how neuromodulatory signaling pathways interface with glial inflammatory outputs, and it generates a coherent set of observations that could serve as a foundation for more mechanistic work.

Weaknesses:

The central claim that Gs, Gq, and Gi signaling broadly and directly constitute a general regulatory code for tumor necrosis factor expression is more expansive than the current evidence fully supports. In particular, the evidence for Gs-dependent effects is indirect, relying on beta-adrenergic receptor activation and forskolin-mediated adenylyl cyclase stimulation rather than direct manipulation of Gs itself, leaving uncertainty about pathway specificity. More generally, the use of different endogenous receptors to represent each G-protein class in microglia complicates interpretation, since individual receptors may engage additional signaling pathways beyond their canonical G-protein coupling, limiting the extent to which the results can be attributed to G-protein class alone.

The in vivo experiment also does not definitively establish the cellular source of the observed increase in tumor necrosis factor, as measurements are taken from bulk cortical tissue following astrocyte-targeted Gi activation. This leaves open the possibility that the observed changes arise indirectly from other cell types, particularly microglia, which are shown elsewhere in the study to be strongly responsive to Gi-related manipulations. In addition, the specificity of chemogenetic expression in vivo is not quantitatively demonstrated, further limiting cell-type attribution.

There are also important issues related to experimental design and statistical interpretation. Across several experiments, it is unclear whether reported sample sizes reflect independent biological replicates, technical replicates, or imaging fields, which is especially consequential for the human induced pluripotent stem cell-derived astrocyte experiments where donor-level independence is not clearly established. The in vivo design also appears to treat hemispheres as independent observations despite their paired nature, which may inflate statistical independence given the small sample size.

Finally, several conclusions would benefit from more cautious framing. The data support differential regulation of tumor necrosis factor relative to interleukin-1 rather than strict cytokine specificity, and measurements based solely on messenger RNA should not be interpreted as direct evidence of cytokine production. The comparison between glial signaling effects and neuronal excitation or inhibition also juxtaposes fundamentally different biological readouts and should not be interpreted as a direct functional opposition. Overall, while the study provides interesting and potentially important observations, the broader pathway-level and cell-type-specific conclusions are not yet fully established by the current experimental evidence.

Reviewer #2 (Public review):

Summary:

Although our visual system is continuously analyzing the current visual scene, its processing proceeds in discrete episodes separated by brief eye movements (saccades). It has generally been assumed that the analysis of the next visual snapshot begins in earnest when the eyes land on a new fixated location just after a saccade, but there have been various studies indicating that at least some amount of processing occurs earlier, as the system anticipates the impending eye movement. Here, the authors use magentoencephalography (MEG) measurements to record visually-driven responses and determine at what point exactly the processing of a new visual snapshot begins.

Strengths:

(1) The work is concise and to the point, and the techniques used are a good way to answer the underlying question about visual processing, since they reflect widespread activity in the brain (rather than activity at a particular location or structure).

(2) The use of natural images and extensive data collection from 5 participants is a nice feature of the experimental design which permits characterization of the common effects and of variance across individuals.

(3) The data are analyzed rigorously, but the results are also understood intuitively; for instance, by visual comparison of responses aligned on fixation onset versus saccade onset.

(4) The results provide a clean characterization of when visual analysis begins relative to saccade onset under natural viewing conditions.

Weaknesses:

(1) There were questions about how the scene-onset condition was established, and how data were selected for it.

(2) The significance of the results is slightly overstated; the text would benefit if some of the claims were phrased with a bit more carefully.

(3) In particular, the issue of how motor-related processes (versus stimulus-related content) may determine the processing of the next visual snapshot should be discussed with a bit more nuance.

These are minor weaknesses. Overall, I found the work to be novel and instructive, as it bridges neurophysiological and psychophysical findings in a satisfactory way.

Reviewer #2 (Public review):

Summary:

The authors extend their previous population-based Ct-value framework for inferring community epidemic trajectories from human infections to vector infections, using mosquitoes as vectors for West Nile virus. They use agent-based modelling to distinguish virus-positive detections arising from non-active infection states from those reflecting active infections, and then apply this framework to mosquito surveillance data from Colorado and Texas.

Overall, this is a well-designed and carefully evaluated study. The manuscript proposes a feasible and potentially valuable framework for vector infection surveillance. The findings are supported by both mechanistic agent-based simulations and applications to real-world mosquito surveillance data, which strengthens the biological plausibility and practical relevance of the proposed approach.

Strengths:

A major strength of the study is its clear methodological extension from human infection surveillance to vector infection surveillance. The agent-based modelling framework provides a useful basis for distinguishing active infections from virus-positive detections that may reflect non-active infection states. The application to surveillance data from two different geographic settings further supports the feasibility of the framework. Overall, the study is carefully designed, and the model schematic and main analyses are generally clear.

Reviewer #2 (Public review):

Summary:

The authors studied cognitive control and attention in response to mnemonic prediction errors (MPEs): situations in which the external reality violates internal memory-based predictions. The behavioral task first established strong versus weak predictions, and then either confirmed or violated these predictions. The authors examined markers of cognitive control (frontal theta) and attention (posterior alpha suppression, pupil response) while strong and weak predictions were confirmed or violated. They found increased cognitive control (frontal theta) for strong MPEs, which correlated with subsequent memory. Markers of attention (alpha suppression, pupil response) also accompanied strong MPEs but did not correlate with subsequent memory. Pupil response was investigated using an interesting approach that decomposes the response into different components, finding that different components respond earlier or later and show different correlations with MPEs and their strength. The authors also investigated how EEG, reaction time, and pupil responses correlated with one another, providing further insight into the mechanism underlying the response to MPEs. Together, the study points toward multiple control and attention mechanisms involved in MPE response and memory.

Strengths:

The study has a clear behavioral paradigm with multiple measures - behavioral, EEG, and pupillometry that offer an investigation into different aspects of MPE response and memory.

The study is also very comprehensive in looking at multiple phases in processing MPEs: the prediction phase (prior to the violation), the response to MPEs, and subsequent memory of MPEs, all within one study. Specifically, the link between neural mechanisms and subsequent memory is a major advancement, as most prior studies did not include this component. Mechanisms underlying subsequent memory of MPEs are theoretically important, as a primary function of MPEs is to promote learning and memory. As the authors mention, the different neural and pupillary signals are not robustly correlated, suggesting multiple mechanisms underlying MPE detections, which is interesting, offers avenues for future research, and can facilitate a better theory of how MPEs are processed in the brain. Finally, the decomposition of pupil response into different components and their correlation with behavior (RT during match/MPE detection) is interesting.

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important and timely question in the molecular simulation of biomolecular condensates. Most residue-level coarse-grained models used for IDP phase separation employ implicit solvent and represent effective interactions through relatively simple pairwise potentials. While these models have been very useful, they usually do not explicitly distinguish direct contacts from solvent-separated interactions, nor do they include an energetic barrier associated with water removal. This manuscript attempts to address that limitation by introducing desolvation-inspired terms into coarse-grained models and examining their consequences for phase behavior, chain conformations, dense-phase packing, and dynamics.

The central idea is physically well motivated. Using a simple homopolymer model, the authors show that increasing the desolvation barrier suppresses phase separation, whereas stabilizing solvent-separated contacts enhances phase separation. They further show that solvent-separated interactions can reduce dense-phase over-compaction, which is a meaningful result given the known challenges in obtaining both accurate single-chain dimensions and realistic dense-phase properties from the same coarse-grained model. The finding that desolvation-like terms can reshape dense-phase packing without simply rescaling the overall interaction strength is interesting and could be useful for future model development. I also found the attempt to connect conformational changes across dilute and dense phases with thermal distance from the critical point to be intriguing. The dynamic analysis, including the FRAP-like simulations and the discussion of kinetic arrest during coarsening, adds another useful dimension to the work.

Overall, I think this is a useful and potentially important contribution.

Comments on revised version.

The authors have addressed my earlier comment regarding conformational changes between the dilute and condensed phases. One small additional suggestion is that they may find two related studies useful in this context: Devarajan et al., Nature Communications (2024), on relationships between dilute-phase conformations and condensate material properties, and Wang et al., Chemical Science (2024), which examines sequence-dependent conformational changes during condensation for both model polyampholyte sequences and naturally occurring IDPs. These studies may provide some complementary context for the discussion. This is simply a literature suggestion and does not affect my overall assessment of the revised manuscript.

Reviewer #2 (Public review):

Summary:

Grichine et al. investigate platelet-mediated fibrin compaction using human donor platelets and propose a novel mechanistic model in which platelets generate contractile forces and wind fibrin fibres into compact, coiled structures. Using a combination of 2D spreading assays, 3D clot imaging via expansion microscopy, live-cell imaging, and computational modelling, the authors present evidence of cage-like fibrin architectures, coiled fibre morphologies, and platelet-centred "rosette" structures that are present during fibre compaction. They suggest the involvement of actomyosin in fibre compaction and, overall, the study addresses an important and longstanding question in thrombosis and haemostasis while offering a conceptually novel perspective on clot compaction.

Strengths:

The integration of multiple imaging modalities is a notable strength. In particular, the 2D fibre-retraction assay provides a useful model for understanding the spatiotemporal dynamics of platelet-mediated fibrin compaction, which could be applied to other systems and may yield detailed mechanistic insights into biological processes. The live-imaging approaches are particularly well executed and provide valuable dynamic insights into fibre accumulation and compaction.

Weaknesses:

The primary weakness of the paper is the absence of direct evidence demonstrating the mechanism of fibre compaction via cytoskeletal swirling. Consequently, the relationship between platelet dynamics and fibrin organisation, including coordinated measurements of platelet motion and fibre rearrangement, is not directly assessed (perhaps due to technical barriers). However, the paper does provide solid evidence through myosin inhibition and computational modelling, demonstrating how platelets might mediate fibre compaction.

Comments on revised version.

Overall, the study addresses an important question in thrombosis and haemostasis and introduces a potentially impactful conceptual framework for understanding clot compaction. The imaging approaches and datasets presented will be valuable to the community, particularly to researchers interested in platelet mechanics and fibrin organisation. The possibility that fibres can be compacted extracellularly through cytoskeletal swirling represents a compelling and relatively unexplored mechanism. Therefore, this paper does a good job of establishing a thought-provoking mechanism with solid supporting evidence, although a direct demonstration of the underlying molecular mechanism requires further investigation.

Reviewer #2 (Public review):

The authors have substantially revised the paper in response to the original review, which is greatly appreciated. It is clear that it will eventually make a nice contribution to the literature. This being said, the following points are somewhere between major and minor in term of their implications for interpretation of the study results. If they were to be addressed, the paper would be again improved.

There are a few remnants of the past language that are not helpful re interpretation of the study results: 1) "Specifically, the observed population gradients could emerge either from the pooled activity of frequency-selective neurons that respond to individual tones or from neuronal subpopulations that integrate information across tones to encode their learned threat-value."; and 2) "Together, these findings suggest that the PL integrates sensory similarity with learned threat value to generate stable representations that support adaptive generalization and discrimination." Neither of these statement follows what has been shown in the study, even with inclusion of the results from the GLM analysis (see point 4 below).

(1) This paragraph in the Discussion is difficult to follow: "Generalization has traditionally been explained by perceptual similarity (Shepard, 1987), whereby stimuli resembling a conditioned cue recruit overlapping sensory representations and evoke similar behavioral responses (Corches et al., 2019; Grosso et al., 2018). Although perceptual similarity clearly influences the extent of generalization, accumulating evidence indicates that it cannot fully account for generalized responding (Verra et al., 2026). More recent frameworks propose that associative learning assigns learned value to novel stimuli by integrating their sensory similarity with previous experience, allowing behavior to scale according to predicted biological significance (Verra et al., 2026; Zaman et al., 2023). Our findings provide a neural framework consistent with these ideas. Sensory similarity promoted consistent neuronal population responses across tones, whereas associative learning organized these responses into graded representations that tracked learned threat value across the stimulus continuum. Thus, sensory similarity appears to define the neuronal substrate upon which associative learning constructs value-based representations that support graded behavioral generalization."

While the revisions have removed the many unnecessary references to inference and integration, this paragraph seems like it is adhering to the original idea of how the authors wished to present their work. If the authors wished to talk about something more than perceptual similarity in the context of generalization, they should have used a task that lends itself to a more-than-perceptual-similarity explanation. Again, the inclusion of the GLM analysis is suggestive for some of what the authors wish to say, but doesn't justify the statements that: "Sensory similarity promoted consistent neuronal population responses across tones, whereas associative learning organized these responses into graded representations that tracked learned threat value across the stimulus continuum." In short, the analysis does not substitute for the design that could have and should have been used to assess learned threat value independently of sensory similarity.

(2) The next paragraph in the Discussion is also confusing. "Such reorganization has been proposed to provide flexibility by allowing new information to be incorporated into existing cortical representations while preserving stable behavioral performance (Mau et al., 2020; Zaki & Cai, 2024). Several mechanisms could contribute to this turnover, including systems consolidation, retrieval-induced reconsolidation or memory updating, and repeated nonreinforced stimulus exposure (Lacagnina et al., 2019; Mau et al., 2020; Sangha, 2015; Zaki & Cai, 2024). Although our experiments cannot distinguish between the first two possibilities, the behavioral data argue against extinction as the primary explanation. Extinction is generally associated with the formation of new CS+-safety associations (Bouton et al., 2021), whereas discrimination ratios increased across retrieval sessions, indicating that animals progressively improved their discrimination between threat-associated and safe stimuli rather than acquiring generalized safety responses. This pattern is consistent with previous work showing that discrimination learning sharpens stimulus representations and narrows behavioral generalization gradients (Dunsmoor & LaBar, 2013; Herzog et al., 2021; Jenkins & Harrison, 1960; Lommen et al., 2017). Importantly, turnover was not uniform across the population. Graded neurons retained remarkably consistent response profiles across retrieval sessions, and their activity remained more strongly associated with learned threat value than with freezing behavior. These observations indicate that stable components of the population code can coexist with extensive reorganization of surrounding neuronal ensembles."

The issue with repeated testing is *not* caused by extinction per se. The issue is that non-reinforcement across the repeated testing should differentially affect the CS+ and CS-. Specifically, it should extinguish responding to the CS- stimulus at a rate that matches its distance from the CS+, thereby sharpening the CS+ versus CS- discrimination in precisely the ways that have been observed. Ergo, the repeated testing *is* a problem for inferences that might be drawn about the way that generalization gradients change with time; and *is* a problem for statements regarding "dynamic reorganization of cortical activity patterns over time." There is nothing in the study that allows one to comment on the reorganization of cortical activity patterns over time. The reorganization can and should be attributed to the repeated testing, which is confounded with time. Nonetheless, the reorganization must be due to the repeated testing and NOT time as the present findings are inconsistent with the well-documented broadening of generalization gradients with time.

(3) In the next paragraph, the authors state: "At the same time, narrower generalization gradients and improved discrimination across retrieval sessions suggests ongoing memory updating. These observations are consistent with contemporary theories proposing that systems consolidation and retrieval-dependent updating are complementary processes through which memories continue to evolve after learning (Mau et al., 2020; Tome et al., 2024; Zaki & Cai, 2024)."

In general, I'm not sure why one would invoke systems consolidation or retrieval-induced reconsolidation as an explanation for any of the present findings: they are not explanations of much at all. In this specific text, the authors seem to be implying an updating process that occurs independently of what is learned across the repeated sessions of testing. Why? The changes that occur in the behaviour and neuronal representations are perfectly explicable in terms of additional learning that occurs - of the sort that I hope to have made clear in my previous comment. Why invoke more than what is needed to explain the observed pattern of results?

(4) Re the GLM analysis - The authors write that: "the fact that the GLM analysis indicates that these neurons reflect learned threat value more than freezing behavior, suggests that they encode an abstract property of the learned stimulus rather than simply mirroring behavioral output."

This is fine if freezing fully indexes the state of conditioned fear and there are no other behaviours in which animals express their fear. If, however, fear is expressed in a range of other behaviours that are likely coordinated by the PL (e.g., startle, vigilance, scanning, orienting to source of danger), this interpretation of the GLM analysis is unwarranted. This is an important point and would be worth noting somewhere in the paragraph where the statement appears.

Reviewer #2 (Public review):

Summary:

This manuscript examines decision-making in a context where the information for the decision is not continuous, but separated by a short temporal gap. The authors use a standard motion direction discrimination task over two discrete dot motion pulses (but unlike previous experiments, fill the gaps in evidence with 0-coherence random dot motion of differently coloured dots). Previous studies using this task (Kiani et al., 2013; Tohidi-Moghaddam et al., 2019; Azizi et al., 2021; 2023) or other discrete sample stimuli (Cheadle et al., 2014; Wyart et al., 2015; Golmohamadian et al., 2025) have shown decision-makers to integrate evidence from multiple samples (although with some flexible weighting on each sample). In this experiment, decision-makers tended not to use the second motion pulse for their decision. This allows the separation of neural signatures of momentary decision-evidence samples from the accumulated decision-evidence. In this context, classic electroencephalography signatures of accumulated decision-evidence (central-parietal positivity) are shown to reflect the momentary decision-evidence samples.

Strengths:

The authors present an excellent analysis of the data in support of their findings. In terms of proportion correct, participants show poorer performance than predicted if assuming both evidence samples were integrated perfectly. A regression analysis suggested a weaker weight on the second pulse, and in line with this, the authors show an effect of the order of pulse strength that is reversed compared to previous studies: A stronger second pulse resulted in worse performance than a stronger first pulse (this is in line with the visual condition reported in Golmohamadian et al., 2025). The authors also show smaller changes in electrophysiological signatures of decision-making (central parietal positivity, and lateralised motor beta power) in response to the second pulse. The authors describe these findings with a computational model which allows for early decision-commitment, meaning the second pulse is ignored on the majority of trials. The model-predicted electrophysiological components describe the data well. Some flexible weighting of the second pulse also described the data well (in line with previous studies), but this explanation suffers from additional model complexity. In particular, this analysis of model-predicted electrophysiology is impressive in providing simple and clear predictions for understanding the data.

Weaknesses:

Behaviour in this experiment is different from previous experiments which use very similar designs (Kiani et al., 2013; Tohidi-Moghaddam et al., 2019; Azizi et al., 2021; 2023). The authors provide some possible explanations for this in the discussion. Overall performance in this experiment was much worse than previous experiments: Participants achieved ~85% correct following 400 ms of 33 - 45% coherent motion. In previous work, performance was ~90% correct following 240ms of 12.8% coherent motion. A second weakness is that, while bounded model can describe the data in this manuscript, it cannot explain the data from previous experiments showing a stronger weight on the second pulse.

Reviewer #2 (Public review):

This study by Anttonen, Christensen-Dalsgaard and Elemans describes the development of hearing thresholds in an altricial songbird species, the zebra finch. The results are very clear and along what might have been expected for altricial birds: at hatch (2 days post-hatch), the chicks are functionally deaf. Auditory evoked activity in the form of auditory brainstem responses (ABR) can start to be detected at 4 days post-hatch but only at very loud sound levels. The study also shows that ABR response matures rapidly and reaches adult like properties around 25 days post-hatch. The functional development of the auditory system is also frequency dependent with a low to high frequency time course. All experiments are very well performed. The careful study throughout development and with the use of multiple time-points early in development is important to further ensure that the negative results found right after hatching are not the result of the experimental manipulation. The results themselves could be classified as somewhat descriptive but, as the authors point out, they are particularly relevant and timely. Since 2016, there has been a series of studies published in high profile journals that have presumably showed the importance of prenatal acoustic communication in altricial birds, mostly in zebra finches. This early acoustic communication would serve various adaptive functions. Although acoustic communication between embryos in the egg and parents has been shown in precocial birds (and crocodiles), finding an important function for pre-natal communication in altricial birds came as a surprise. Unfortunately, none of those studies performed a careful assessment of the chicks' hearing abilities. This is done here, and the results are clear: zebra finches at 2 and 6 days post hatch are functionally deaf. Since, it is highly improbably that the hearing in the egg is more developed than at birth, one can only conclude that zebra finches in the egg (or at birth) cannot hear the heat whistles. The paper also ruled out the detection on egg vibrations as an alternative path. The prior literature will have to be corrected, or further studies conducted to solve the discrepancies. For this purpose, the "companion" paper on bioRxiv that studies the bioacoustical properties of heat calls from the same group will be particularly useful. Researchers from different groups will be able to precisely compare their stimuli.

Beyond the quality of the experiments, I also found that the paper was very well written. The introduction was particularly clear and complete (yet concise).

Weaknesses:

My only minor criticism is that you don't discuss potential differences between behavioral audiograms and ABRs. Optimally, one would need to repeat the work of Okanoya and Dooling with your set up and using the same calibration. The ~20dB difference might be real or it might be due to SPL measured with different instruments, at different distances, etc. Either way, you could add a sentence in the discussion that states that even with the 20 dB difference in audiogram heat whistles would not be detected during the early days post-hatch, but that adding a (novel) behavioral assay in young birds could further resolve the issue.

More Minor Points.

(1) As mentioned in the main text, the duration of pips (form pips to bursts) affects the effective bandwidth of the stimulus. I believe that you could give an estimate of this effective bandwidth given what is know from bird auditory filters. I think that this estimate could be useful to compare to the effective bandwidth of the heat-call which you can now also estimate.

(2) Fig 5b. label the green and pink areas as song and heat-call spectrum. Also note that in the legend you say: "Green and red areas display the frequency windows related to the best hearing sensitivity of zebra finches and to heat calls, respectively". I don't think this is what you meant. I agree that 1-4 kHz is the best frequency sensitivity of zebra finches but you probably meant green == "song frequency spectrum" and pink == "heat call spectrum". In either case the figure and the legend need clarification.

(3) Fig 5c. Here also, I would change the song and heat-call labels to "song spectrum", "heat call spectrum". You don't want readers to think you used song and heat calls in these experiments (maybe next time?). For the same reason, maybe in 5a you could add a cartoon of the oscillogram of a frequency sweep next to your speaker.

(4) Methods. In your description of the stimulus, you describe "5ms long tone bursts" but these are the tone pips in the main part of the manuscript. Use the same terms.

Comments on revisions:

In the latest version of this manuscript, Anntonen et al have diligently addressed all the issues raised by the reviewers. As I mentioned in our discussions among reviewers, it is impossible to "prove" a null hypothesis and both methods and experimental design could always be improved. At this stage however, they provide convincing evidence that the sound intensity of heat calls are below the hearing thresholds of zebra finch chicks.

Reviewer #2 (Public review):

Summary:

The authors studied aggregation, buckling, and particle collection by the filamentous cyanobacterium Fluctiforma draycotensis, as well as by the filamentous Pseudanabaena sp. (order Pseudoanabenales). They performed a range of experiments, from imaging individual gliding filaments to multiple-day experiments showing the formation of large aggregates around a particle formed from a precipitate. They also developed a model of buckling filaments to argue that the ability of elastic filaments to collect particles and form macrostructures is confined to a part of the filament phase space in terms of length and flexibility, meaning that gliding combined with certain filament length and flexibility naturally reproduces the observations.

Strengths:

This is an impressive study that uses multiple tools to connect macrostructure formation with filaments' gliding motility and buckling. It adds an important perspective on the biological and physical factors at play in the emergence of aggregates.

Weaknesses:

The authors ignore the possibility that filament behavior plays an important role in the emergence of the observed patterns. Cyanobacteria have been shown to control their gliding motility (Pfreundt et al Science 2023; Kurjahn et al Nature Comm 2024), and their molecular motors are known to be regulated by chemotaxis-like signaling pathways (Risser ARM 2025). As far as I know, how the coordination between the pulling agents along an individual filament works is actively debated, but there seems to be little doubt that it exists. 

To illustrate this point better, note that the aggregation observed by the authors is consistent with the length-dependent ability of filaments to coordinate gliding (I'm not saying this is how it works in Fluctiforma draycotensis; I'm saying it's consistent). Suppose the coordination requires sufficiently long filaments, which could be the case when signaling molecules travel along the filament, propagating information about when individual pulling agents should reverse. In such a model, short filaments act randomly because they fail to coordinate gliding by the time they glide off nascent aggregates, whereas longer filaments can perform informed reversals because they have more time for coordination. Such behavior then explains the lack of aggregation in Pseudanabaena sp. (via behavior, not lack of stiffness). Note that Trichodesmium is stiff; its filaments do not buckle, yet Trichodesmium forms organized aggregates via tightly controlled motility. Note also that, as the authors report, since Pseudanabaena sp. is both shorter and faster, its filaments have relatively (to the time needed to glide the filaments' length) little time to coordinate reversals. In my opinion, whether the observed patterns passively emerge from gliding and buckling or result from active behavior remains an open question.

I also have a small suggestion regarding this statement on model novelty:

The essential novelty of this model is that the filament itself is active and out of equilibrium, and additionally, the forces and torques are applied locally along its centreline, and not at its extremities as in previous steady-state mechanical studies of elastic, twistable filaments such as DNA [31-33] (see Methods and SI).

This statement needs to be revised as it ignores a substantial body of work on self-organization of active filaments: (R. E. Isele-Holder, J. Elgeti, G. Gompper, Soft Matter 2015; Pfreudnt et al, Science 2023; Faluweki et al PRL 2023; Kurjahn et al Nature Comm 2024).

Last point: the authors often say that their observations are reproducible ('...reproducibly forms macroscopic granules...'). What is meant? Different experiments on different days, different aliquots?

Reviewer #2 (Public review):

Summary:

In this study, the authors used genetic, transcriptomic, imaging, and electrophysiological approaches to investigate the role of P2X7R in retinal remodeling in rd1 mice. The authors propose that RA signaling upregulates P2X7R, leading to altered Ca²⁺ signaling, HCN1 expression, and spontaneous RGC hyperactivity.

Strengths:

Multiple lines of experiments were conducted, focusing on an important question in the field.

Weaknesses:

However, some aspects of the experimental design, statistical analysis, and interpretation require clarification. In particular, the genetic controls and causal evidence should be strengthened before the proposed RA-P2X7R-Ca²⁺-HCN1 pathway can be fully supported.

Some major issues:

(1) The Abstract states that P2X7R deletion prevents the upregulation of RA-responsive genes, whereas the Results state that RAR-dependent genes were not consistently changed by P2rx7 deletion. This central statement should be corrected and clarified.

(2) The P2rx7 knockout model requires further discussion. JAX strain 005576 targets exon 13 and has previously been reported to retain truncated P2X7 transcripts with residual activity (Masin et al., 2012). The authors should avoid describing this allele as complete loss of the entire P2rx7 gene unless additional isoform-specific validation is provided.

(3) The evidence that RAR directly regulates P2rx7 transcription remains incomplete. Predicted promoter motifs and reduced P2X7R protein after BMS-493 treatment are supportive, but they do not establish direct transcriptional regulation. Measurement of P2rx7 mRNA, RAR promoter occupancy, or promoter mutagenesis would strengthen this conclusion.

(4) Several statistical results require verification. In Figure 2H, four paired eyes are analyzed using an unpaired Mann-Whitney test, and the reported p<0.001 is difficult to reconcile with n=4. Similarly, the significance levels in Figure 5A-D are not compatible with a two-sided Wilcoxon rank-sum test using n=3 mice per group. The statistical unit, exact P values, and number of biological replicates should be rechecked.

(5) The overall causal pathway remains partially inferential. The study does not directly show that increased Ca²⁺ causes HCN1 upregulation or that HCN1 is required for RGC hyperactivity. These steps should either be experimentally tested or presented as a proposed model rather than an established mechanism.

Reviewer #2 (Public review):

Summary:

The study's aim was to establish whether stability in thought patterns relates to the particular thought pattern, the task context, or their interaction. And further, whether stable thought patterns could be linked with distinct brain patterns.

Strengths:

The core reliability framing is novel, and the trait/state/interaction decomposition is a fruitful way to pose the question, leading to the finding that stability is neither a pure trait nor a pure task property, but emerges from their interaction, which is a solid contribution.

The aim to characterise aspects of stability across individuals and across tasks was achieved and is well supported.

Weaknesses:

While the paper makes excellent use of existing data sets, the independent samples, i.e., one study sample for the cognitive/thought-sampling data, and several different study samples to generate the brain maps, do limit the brain-behaviour conclusions that can be drawn.

Reviewer #2 (Public review):

Summary:

In this paper, the authors use electrophysiological recordings from the olfactory tubercle (OTu) and ventral tegmental area (VTA) of mice learning an olfactory Pavlovian conditioning task to demonstrate the consistency of changes in OTu odour responses with gradient descent updates minimising reward prediction error (RPE). The paper is clearly written and the work well motivated. The authors address a gap in the literature on animal reinforcement learning by providing neural evidence for gradient-based representation learning, something that had been proposed but not yet tested. The results are convincing and the limitations comprehensively addressed. Of particular interest is the proposal that OTu SPNs could solve the weight transport problem through knowledge of the sign of their downstream connections from their expression of either D1/D2 receptors. This makes a concrete experimental prediction that future research could test.

Strengths:

The paper provides one of the first demonstrations backed by neural recordings that representation learning in the brain is consistent with gradient descent. It shows how, although weight transport may be biologically implausible, the brain appears to find other ways to compute a gradient in multilayered networks for efficient learning. The study builds nicely on recent work in systems neuroscience and provides evidence for a concrete implementation in the OTu-VTA circuitry of mice.

The paper demonstrates that changes in OTu striatal projection neuron (SPN) activity over trials are proportional not only to the RPE relayed by VTA dopamine neurons, but also account for the influence of each particular SPN on the RPE. If an SPN decreases the RPE when active, its activity will increase on the next trial after a positive RPE. The activity will instead decrease for an SPN that increases the RPE. This relation is encapsulated in the update rule of Equation 5.

Weaknesses:

The main weakness of the paper is that it provides only indirect evidence for the update mechanism by inferring synaptic weights based on the (justified) assumption of VTA dopamine neurons encoding RPE. This is still a substantial contribution to understanding representation learning in the brain, though I do think that the authors could provide some additional evidence to further convince readers. One idea could be to analyse the distribution of inferred weights and validate whether it agrees with known statistics of connectivity between OTu SPNs and their downstream projections (e.g. fraction of D1/D2 SPNs).

Further, as dopamine neurons are known to have asymmetrical responses for positive and negative RPEs (with the dips in activity related to negative RPEs being generally smaller) I'd expect an improvement in the correlation of the learning updates particularly after the reversal if the authors account for this in the model.

Reviewer #2 (Public review):

Summary:

In this study, Benamar et al. investigate whether DNA double-strand breaks induce extensive abnormal DNA synthesis in cancer cells and whether this response contributes to the cytotoxic effects of ionizing radiation and other DNA-damaging treatments. The authors refer to this process as double-strand-break-induced genomic amplification (DIGA). Mechanistically, they propose that insufficient protection of broken DNA ends permits excessive resection, followed by RAD51-dependent strand invasion and RAD52-POLD3-POLD4-dependent DNA synthesis resembling break-induced replication.

Overall, the study addresses an interesting and potentially important question. Break-induced replication was originally defined as a pathway that repairs one-ended DNA breaks through extended synthesis from an invaded homologous template. Studies in yeast established that this process involves a migrating DNA-synthesis bubble, conservative inheritance of newly synthesized DNA, frequent template switching, and high mutagenicity. Related forms of DNA synthesis have subsequently been described in mammalian cells at collapsed replication forks, telomeres, under-replicated mitotic regions, and some transcription-associated DNA breaks.

The important advance of this study is the proposal that double-strand-break-associated DNA synthesis, with several features of break-induced replication, can become sufficiently extensive to cause a measurable increase in total cellular DNA content and that this synthesis correlates with radiation-induced cell death. However, the physical structure, genomic distribution, and extent of the additional DNA have not yet been directly established.

Strengths:

Radiation-induced breaks are generally considered in relation to DNA repair, chromosome rearrangements, checkpoint activation, mitotic failure, senescence, and cell death. The possibility that broken DNA ends can also initiate extensive DNA synthesis provides a potentially important additional link between defective break repair, genome amplification, and treatment-induced cytotoxicity.

The authors examine this phenomenon using several complementary approaches and comparisons across multiple cancer cell lines. The finding that several double-strand-break-inducing treatments produce a similar response suggests that the phenotype is not restricted to ionizing radiation or to a single cellular background.

The authors also make efforts to distinguish DIGA from canonical origin-dependent re-replication, such as that caused by CDT1 stabilization and inappropriate origin relicensing. They find that, following irradiation, CDT1 is degraded rather than stabilized and that CDT1 depletion does not suppress DIGA. Perturbation of ORC1 or ORC2 also has little effect on the phenotype. In addition, depletion of SET8 or loss of SUV4-20H1 increases, rather than decreases, DIGA.

The authors carry out a systematic analysis of DNA-end protection and processing. Loss of ATM, RNF8, RNF168, 53BP1, RIF1, or Shieldin components enhances DIGA, whereas depletion or inhibition of CtIP, MRE11, EXO1, RAD51, RAD52, POLD3, or POLD4 suppresses it. These experiments support a model in which insufficient end protection permits excessive DNA-end resection, followed by strand invasion and recombination-associated DNA synthesis involving factors linked to break-induced replication.

Overall, the findings place DIGA within a growing body of evidence that recombination-associated DNA synthesis can extend well beyond short repair patches. The study attempts to connect the processing of double-strand breaks with abnormal DNA synthesis, increased cellular DNA content, and the cytotoxic response to radiation.

Major weaknesses and suggested experiments:

(1) The main evidence for DIGA is the appearance of cells with greater-than-G2/M DNA content by flow cytometry. BrdU incorporation shows that active DNA synthesis occurs within this population, and the density-gradient experiments provide further evidence for newly synthesized DNA. However, these methods do not reveal the physical nature of the proposed genomic amplification-for example, which genomic regions are copied or how long the synthesis tracts are.

This is important for the central conclusion of the study because mammalian break-induced replication can produce genomic duplications, but the extent of synthesis depends strongly on the type of DNA lesion. Previous work has shown that repair of damaged replication forks can produce segmental genomic duplications (Costantino et al., 2014; PMID: 24310611). By contrast, recent measurements at defined two-ended double-strand breaks suggest that mammalian break-induced-replication tracts can be relatively restricted (Li et al., 2021; PMID: 33470420; Shah et al., 2024; PMID: 39368985).

Thus, the large increase in total DNA content detected by flow cytometry in this study would require many simultaneous synthesis events, very long synthesis tracts, or an alternative process such as whole-genome duplication. The authors should therefore characterize the additional DNA directly. Whole-genome sequencing of sorted cells with greater-than-G2/M DNA content could be particularly informative.

(2) The genetic dependencies are consistent with synthesis initiated from resected DNA ends, but they do not directly demonstrate that the new DNA synthesis begins at double-strand breaks. DNA damage could indirectly alter replication-origin activity, cell-cycle progression, or DNA synthesis at genomic regions distant from the original lesions. Increased DNA content alone therefore does not establish amplification initiated directly at DNA breaks.

(3) MLN4924-induced re-replication is used throughout the study, but it is unclear whether the authors have combined MLN4924 with ionizing radiation and measured the resulting DNA synthesis. This experiment could clarify whether conventional re-replication and DIGA are independent, overlapping, or mechanistically connected.

(4) The results involving canonical non-homologous end joining are complex. Loss or inhibition of DNA-PKcs increases DIGA, whereas loss of XRCC4 or XLF strongly suppresses it, and loss or inhibition of LIG4 has a weaker suppressive effect. The authors suggest that XRCC4 and XLF stabilize broken DNA ends and thereby permit the synthesis reaction independently of final ligation. This is an interesting model, but the current evidence does not yet establish it. XRCC4 and XLF can affect end synapsis, break persistence, chromosome fusion, resection, and cell-cycle progression. Their loss could therefore reduce DIGA through several indirect mechanisms. The authors should measure DNA-end resection, RAD51 loading, persistence of double-strand breaks, and recruitment of RAD52 or POLD3 in XRCC4-, XLF-, and LIG4-deficient cells. Complementation with separation-of-function mutants that differentially affect XRCC4-XLF end bridging and LIG4 recruitment could test whether end stabilization, rather than ligation, is important. Without this, the opposing effects of upstream and downstream non-homologous end-joining components remain difficult to understand.

(5) The correlation between the amount of DIGA and radiation sensitivity across cancer cell lines is interesting. However, cell lines differ in many factors that influence radiation responses, including p53 status, apoptosis, checkpoint activity, ploidy, homologous recombination capacity, and proliferation rate. Matched models would provide stronger evidence. Conversely, restoring DNA-end protection in a DIGA-prone cancer cell line could test whether this reduces susceptibility. These experiments would help determine whether DIGA is a general property of cancer cells or a feature of particular repair-defective genetic backgrounds.

Reviewer #2 (Public review):

Summary:

This study by Dong et al characterizes the roles of highly-expressed Rab GTPases Rab5, Rab7 and Rab11 in the development and wiring of olfactory projection neurons in Drosophila. This convincing descriptive study provides complementary approaches of Rab expression and localization profiling, conventional dominant negative mutants, and clonal loss of function mutants to address the roles of different endosomal trafficking pathways across circuit development. They show distinct distributions and phenotypes for different Rabs. Overall, the study sets the stage for future mechanistic studies in this well-defined central neuron.

Strengths:

Beautiful imaging in central neurons demonstrates differential roles of 3 key Rab proteins in neuronal morphogenesis as well as interesting patterns of subcellular endosome distribution. These descriptions will be critical for future mechanistic studies. The manuscript is well-written and explanatory, very accessible to a wide audience without sacrificing technical accuracy.

Comments on revised version.

The paper is greatly improved with new data and better-nuanced interpretation. The clarity of explanations for a non-Drosophila reader have been redone particularly well.

Reviewer #2 (Public review):

Summary:

By analyzing cells' frequency-dependent viscoelastic properties and intracellular activity through microrheology, Münker et al simplify the complex active mechanical state into six key parameters that constitute the mechanical fingerprint. They apply this concept to cells treated with cytoskeleton-inhibiting drugs. Additionally, a comprehensive statistical analysis across various cell types shows how cells coordinate their mechanical properties within a defined phase-space marked by activity, mechanical resistance, and fluidity.

Strengths:

(1) The distribution of the six parameters: they have been well characterized based on established theories, and they can be used to understand cell-type-specific biomechanical differences. The examples of muscle cells and immune cells were profound and informative.<br /> (2) Efforts to perform dimension reduction of parameter space into activity (E), fluidity (C1) and resistance (A) are insightful and will be helpful for future characterization of cell mechanics.

Comments on revised version.

In the original submission, cytochalasin B alone showed little effect on viscoelastic and active energy parameters, and it was unclear whether this reflected a true absence of actin's role or an artifact of the perturbation method used. In the revised manuscript, the authors addressed this by repeating the cytochalasin B measurements with larger sample sizes and adding latrunculin A, a mechanistically distinct and more potent actin-depolymerizing drug, together with immunostaining to confirm cytoskeletal disruption. This convincingly shows that actin depolymerization does affect the solid-like prefactor and fluidity, resolving the original concern.

Nocodazole-induced microtubule depolymerization previously did not appear to reduce the solid-like property A, which was unexplained. The revised manuscript removes the speculative compensation-mechanism explanation, adds a discussion comparing the results to prior AFM literature (explaining the discrepancy as reflecting different mechanical compartments probed - cortex vs. intracellular), and the new data now show a significant reduction of A with nocodazole treatment as well. This weakness is resolved.

Reviewer #2 (Public Review):

Summary:

In this study, Styer et al. impose artificial selection on root-associated microbiomes to increase drought tolerance in rice plants using different soils as starting microbiomes. Using NDVI and biomass as a proxy for plant health, they find that iterative passaging of the microbiomes of the best-performing plants increased plant resilience to drought stress in a soil-dependent manner. The study makes use of numerous controls. The authors survey the microbiota of the plants across generations, using an array of interesting analyses to characterize their observations. Firstly, the authors find that the acquired microbiomes are divergent towards the beginning of the selection experiment, but nearly converge later suggesting that the selected communities become more similar over time. One reason is that the diversity of the microbiomes severely decreases after only one or two generations of selection AND that microbes from each inoculation source appear to easily disperse across the experiment, leading to microbiome homogeneity. The authors then present an analysis to correlate ASVs with the NDVI and Biomass over the course of the experiment (using the rice soil selection lines) to develop hypotheses about which ASVs may impact plant traits.

Strengths:

The authors set out to refine the understanding of microbiome artificial selection, a topic of recent interest to the plant microbiome field. The authors use an established approach (Mueller et al), expanding upon it by including multiple starting soil inocula to ask whether the strength of selection varies by input microbiome. This is an important and novel question. Using drought resilience as measured by NDVI and plant biomass to select upon was a wise choice for this type of study, given their relative ease and quickness to assess. The inclusion of several types of controls, multiple selection lines, and several starting soil inocula showed a thoughtful experimental design. The analyses were diverse, non-standard, and attempted to address microbiome dynamics on multiple fronts. I am not necessarily convinced by some of the conclusions (see below), however, I think this study examines an important and exciting topic in the area of plant microbiomes. I predict the findings of the experiments will inform a wide audience of researchers attempting similar studies and be helpful in their designs.

Reviewer #2 (Public Review):

The revised manuscript has substantially improved, and the authors have satisfactorily addressed most of the concerns raised in my original review. Overall, the experimental evidence and its relationship to the computational model are now presented more clearly and rigorously, substantially strengthening the manuscript.

My main reservation in the original review concerned the role of the initial topographic connectivity bias in the computational model. The revised manuscript provides a clearer interpretation of this aspect. The initial bias represents a coarse activity-independent scaffold, while the final organization of the maps depends on its interaction with the structure and degree of correlated spontaneous activity. Importantly, the simulations show that the presence of the initial bias alone does not determine the final connectivity pattern. I therefore consider the computational results substantially better supported and interpreted in the revised manuscript. Nevertheless, as the model starts from a predefined coarse topographic organization of the projections from the primary sensory cortices to RL, in my opinion, the results demonstrate how structured spontaneous activity can refine and align an initially organized connectivity scaffold, rather than showing that spontaneous activity itself establishes this topographic organization.

This distinction is relevant when interpreting the central mechanistic conclusion of the study. The work provides convincing support for the idea that correlated spontaneous activity can contribute to the refinement and alignment of multisensory cortical maps, conditional on the existence of an initial coarse topographic organization. Establishing experimentally how this initial connectivity is organized during the relevant developmental period, and how spontaneous activity modifies it, remains an important question for future work.

Overall, I consider the revised manuscript considerably stronger than the original submission. Most of my previous concerns have been adequately resolved, and the study provides valuable experimental and computational insight into how spontaneous activity may contribute to the development of aligned multisensory representations.

Reviewer #2 (Public review):

Recent work from the authors identified the synaptic changes and glial reaction that occurs during exposure of a Drosophila odorant receptor neuron population to continued exposure of a stimulating odorant. This work markedly advanced our understanding of cellular response to critical periods. This current Advance manuscript carries that work forward and examines the non-autonomous responses to constant odorant exposure. The authors discover that the changes to ORN populations are not accompanied by changes to either PN dendrite or PN axon volume, nor are they concurrent with changes in postsynaptic PN structures. These changes are, however, notable accompanied by changes in Ca2+ and voltage responses in ORNs. Importantly, this set of responses is specific for the Or42a ORNs (that are highly sensitive to the odorant in question, ethyl butyrate) and not the Or43b ORNs (which respond to ethyl butyrate, but not as drastically). Finally, the authors include connectomics analyses showing that Or43b and Or42a ORNs differ in their synaptic input/output relationships.

This is an excellent use of the Advance mechanism for the journal as these are important follow-up findings for the parent story. The non-autonomous effects (or lack thereof) on PNs is an important part of the story as is the functional response of Or42a ORNs and the differing response of similarly (but not identically) sensitive Or43b ORNs. The experiments are well conceived, controlled, and conducted. Where the story falters a bit, though, is with the connectomics analysis. The authors show distinct differences between Or43b and Or42b ORN input output relationships and suggest that those differences may underlie the differences observed in their response to ethyl butyrate exposure during the critical period. This is certainly a possibility, but as it stands now, it is too disconnected to offer significant proof. There would have to be additional experiments to address this. Right now, the inclusion of the connectomics work feels like a distraction at best, and a complete non sequitur at worst. To be clear, the connectomics work is well done and I have no issues with its validity, but is not helpful to the central thesis of the work. I would suggest the authors either remove it entirely or strongly rethink how it fits into the paper.

Comments on revised version:

I appreciate the consideration of my comments and the authors' responses. The additional data on PN synapse number is intriguing (and welcome) as is the text discussing potential postsynaptic compensatory mechanisms. I respect the authors' decision in retaining the connectivity analysis, but despite the textual changes, I still feel that it is peripherally related to the main thesis of the work and would best be omitted from the paper and included in a separate, more relevant study. Ultimately, though, that is their choice.

Reviewer #2 (Public review):

Summary:

This manuscript examines how GATA6-dependent programming of resident peritoneal macrophages regulates their lipidome and, in turn, eosinophil homeostasis, combining lipid imaging, mass spectrometry, transcriptional analysis and in vivo pharmacology. BODIPY/CARS microscopy with targeted lipidomics convincingly shows substantial lipid changes following myeloid GATA6 deficiency, particularly in sphingolipids, with Smpd1 and Gba2 manipulations providing mechanistic support.

Strengths:

The authors also connect these changes to eosinophil biology, confirming increased peritoneal eosinophils in Gata6-deficient mice with reduced apoptosis (via two methods) rather than increased production. Testing of alternative explanations (chemokines, IL-5, prostaglandins, 12/15-LOX products) strengthens the argument by narrowing candidate mechanisms. The identification of increased LTE4 is notable as it correlates with eosinophil abundance, and zileuton reduces LTE4, eosinophil numbers, and increases apoptosis. This supports a role for 5-LOX/cysteinyl-leukotriene signalling.

Weaknesses:

The principal weakness is specificity: zileuton affects the broader leukotriene pathway, not LTE4 alone, so correlation with LTE4 doesn't establish causality. This matters more given the LTE4 receptor remains unidentified (to the best of my knowledge). Similarly, the proposed transcellular biosynthesis mechanism (ImmGen data suggesting complementary enzyme expression across cell types converting LTC4 to LTE4) is inferential; direct evidence of cellular source and transfer is lacking.

Design limitations include reliance on pooled animals in some lipidomic measurements, small replicate numbers, and a stronger eosinophil phenotype in females that shifts subsequent analysis toward females. Indeed, this sex dependence deserves more discussion given it limits generalisability.

Overall, this is a technically strong, conceptually interesting study. The core conclusions, that GATA6-dependent regulation of the macrophage lipidome and a role for cystl Lts in eosinophil survival, are well supported. The more specific claim that LTE4 is the causal factor via a defined transcellular pathway is plausible but not yet firmly established. Experimental strengthening or moderated claims would improve the study.

Reviewer #2 (Public review):

Summary:

Koh and colleagues investigate the broader sensory role of LITE-1, a gustatory receptor previously linked to UV light detection in C. elegans. Their study explores whether LITE-1 also mediates avoidance of specific chemical stimuli-namely, high concentrations of diacetyl and 2,3-pentanedione. They show that LITE-1 is required in the ADL and ASK neurons for calcium responses to diacetyl, and that its expression in body-wall muscles is sufficient to trigger hypercontraction upon odorant exposure. Molecular docking suggests both odorants may directly bind to LITE-1 with micromolar affinity. These findings suggest LITE-1 may act as a multimodal receptor for both light and chemical stimuli.

Strengths:<br /> • Methodological Precision: The study is technically strong, with well-executed calcium imaging and quantitative behavioral assays that clearly show neural and muscular responses to chemical stimuli.<br /> • Novelty and Scope: The work presents a compelling case for LITE-1 functioning as a multimodal sensor, which is an intriguing expansion of its known role.<br /> • Potential Impact: If validated, the findings could significantly advance the understanding of sensory integration in C. elegans, and the tools developed may be broadly useful to the research community.<br /> • Relevance to the Field: The study adds to evidence that C. elegans uses non-canonical sensory pathways and may inspire further exploration of multimodal receptor functions in other systems.

Weaknesses:<br /> • Lack of Rescue Experiments: The absence of rescue experiments makes it difficult to definitively link the observed phenotypes to loss of lite-1.<br /> • Single Loss-of-Function Approach: The reliance on a single genetic mutant limits interpretability. Additional strategies such as RNAi (e.g., neuron-specific knockdown) would provide stronger evidence.<br /> • Unclear Neuronal Contribution: While calcium responses in ADL and ASK are reduced, it's unclear which neuron(s) are necessary for behavioral avoidance. Cell-specific rescue or knockdown experiments are needed.<br /> • Unvalidated Docking Data: The molecular docking predictions lack experimental validation. Site-directed mutagenesis would be needed to support claims of direct interaction.<br /> • Limited Odorant Specificity Testing: Docking analysis does not include non-binding odorants, making it difficult to assess binding specificity.<br /> • Incomplete Quantification: Some calcium imaging results (e.g., in AWA neurons of unc-13 mutants) lack statistical comparisons, which limits their interpretive value.

Comments on revisions:

I thank the authors for their thorough revision. The manuscript is substantially improved, and most of the concerns raised in my original review have been addressed.

The strongest improvement is the addition of new genetic evidence supporting a role for LITE-1 in high-concentration diacetyl avoidance. The use of multiple independent lite-1 alleles strengthens the conclusion that the phenotype is specifically due to loss of lite-1 function, and the ADL-specific rescue experiment is an important addition. While the rescue is not complete, it is convincing and supports the idea that LITE-1 activity in ADL contributes to the avoidance response.

The neuronal analysis is also stronger. The new statistical analysis across the sensory neurons addresses my previous concerns regarding quantification, and the revised interpretation of the calcium imaging data is more balanced. The revised title of this section is also more consistent with the data and appropriately focuses on ADL and ASK rather than ASH.

I also appreciate the additional controls addressing possible effects of ambient light. The light versus dark experiments make it unlikely that the observed behavioral phenotypes are secondary to unintended activation of LITE-1 by environmental illumination.

The expanded odorant analysis and inclusion of docking predictions for additional compounds are useful additions. Together with the ectopic expression experiments in body-wall muscles, these data strengthen the argument that LITE-1 can respond to diacetyl and related compounds.

My main remaining concern is the same one raised in the initial review: the docking results remain largely computational predictions and have not been tested experimentally through binding-site mutagenesis or other functional validation. As a result, the manuscript still does not demonstrate direct ligand binding to LITE-1. However, I think the authors have strengthened the indirect evidence considerably, and the conclusions are now generally written in an appropriately cautious manner. I would encourage the authors to continue framing the docking results as supportive of a direct interaction rather than definitive proof of one.

Overall, I believe the manuscript has been significantly strengthened by the revision. The remaining limitation is largely mechanistic and does not, in my opinion, undermine the central conclusions of the study.

Reviewer #2 (Public review):

The manuscript by Hajimohammadi, Mohr, O'Connell and Kelly is intended to demonstrate that participants integrate evidence over time to make a decision, even in a noise-free, static decision context. This is validated by the observation that 1) participant accuracy improves with increased exposure to the stimulus; and 2) there is a correlation between participant accuracy and a neural index of evidence accumulation, as measured by centro-parietal positivity (CPP).

Strengths:

(1) Joint modelling of accuracy and CPP dynamics is a significant achievement, as behaviour alone often cannot distinguish between competing theories of decision-making. In the case of protracted sampling in particular, the absence of reaction times (RT) due to the delayed nature of the response makes this method highly appealing.

(2) The experimental manipulations and the method used to extract the different neural indices are well chosen, enabling the mapping of putative cognitive processes such as evidence accumulation and motor preparation onto the recorded EEG with clarity.

(3) The in-depth discussion of the results clearly articulates those reported by the authors and in previous works.

Weaknesses:

(1) Regarding the first point I raised in the first version of the manuscript, I'm satisfied with the author's response.

(2) For the second point, however, I'm still unconvinced, noting that my understanding of the fitting routine is relatively limited. To me, the comparison of the behavioral and the joint-modelling is relatively weak in terms of evidence. Despite the revision, it is still unclear whether the behavioral models are well conditioned given the very weak constraint exerted by (aggregated, see below) binary decision data only. To my previous comment, the authors reply, "We can instead address identifiability somewhat indirectly through parameter estimate consistency across the 10 fits we conducted with different instantiations of noise". I'm worried that the different instantiations of noise (limited to 10), generated for the parameter search, have any impact at all. A better quantification of the uncertainty in parameter estimates, as well as the AIC, is needed, for example through a cross-validation scheme, or bootstrapping/jack-knifing at the level of the participants. Without this, I remain unconvinced that the behavioral models are suited for the comparison with the joint-neural models:

(3) Relatedly, regarding the response of the authors to my minor comment 4, after the revision of the manuscript, it is clearer that the behavioral models were fitted only on 6 data points (accuracies averaged over participants for each condition) for models with up to 3 parameters. I understand that the authors average behavior to make a comparison with the joint neural model, whose neural signals are noisy at the participant level. However, if the neural model, or the chosen linking function, needs this aggregation to outperform the behavioral model, that questions a bit whether the joint model is really useful in practice.

While I see these two last points as serious for the comparison between behavioural and joint-neural models, this does not change the main addition of the paper: that is, the joint model and the evidence for protracted sampling.

Reviewer #2 (Public review):

Summary:

The authors aimed to identify the specific neurons, neurotransmitters, and neuropeptides that mediate the longevity effects of the hypoxic response in C. elegans. By genetically dissecting the pathway downstream of HIF-1, they define a neural circuit involving ADF serotonergic neurons, the SER-7 receptor in the RIS interneuron, tyraminergic signaling from RIM, and neuropeptide NLP-17, ultimately linking neuronal hypoxic sensing to pro-longevity signaling in the intestine.

Strengths:

The study employs a diverse genetic toolkit, including neuron-specific transgenes, tissue-specific knockouts and rescues, RNAi knockdowns, allowing the authors to pinpoint causality, sufficiency, and necessity with high resolution. The comprehensive mapping of cell-nonautonomous signaling adds depth to our understanding of how HIF and serotonin signaling interface with aging pathways. The conclusions are supported by consistent survival assays and fmo-2 gene expression analyses.

Reviewer #4 (Public review):

Summary of General Strengths & Weaknesses:

The studies here are highly informative for anatomical tracing and sympathetic nerve function in the liver in relation to glucose levels, but because they are conducted in a single species, it is challenging to translate them to humans or determine whether these neural circuits are evolutionarily conserved. Dual-labeling anatomical studies are elegant, and the addition of chemogenetic and optogenetic studies provides mechanistically informative. Denervation studies lack proper controls, and sensory innervation in the liver is overlooked.

Specific Weaknesses - Major:

(1) The species name should be included in the title.

(2) Tyrosine hydroxylase was used to mark sympathetic fibers in the liver, but this marker also labels a portion of sensory fibers that need to be ruled out in whole-mount imaging data.

(3) Chemogenetic and optogenetic data demonstrating hyperglycemia should be described in the context of prior work demonstrating liver nerve involvement in these processes. The Discussion currently mentions this only briefly, but comparing methods and observations would be helpful.

(4) Sympathetic denervation with 6-OHDA can drive compensatory increases in tissue sensory innervation, and this should be measured in the liver denervation studies to implicate potential crosstalk, especially given the increase in LPGi cFOS that may be due to afferent nerve activity. Compensatory sympathetic drive may not be the only culprit, though that is clearly assumed. The sensory or parasympathetic/vagal innervation of the liver is altogether ignored in this paper and could be better described in general.

Comments on the revised version.

Across all reviewer comments, the revised resubmission has adequately addressed all concerns.

Reviewer #2 (Public review):

Summary:

Zhang et al. investigate how blood feeding and dietary protein influence sleep in the mosquito Aedes aegypti. The authors first establish a behavioural definition of sleep using postural analysis and arousal threshold measurements, then demonstrate that both blood meals and a bovine serum albumin (BSA)-based protein diet increase sleep for several days. They further show that RNAi-mediated knockdown of the leucokinin receptor (Lkr) enhances sleep, implicating neuropeptide signalling in the regulation of postprandial sleep.

Strengths:

The central question is well-motivated, and the experimental approach is systematic. The use of multiple independent methods to characterise sleep - postural analysis, infrared activity monitoring, videography, and arousal threshold - provides converging evidence. The 10-minute immobility criterion is grounded in the arousal threshold data, bouts exceeding 10 minutes corresponding to the first bin at which a significant effect emerges. The demonstration that the sleep increase is already detectable before oviposition establishes that the phenotype begins with feeding rather than with the completion of the reproductive cycle. The BSA feeding experiment is a particularly effective demonstration that dietary protein, rather than other blood components, is a key regulator of the sleep increase. The conservation of leucokinin signalling in sleep regulation between Drosophila and Ae. aegypti is a noteworthy finding that adds comparative depth. The "opportunistic versus determined" host-seeking distinction is appropriately framed as a hypothesis for future testing rather than as a conclusion drawn from the present data, and the limits of the design with respect to reproductive physiology are stated explicitly.

Weaknesses:

(1) Confound of reproduction and sleep. Blood and BSA both support egg development, so neither condition isolates nutrient sensing from reproductive physiology. The relative contributions of diet, egg development and post-reproductive recovery remain undetermined.

(2) Sleep versus reduced locomotion. The pDoze and pWake measures are defined here as proportions of time above or below a velocity threshold, rather than as the per-minute transition probabilities of the established definition (Wiggin et al. 2020, PNAS). So defined, they are equivalent to percent sleep and percent wake and cannot distinguish a sleep-like state from the mechanical consequences of engorgement.

(3) Data availability. Raw data are stated to be available on request rather than deposited in a public repository, which makes independent reanalysis less straightforward than it need be.