Reviewer #2 (Public review):

Pescher and colleagues present a revised manuscript detailing the multi-omic characterisation of Leishmania donovani amastigote to promastigote differentiation and integration of this data. The molecular pathways that regulate Leishmania life-stage transitions are still poorly understood, with many approaches exploring single proteins/RNAs etc in a reductionist manner. This paper takes a systems-scale approach and does a good job of integrating the disparate -omics datasets to generate hypotheses about the intersections of regulatory proteins that are associated with life-cycle progression. The differentiation step studied is from amastigote to promastigote using hamster-derived amastigotes which is a major strength. The use of hamsters permits the extraction of parasites that are host adapted and represent "normal", host-adapted Leishmania ploidy; the promastigote experiments are performed at a low passage number. Therefore, this is a strength or the work as it reduces the interference from the biological plasticity of Leishmania when it is cultured outside the host for prolonged periods. The multi-omics datasets presented are robust in their acquisition and analysis and will form an excellent resource for researchers studying the molecular events (particularly proteasomal protein degradation, and phosphorylation) during life-stage progression.

General comments on the revisions:

My view is that the authors have made significant, satisfactory changes that address the comments and queries I made on the original manuscript (Review Commons).

There are two areas where the authors had to make major changes/justifications where further comment is merited, these were:

RNA-seq.<br /> The most significant issue was the originally underpowered RNA-seq which had only two replicates. This has been repeated with four replicates now. This has not led to changes in the interpretation of the data between the original study and this one. One comment that the authors make in the response to this was : "Given the robustness of the stage-specific transcriptome, and the legal constrains associated with the use of animals, we chose to limit the number of replicates to the necessary". Ensuring that animal experiments are properly powered and that maximum robustness of the data from the minimum sample size is an important part of experimental design for ethical use of animal models. Essentially the replication here could have been avoided if the original study had used 1 more animal. However, the new version of RNA-seq brings appropriate confidence to the interpretation of the data.

Phosphoproteomics.<br /> The authors provide a robust justification of their strategy for the phosphoproteomics and highlight the inclusion criteria for phosphosites: "Phosphosites were only considered if detected with high confidence (identification FDR<1%) and high localisation confidence (localisation probability >0.75) in at least one replicate". The way missing values were dealt with is explained "For statistical analyses, missing values within a given condition were imputed with a well-established algorithm (MLE) only when at least one observed value was present in that condition." This fills in some of the gaps I was missing from the original manuscript, and I am satisfied that the data analysis is entirely appropriate for a discovery/system -based approach such as this one. The authors also edit the manuscript to reflect that "occupancy" or "stoichiometry" might not be the best description of what they were presenting and switched to the terminology of "normalised phosphorylation level" - I think this is an appropriate response.

Overall, in the absence of follow up experiments on specific individual examples, some of the claims in the original submission were toned down and reflect a more neutral description of the data now. Significantly, the data still underpin a key role for regulation of the ribosome between the amastigote and promastigote stages (and during the differentiation process). The recursive and reciprocal links between the phosphorylation and ubiquitination systems are interesting and present many opportunities for future investigation.

Reviewer #2 (Public review):

This is an extremely important work that shows how one can use generative models to construct specificity-switching mutational paths in complex fitness landscapes. The experimental evidence is very clear, and the theoretical tools are innovative.

The work will likely have a deep impact on future research aimed at understanding how evolution navigates fitness landscapes as well as reconstructing ancestral sequences.

The manuscript is extremely clear and well written, the experimental evidence is strong, and the methods are clearly described, so I do not have major issues to raise. A few minor issues are listed below.

(1) I consider the WW domain as an 'easy' case from the point of view of generative modelling. The domain is rather short, epistatic effects are not very strong (e.g. Boltzmann learning usually converges very quickly to a very paramagnetic state), and the resulting models are well interpretable (e.g. the hidden units of the RBM correlate well with subclasses).

This is not always (not often?) the case, however. In more complex proteins, the learning procedures can be slower and the resulting models less interpretable. Just for completeness, perhaps the authors could comment on the generality of the results and what they would expect for other systems based on their experience.

(2) In Section 3.3, the authors say that direct paths connecting Class I and Class IV behave similarly to indirect paths, despite having lower scores according to the RBM. How generic is this? Does it also happen for other classes? This might be an important point to address, as direct paths are easier to sample.

(3) The path shown in Figure 4 goes through a region of non-functionality around sequences 18-19. It seems that the sample path is basically exploring the functional regions for Class I and Class II/III separately, trying to approach the other class, but then it can't really make the switch.

By contrast, the path going from Class I to Class IV seems able to perform the functional switch in a single step (20-21) without losing too much of the function.

Perhaps the authors could better comment on this? Is this a limitation of the sampling method, or a fundamental biological fact?

(4) On page 12, it is stated that the temperature was chosen to 1/3 to maximize the score. This is important and should be mentioned earlier (I didn't notice it until that point).

(5) On page 13, it is stated that: "However, the scores of the ancestral sequences along the phylogenetic pathways assigned by the RBM are significantly lower than the ones of the RBM-designed sequences. This result is expected as ASR reconstruction does not take into account epistasis, differently from RBM, and we expect ASR sequences to generally be of lesser quality."

I was very surprised by this result. My own experience with ASR shows that, on the contrary, sequences found by ASR (via maximum likelihood) tend to have high scores in the (R)BM, and tend to be more stable than extant sequences. I attribute this to the fact that ASR typically finds a "consensus" sequence that maximizes the contribution to the score coming from the fields (the profile), which is typically dominant over the epistatic signal, resulting in a bigger score. Maybe the authors did not use maximum likelihood in the ASR? Some clarification might be useful here.

Reviewer #2 (Public review):

Summary:

This study identifies the outer‑mitochondrial GTPase MIRO1 as a central regulator of vascular smooth muscle cell (VSMC) proliferation and neointima formation after carotid injury in vivo and PDGF-stimulation ex vivo. Using smooth muscle-specific knockout male mice, complementary in vitro murine and human VSMC cell models, and analyses of mitochondrial positioning, cristae architecture and respirometry, the authors provide solid evidence that MIRO1 couples mitochondrial motility with ATP production to meet the energetic demands of the G1/S cell cycle transition. However, a component of the metabolic analyses are suboptimal and would benefit from more robust methodologies. The work is valuable because it links mitochondrial dynamics to vascular remodelling and suggests MIRO1 as a therapeutic target for vasoproliferative diseases, although whether pharmacological targeting of MIRO1 in vivo can effectively reduce neointima after carotid injury has not been explored. This paper will be of interest to those working on VSMCs and mitochondrial biology.

Strengths:

The strength of the study lies in its comprehensive approach assessing the role of MIRO1 in VSMC proliferation in vivo, ex vivo and importantly in human cells. The subject provides mechanistic links between MIRO1-mediated regulation of mitochondrial mobility and optimal respiratory chain function to cell cycle progression and proliferation. Finally, the findings are potentially clinically relevant given the presence of MIRO1 in human atherosclerotic plaques and the available small molecule MIRO1.

Weaknesses:

(1) High-resolution respirometry (Oroboros) to determine mitochondrial ETC activity in permeabilized VSMCs would be informative.

(2) Therapeutic targeting of MIRO1 failed to prevent neointima formation, however, the technical difficulties of such an experiment is appreciated.

Comments on revisions:

The authors have addressed the concerns I previously raised.

Reviewer #2 (Public review):

Summary:

The author investigates how the HIV-1 Env glycoprotein modulates the nanoscale organisation and dynamics of the CXCR4 co-receptor on CD4⁺ T cells. The author demonstrates that HIV-1 Env induces CXCR4 clustering distinct from that triggered by its natural ligand (CXCL12), implicating spatial receptor organization as a determinant of infection. This study investigates how HIV-1 Env (specifically X4-tropic gp120) alters the membrane organization and dynamics of the chemokine receptor CXCR4 and its WHIM-associated mutant, CXCR4R334X, in a CD4-dependent manner. Using single-particle tracking total internal reflection fluorescence microscopy (SPT-TIRF-M), the authors demonstrate that both soluble gp120 and virus-like particles (VLPs) displaying gp120 induce CXCR4 nanoclustering, reduce receptor diffusivity, and promote immobile nanoclusters of CXCR4 at the membrane of Jurkat T cells and primary CD4⁺ T cell blasts. The work offers new insights into the spatial organisation of receptors during HIV-1 entry and infection. The manuscript is well-written, and the findings are significant.

Significance:

Nature and significance of the advance:<br /> This work marks a conceptual and mechanistic breakthrough in understanding HIV-1 entry. It goes beyond the static view of Env-co-receptor interaction to show that nanoscale reorganization of CXCR4, distinct from chemokine-induced clustering, occurs during HIV-1 Env engagement and may be essential for infection.

Context within existing literature. Previous studies established Env-induced CD4 clustering (Yin et al., 2020) and chemokine-induced CXCR4 nanocluster formation (Martínez-Muñoz et al., 2018), but the exact nanoscale rearrangement of CXCR4 in the context of HIV-1 Env and physiological Env densities remains unquantified. This study addresses this gap using SPT-TIRF, STED microscopy, and functional assays.

Audience and influence: The findings will be of interest to researchers in HIV virology, membrane receptor biology, viral entry mechanisms, and therapeutic target development. The receptor-clustering aspect could also influence broader fields of study, such as GPCR organization and immune receptor signalling.

Reviewer expertise: I can evaluate HIV-1 entry mechanisms, viral glycoprotein-host-host-host receptor interactions, single-molecule fluorescence microscopy, and membrane protein dynamics. I am less equipped to evaluate the deep structural modelling aspects, though the in silico AlphaFold results are straightforward to interpret in context.

Reviewer #2 (Public review):

Summary:

This manuscript aims to identify the central nervous system circuitry, specifically within the mushroom body (MB), that mediates nociception-induced escape behavior in adult Drosophila. The authors provide a detailed map of the neural pathways underlying defensive actions in flies. Overall, the study is technically solid, clearly written, and conceptually<br /> interesting.

Strengths:

The authors present compelling evidence by integrating multiple complementary approaches. The ALTOMS laser system enables precise, automated measurement of escape latency, allowing for high-throughput and objective behavioral quantification. Neuronal silencing experiments assess functional necessity and demonstrate that specific dopaminergic neurons (DANs) and mushroom body output neurons (MBONs) are critical for escape behavior. Trans-Tango anatomical mapping further supports the proposed circuit by identifying putative synaptic connections consistent with the authors' model.

Weaknesses:

A central limitation of the study is its heavy reliance on chronic Kir2.1-mediated neuronal silencing as the primary functional manipulation. This approach raises concerns about potential developmental compensation and indirect network effects. The authors could strengthen their conclusions by incorporating more temporally precise, reversible silencing strategies, such as recently developed optogenetic- or chemogenetic-based methods.

In addition, the study relies on the trans-Tango system to identify downstream synaptic partners, which has several inherent limitations. Trans-Tango detects only chemical synapses and cannot reveal electrical coupling. The system may also yield false negatives due to reporter sensitivity, and anatomical labeling alone does not establish functional connectivity in the context of the specific behavior examined.

Reviewer #2 (Public review):

Summary:

This is a bold and important study and addresses an important question in the field: how species-specific variation in brain oxytocin receptor expression relates to differences in social behavior.

Tsukamoto et al. generated eight independent transgenic mouse lines (Koi lines) carrying a bacterial artificial chromosome (BAC) encompassing the prairie vole Oxtr locus along with flanking intergenic regions, with the goal of probing the behavioral consequences of species-specific variation in brain Oxtr expression. Across these "volized" lines, the authors claim conserved Oxtr expression in the mammary gland but strikingly divergent patterns of brain expression, none of which fully recapitulate endogenous prairie vole Oxtr distribution, and instead exhibit expression patterns that diverge from both mouse and prairie vole brain Oxtr distribution. Nevertheless, some lines exhibit partial overlap with vole Oxtr expression pattern reported in the literature within specific brain regions, and one line displays partner preference behavior reminiscent of prairie voles. The authors further report line-dependent differences in maternal pup retrieval and crouching behaviors, which they interpret as evidence that variation in brain Oxtr expression can drive variation in social behaviors. Together with analyses of topologically associating domain (TAD) architecture, the authors conclude that brain, but not peripheral- Oxtr expression, is shaped by distal regulatory elements beyond the BAC insert, and propose that such regulatory flexibility underlies evolutionary diversification of social behavior.

Strengths:

A particular strength of the study is the generation of multiple independent transgenic lines, which provides a valuable resource for probing regulatory influences on Oxtr expression.

Weaknesses:

While the study addresses an important question, I have several methodological and conceptual concerns regarding the study in its current form. Some aspects of the study fall outside my primary area of expertise, and I am therefore not in a position to fully evaluate the technical difficulty or rigor of those components, or to judge whether my suggestions would be feasible to implement. I defer to reviewers with relevant expertise for a more detailed assessment of these aspects.

(1) Each independent Koi line exhibits a distinct brain expression pattern that differs from both wild-type mouse and prairie vole Oxtr expression, complicating the interpretation of the results. The manuscript does not include a direct comparison of brain Oxtr expression patterns in these transgenic lines with those of prairie voles. Instead, expression similarity is inferred primarily from regional localization and compared indirectly with prior literature (Figures 2-5). For those lines that show partial resemblance to prairie vole Oxtr expression patterns, the authors do not assess whether Oxtr-expressing neurons share comparable anatomical projections or transcriptomic identity with prairie vole Oxtr-expressing neurons. Quantification of expression remains largely descriptive, illustrating expression patterns (Figure 2), OXTR protein distribution (Figure 3; images are difficult to evaluate due to low contrast), or Oxtr mRNA levels across selected brain regions in Koi lines, wild-type mice, and mOxtr-/- mice (Figures 4-5), without directly testing similarity to prairie vole expression. In addition, whole-brain expression data are lacking, with analyses restricted to selected sections. While such analyses may be beyond the scope of the present study, these limitations nonetheless complicate interpretation of the central question - namely, whether the observed behavioral phenotypes arise from vole-like Oxtr circuits rather than from distinct, line-specific expression configurations.

(2) The authors state that Oxtr expression in the mammary gland is similar across all Koi lines and the mOxtr-IRES-Cre knock-in line. However, the images presented in Figure 2 appear to show differences in anatomical detail across lines, and no quantitative analysis is provided to support the claim of equivalence.

(3) The conclusion that integration site rather than copy number determines the observed BAC transgene expression patterns (Lines 202-203) is not fully supported by the data. First, the authors did not compare multiple copy numbers at the same genomic insertion site, making it impossible to disentangle copy-number effects from position effects. Second, BAC copy number does not necessarily scale linearly with expression; higher copy numbers can have a repressive effect on gene expression (Garrick et al, Nat Genet, 1998).

(4) While I am not an expert in TAD analysis, the observed differences in 3D architecture around Oxtr are consistent with a role for long-range regulatory interactions. However, these analyses appear largely descriptive and correlative, and establishing a causal contribution of 3D chromatin organization to Oxtr regulation by distal elements would likely require direct perturbation of TAD boundaries or looping interactions. I recognize that such experiments may be beyond the scope of the present study, but clarifying this limitation in the interpretation would be helpful.

Reviewer #2 (Public review):

Summary:

The authors have developed an elegant, lightweight, open-source system that should be able to be widely disseminated to the community. They have used this system in multiple experimental paradigms and demonstrate its functionality quite elegantly. One of these experiments involves two of three animals in the arena being stimulated, a situation that clearly requires an untethered approach. They have appropriately quantified key system parameters (latency and battery life).

Strengths:

The introduction places this work in a broader context. That context includes a number of previous solutions, many of which are smaller or more technically complex. However, I agree with the authors that there is a need for something that is easy for labs to acquire and deploy in terms of both what goes on the head and the broader infrastructure (i.e., not needing complex wireless power delivery approaches).

The paper does an excellent job of describing the system architecture. And the architecture is good! Their system comprises more than just the bluetooth enabled head-mounted devices - they also have built an interface that allows for TTL triggers that link into existing workflows.

The key metrics for a device like this are weight, battery life, and latency. The weight is 1.4g, which is appropriate for adult mice; the battery life is ~100 minutes of continuous stimulation, which should be sufficient for many experiments, and the latency is typically less than 30 ms, which is fine for all but the most demanding closed-loop experiments.

Performance is demonstrated in two experiments, a continuous Y-maze, which elegantly demonstrates how transfected animals learn to sense optogenetic closed-loop stimulation to drive their choice behavior in a way that control-stimulated animals do not. While authors claim that the ~2m diameter apparatus is "large scale", the second behavior more convincingly demonstrates the need for wireless stimulation.

They used closed-loop monitoring of animal pose to selectively stimulate animals for approaching the tails of a dominant conspecific (based on pre-experimental pairwise assessments). It seems that the original hope was that the increases in following that they observe would result in long-lasting changes in the hierarchy of a cage, but as they report, this was not observed. Critically, their supplementary video demonstrates that they conducted this experiment with two instrumented animals simultaneously. This is a situation where a tether would have been hopelessly tangled within a few moments!

The online documentation seems complete, and it seems quite possible for other labs to adopt and deploy the system.

Weaknesses:

The battery life is highly dependent on the stimulation paradigm. It makes sense that the LED is a major component of power consumption. It would have been elegant to measure the total optical energy that can be provided by the system. In addition, Bluetooth transmission is probably a major consumer of power, and receiving may not be "free". Quantifying power as a function of Bluetooth message rates would have been useful.

Presumably, the major constraint on latency is that the Bluetooth receiver polls at ~10 Hz, resulting in latency blocks of 20+, 30+, or 40+ ms. Why latency is never less than 10 ms is unclear. Could latency be reduced by changing a setting? Having a low-latency option would be very helpful for some experimental situations. Latency is probably the primary weakness of the system.

The programming process sounds quite complicated. It would be nice if they had OTA updates. But described and open source. Similarly, the configuration process (Arduino IDE) seems a bit complex. It would be nice if there were a dedicated cross-platform application.

It is unclear what the maximum number of devices that could be used without wireless interference is. The base station has two charging stations, but it would have been nice to understand the limits beyond this number.

There is a very nice website for the system, but there is some concern that the code and design files are not archived. Could they be deposited with the paper?

Reviewer #2 (Public review):

Summary:

The manuscript describes various conformational states and structural dynamics of the Insulin degrading enzyme (IDE), a zinc metalloprotease by nature. Both open and closed state structures of IDE have been previously solved using crystallography and cryo-EM which reveal a dimeric organization of IDE where each monomer is organized into N and C domains. C-domains form the interacting interface in the dimeric protein while the two N-domains are positioned on the outer sides of the core formed by C-domains. It remains elusive how the open state is converted into the closed state but it is generally accepted that it involves large-scale movement of N-domains relative to the C-domains. Authors here have used various complementary experimental techniques such as cryo-EM, SAXS, size-exclusion chromatography and enzymatic assays to characterize the structure and dynamics of IDE protein in the presence of substrate protein insulin whose density is captured in all the structures solved. The experimental structural data from cryo-EM suffered from high degree of intrinsic motion amongst the different domains and consequently, the resultant structures were moderately resolved at 3-4.1 Å resolution. Total five structures were generated in the originally submitted manuscript using cryo-EM. Another cryo-EM reconstruction (sixth) at 5.1Å resolution was mentioned after first revision which was obtained using time-resolved cryo-EM experiments. Authors have extensively used Molecular dynamics simulation to fish out important inter-subunit contacts which involves R668, E381, D309, etc residues. In summary, authors have explored the conformational dynamics of IDE protein using experimental approaches which are complemented and analyzed in atomic detail by using MD simulation studies. The studies are meticulously conducted and lay the ground for future exploration of the protease structure-function relationship.

Strengths:

The manuscript presents a powerful integrative structural biology study that combines high-resolution cryo-EM, particle heterogeneity analysis, time-resolved cryo-EM, multiscale molecular dynamics simulations, SAXS, and biochemical assays to dissect the conformational dynamics of human insulin-degrading enzyme. A major strength is the identification of a previously unappreciated rotational component of IDE-N relative to IDE-C and the discovery of R668 as a molecular latch governing the open-close transition, supported consistently by structural, computational, mutational, and functional data. The work provides a coherent mechanistic framework linking IDE dynamics to substrate unfolding, allostery, and substrate-dependent catalysis, with clear relevance to diabetes and Alzheimer's disease biology.

Weaknesses:

Despite its depth, several key mechanistic conclusions-particularly substrate unfolding and the proposed "β-grabbing" mechanism-rely heavily on coarse-grained and all-atom MD simulations rather than direct experimental observation. Cryo-EM density for insulin is limited and heterogeneous, restricting definitive structural interpretation of substrate binding modes. The time-resolved cryo-EM experiment captures only a single dominant state at modest resolution, limiting insight into transient intermediates. In addition, the study focuses primarily on insulin, leaving the generality of the proposed mechanism for other IDE substrates insufficiently tested, and the therapeutic implications remain largely speculative without direct pharmacological modulation data.

Reviewer #2 (Public review):

Summary:

The manuscript introduces Neuroplex, a pipeline that integrates miniscope Ca²⁺ imaging in freely moving mice with multiplexed confocal and spectral imaging to infer projection identities of recorded neurons. This technical approach is promising and could broaden access to projection-resolved population imaging. However, the core quantitative analyses apply a winner-take-all single-label assignment per neuron even when multiple fluorophores exceed threshold, with additional labels treated descriptively as "secondary hits." While the authors acknowledge and simulate dual labeling, the extent to which this single-label decision rule affects subtype fractions and behavioural comparisons remains uncertain without a multi-label (or probabilistic) sensitivity analysis and propagation of classification uncertainty.

Strengths:

(1) Conceptual advance and practicality: Decoupling acquisition from identity readout constitutes an innovative approach that is, in principle, applicable in laboratories currently using single-color miniscopes.

(2) Engineering thoroughness: The manuscript offers detailed consideration of GRIN optics, spectral libraries, registration procedures, and simulations that address signal-to-noise ratio, background, and class imbalances.

(3) Immediate community value: If demonstrated to be robust, the pipeline could enable projection-resolved analyses without reliance on specialized multicolor miniscopes.

Comments on revision:

The authors have addressed my comments, and I have no further remarks.

Reviewer #2 (Public review):

Summary:

In this manuscript, Malfatti et al. study the role of Chrna2 Martinotti cells (Mα2 cells), a subset of SST interneurons, for motor learning and motor cortex activity. The authors trained mice on a forelimb prehension task while recording neuronal activity of pyramidal cells using calcium imaging with a head mounted miniscope. While chemogenetically increasing Mα2 cell activity did not affect motor learning, it changed pyramidal cell activity such that activity peaks become sharper and differently timed than in control mice. Moreover, co-active neuronal assemblies become more stable with a smaller spatial distribution. Increasing Mα2 cell activity in previously trained mice did increase performance on the prehension task and led to increased theta and gamma band activity in the motor cortex. On the other hand, genetic ablation of Mα2 cells affected fine motor movements on a pasta handling task while not affecting the prehension task. While overall this study addresses an important and timely question, limitations in the design of the motor learning task and data analysis significantly weaken the conclusions drawn in this manuscript.

Strengths:

The proposed question of how Chrna2-expressing SST interneurons affect motor learning and motor cortex activity is important and timely. The study employs sophisticated approaches to record neuronal activity and manipulate the activity of a specific neuronal population in behaving mice over the course of motor learning. The authors analyze a variety of neuronal activity parameters, comparing different behavior trials, stages of learning, and the effects of Mα2 cell activation. The analysis of neuronal assembly activity and stability over the course of learning by tracking individual neurons throughout the imaging sessions is notable, since technically challenging, and yielded the interesting result that neuronal assemblies are more stable when activating Mα2 cells.

Overall, the study provides compelling evidence that Mα2 cells regulate certain aspects of motor behaviors, likely by shaping circuit activity in the motor cortex.

Weaknesses:

While the authors addressed some of the concerns raised by the reviewers, several major limitations still exist in the revised manuscript.

(1) I appreciate the authors now showing more measures of the prehension task (total reaches, success reaches/min, and success ratio) and providing more details on the task design. However, it is unclear why the authors chose a task design that is somewhat different from the commonly used approach. Here they increase the distance of the food pellet each session and are thus making the task increasingly harder, whereas commonly the target distance is kept stable (See 10.1038/nature08389 for example). The result is that important readouts of learning (e. g. success rate) thus remain stable, making it impossible to judge if learning has occurred, without a control group of non-trained mice. This makes it impossible to judge if the task is affected by increased Mα2 cell excitability, since there is no reference of how these measurements are supposed to change in a mouse that learns or doesn't learn the task.

(2) Regarding the analysis of the calcium imaging data, it is still unclear why the authors cannot report a commonly used dF/F0 or z-score value, as recommended by both reviewers. The authors state the 1 sec time window prior to the prehension cannot be used as a baseline (F0), as there might be preparatory motor activity. In that case an even earlier window (such as -2 to -1sec) or z-scores should be used. The current version relabeling the background subtracted fluorescence signal as dF/F0 is misleading. Relatedly, it is unclear why the authors don't think the 1 sec window before prehension cannot be used as baseline, but at the same time use the difference in calcium activity before and after prehension onset as a cut-off criterion for defining cells as modulated during prehension and including in the analysis.

(3) While the authors have improved their statistical reporting, key information is still missing in several places. For example, no N-numbers are listed in legends for figure 3, and there is no mention of the number of mice for analysis in figures 2 and 3. For clarity, the authors should also include the statistical test performed in the figure legends for any p-values shown in the figure.

Reviewer #2 (Public review):

Summary:

The authors aim to map neurons encoding negative valence at the whole-brain scale in larval zebrafish. Using two-photon light-sheet imaging combined with various aversive stimuli, they visualize and quantify stimulus-evoked neural responses, identify the anatomical locations of responsive neurons, and explore the possibility of genetically accessing Rl neurons that respond preferentially to strongly noxious stimuli.

Strengths:

The major strength of this study lies in its use of two-photon light-sheet imaging, which provides a system-level characterization of neuronal response to aversive stimuli. The authors systematically compare multiple classes of aversive stimuli (heat, electric shock, looming, etc.), showing that strongly threatening stimuli converge on a compact neuronal population in the Rl, supporting the robustness of the finding. Finally, the identification of Tiam2a expression in these neurons provides a potential genetic handle for future functional studies.

Weaknesses:

The main weakness of the study is the lack of causal evidence supporting the functional role of the identified neurons. Without optogenetic, chemogenetic, or ablation experiments, it is difficult to determine whether these neurons are required for or sufficient to encode negative valence. In addition, the study does not include positive-valence or neutral stimuli controls, making it difficult to distinguish whether the observed neural responses reflect valence per se or more general downstream response such as motor output. Finally, the lack of behavioral readouts limits the ability to directly link the identified neural populations to defensive behaviors.

Reviewer #2 (Public review):

Summary:

This paper investigates how a combination of spike-timing-dependent plasticity rules in recurrent spiking networks leads to the spontaneous emergence of repeating neuronal sequences. The authors show that despite the weight distribution reaching a steady state, individual synaptic connections undergo constant turnover with timescales that depend on connection strength. The plasticity rules promote fan-in/out connectivity motifs that appear to support sequence generation.

Strengths:

The question addressed is important and biologically relevant. The most interesting finding of the paper is the coexistence of a stable weight distribution with constant turnover of individual synaptic connections.The simulations seem to be carefully executed.

Weaknesses:

The paper does not make a sufficient attempt to explain why the observed phenomena arise under the specific learning rules employed. There is no theoretical reduction, no analytical argument, and no mechanistic intuition. As it stands, this reads as a descriptive simulation study.

It is never made clear which results reflect robust qualitative phenomena and which are specific to the particular hyperparameter choices of these simulations. Specific percentages and parameter values are reported throughout the main text without justification of their importance or generality.

The finding that sequence composition undergoes continual turnover while the global weight distribution remains stable is interesting, but the authors should more carefully situate this result within the existing theoretical literature on synaptic drift and sequence stability under ongoing plasticity. Several modeling papers have addressed related phenomena, and the novelty of the present contribution relative to this body of work is not clearly established.

Reviewer #2 (Public review):

Summary:

The authors examine the effects of activating the dorsal raphe nucleus serotonergic system using a combination of calcium imaging and optogenetics in freely moving larval zebrafish. Their findings show that optogenetic stimulation induces a state of behavioral quiescence.

They further investigate whether this state corresponds to sleep or reduced motor activity. Analyses of posture and sleep-related paradigms indicate that serotonergic activation primarily suppresses motor output rather than promoting sleep. Notably, this suppression appears to be bout type-dependent, with stronger effects on neurons associated with larger tail amplitudes and turning angles.

In addition, auditory stimulation experiments reveal no significant impact of serotonin on sound encoding.

Strengths:

The study combines advanced experimental techniques with state-of-the-art analytical methods, enabling precise and compelling insights into the role of serotonergic modulation. The experiments and analyses are well aligned with the questions being addressed, and the results appear robust and reliable.

Moreover, the implementation of experiments that combine calcium imaging and optogenetics in freely moving animals is technically challenging and appears well justified in the context of the research questions.

Weaknesses:

While the analytical techniques employed are sophisticated and appear to be appropriately applied, their presentation makes the manuscript difficult to follow. Although the explanations are provided in the Methods section, including more guidance in the main text, such as how to interpret each analytical approach and what outcomes would be expected under different scenarios, would help readers who are less familiar with these techniques.

Providing this context would better guide the reader in navigating the figures, broaden the accessibility of the work, and ultimately increase its impact.

While the authors discuss different quiescent states mediated by serotonin reported in previous studies, their interpretation is limited to stating that "a common feature shared by these distinct behavioral states is a pronounced reduction in movement," and consequently proposing that activation of dorsal raphe nucleus is not sufficient to specify a particular behavioral state, but rather plays a primary role in driving motor suppression.

In my view, a more thorough attempt to determine whether the observed state corresponds to any of the previously described forms of quiescence, or represents a subset or variant of them, would strengthen the manuscript. This would help better integrate the findings with the existing literature.

For example, given that the authors have access to whole-brain activity data, it would be valuable to examine and discuss whether there are shared patterns of activation with previously reported quiescent states.

The manuscript largely avoids discussing the mechanisms underlying the observed motor suppression. For instance, is this effect driven directly by serotonin release onto target neurons? Is it mediated by glial activity, as suggested in other studies? Are additional neuromodulatory systems being recruited?

While addressing these questions may require substantial further work, potentially beyond the scope of the present study, the availability of whole-brain data provides an opportunity to at least explore or discuss these possibilities. In particular, it would be interesting to examine the recruitment of regions not directly stimulated but known to be associated with other neuromodulatory systems or promoting glial activation (e.g., the locus coeruleus).

Reviewer #2 (Public review):

Summary:

This paper nicely demonstrates that "speech tracking" in the auditory cortex extends all the way up to 100Hz-150Hz. Specifically, the study asks whether the fluctuations in sound amplitude found in speech at various time scales relate to fluctuations found in similar time scales in intracranial recordings in auditory brain areas. First, it analyzes amplitude fluctuations in speech of 17 different languages, and characterizes fluctuations due to syllabic rate (2-6Hz), vocalic features (30-50 Hz), and fundamental frequency (100-150 Hz, in male speakers). It then analyzes whether neural activity occurs while listening to male and female speakers in French. By measuring changes in power spectrum relative to rest, it links the sound amplitude fluctuations to fluctuations in neural activity in the same frequency bands, referring to them as "theta", "low-gamma", and "high-gamma". Using Grange "causality," it clearly shows that the neural fluctuations can be predicted linearly from the sound fluctuations. Using a cross-frequency coupling measure, they further show that, in the neural dynamic, high-gamma fluctuations precede theta fluctuations.

Strengths:

(1) Analysis of neural activity (Figure 2 is a very compelling account of how theta, low, and high gamma observed in neural recordings closely follow the properties of the acoustic speech signal itself.

(2) This includes phase amplitude coupling, a property that I had not previously seen described for the speech signal itself, and is here nicely demonstrated in Figure 1.

(3) The Grange "causality" analysis makes a compelling case that neural fluctuations in these frequency bands are driven by the stimulus itself.

(4) The finding in Figure 4 that female fundamental emerges at half the frequency in the neural activity is, to my knowledge, an entirely novel observation, not just in speech but in amplitude modulated sounds in general. This non-linear phenomenon is very interesting and prompts a host of interesting questions for future research: Does this happen only for voiced speech, does it depend on the harmonic stack of speech, or can it be produced with a single AM frequency? Are there preferred frequencies for this phenomenon?

(5) The cross-frequency coupling measure shows a number of directed effects in the neural signal which seem to counter the predominant view in neuroscience, namely, that the phase of the slower fluctuations "organize" or "drive" the faster fluctuations seen in power, e.g. theta→gamma coupling, which here is seen to be reversed as gamma→ theta coupling, and this is not a property of sound itself. This, too, should lead to a number of follow-up studies (although there are some potential confounds here).

Weaknesses:

(1) The claim that different frequency bands are processed in different locations, referred to in the abstract as "multiplexing" is less well supported. The neural analysis is performed on independent components that are spatially distributed, making this claim less transparent than it could be, with other, more direct ways of treating electrode location, such as bipolar referencing.

(2) The writing in the Introduction and Results section obscures the source of sound amplitude fluctuations at different timescales. Instead, it treats these fluctuations as some sort of discovery. This is strange because the abstract and discussions are fairly accurate on this point - namely, they are all due to well-known properties of speech. The descriptions are accurate, although I would put it slightly differently: fluctuations below 6Hz are due to varying length of sentences and words, 25Hz-50Hz are well-established stationary times of the vocal tract, and 100-150Hz are the vibration of the vocal cords in male speakers.

(3) The problem of guiding the analysis of sound by notions from neural signals is most glaring when they restrict their analysis to less than 150Hz, which leaves out female-voiced speech.

(4) Along with this, there is a heavy emphasis on notions of "rhythms" and "oscillations" when clearly, aside from the vocal cords, there is no evidence for rhythmic fluctuations. Any reasonable definition of a rhythm would need at least 2 or 3 cycles of a repeated pattern. A spectral "peak" for the sound envelope is shown at 5Hz. But this is not indicative of a regular rhythm. Instead, the peak appears to be an artifact of displaying power per octave rather than power spectral density. A peak in a power per octave is not a reliable indicator of a coherent oscillation, and the speech envelope does not exhibit a clear 5Hz rhythm. Unfortunately, prior literature has not been clear on this. It would be more accurate if the word "rhythm" were replaced with "fluctuation" and/or "activity" for the case of speech envelope and neural activity, respectively.

(5) The Introduction also omits the literature on neural responses to amplitude-modulated sounds that go up at least to 200Hz and more. So the findings here on "high-gamma" are well in line with prior literature.

(6) The fact that neural analysis was cut off at 150Hz to me is a missed opportunity to test if neural speech tracking goes all the way up to 200Hz of the typical female fundamental.

(7) The gamma→theta effects reported here could be confounded by a simple longer delay in the analysis of theta. In fact, Figure S5 confirms that delay. It is unclear whether the CFD metric captures anything more than a temporal delay between the two signals. The term "functionally interconnected" in the abstract is a bit of a stretch; it may be essentially delayed correlation.

(8) There is a minor concern with the claim that low-gamma drives theta amplitude. While statistics on this are reported, the corresponding figure may be suggesting an alpha-harmonic instead of theta (Figure 5c).

Reviewer #2 (Public review):

Summary:

This study combines EEG with frequency-tagging and broadband stimulation paradigms to investigate the developmental precursors of brain rhythms in 8-month-old human infants. The manuscript employs state-of-the-art methods, focusing on theta and alpha rhythms to assess their functional significance in visual information processing.

By evaluating responses to visual stimulation at different frequencies and broadband stimulation presented simultaneously with sounds, the authors report a stimulation frequency-independent response at ~4 Hz. They interpret this as the precursor of the adult alpha rhythm involved in perceptual echo mechanisms. However, I have a number of questions regarding the hypotheses, experimental framework, and analytical approach that need to be addressed before confirming the conclusions.

Strengths:

(1) The analyses are innovative, and the frequency-tagging paradigm is particularly well-suited for studying challenging populations with short protocols.

(2) The sample size is adequate.

Weaknesses:

There is a gap between the hypotheses and the experimental paradigm, as well as between the hypotheses and the analytical choices. These gaps could alter the interpretation of the findings and thus require clarification (or perhaps a reformulation of the theoretical framework).

I am not convinced that the conclusion - that the theta rhythm is the functional precursor of the alpha rhythm in the infant visual system - holds without addressing the following questions.

In brief, my specific concerns are the following:

(1) Gap Between Hypotheses and Experimental Paradigm:

The experimental paradigm involves the simultaneous presentation of sound and image, i.e., cross-modal sensory information, which contrasts with the manuscript's theoretical framework and conclusions, all of which are grounded in visual information processing. Previous work has shown that preverbal infants spontaneously engage in cross-modal associative learning in such audiovisual paradigms (e.g., Kabdebon et al., 2019). This raises the question of whether the paradigm taps into different mechanisms - such as associative learning - rather than those hypothesized, and whether these mechanisms might better explain the observed 4 Hz response. Associative learning mechanisms are particularly relevant to theta rhythm, involving hippocampal learning and the engagement of wider networks, including frontal areas.

Given this cross-modal design, I question whether it might alter the interpretation of the paradigm and the conclusions drawn. The current framing of the manuscript suggests that theta/4 Hz is the functional equivalent of the alpha rhythm for visual processing in the 8-month-old brain. However, the use of multisensory input complicates this conclusion for the visual domain and the parallel to adult mechanisms.

Kabdebon, C., & Dehaene-Lambertz, G. (2019). Symbolic labeling in 5-month-old human infants. Proceedings of the National Academy of Sciences, 116(12), 5805-5810.

(2) Analytical Focus - Gap Between Hypothesis and Analysis Choices:

The link between the literature described in the introduction and the hypothesis of a 4 Hz inherent rhythm in the visual system remains unclear. This puzzles me as to why the analyses focused on 4 Hz and a control band that is not adapted to the infant population. The focus of the analyses on 4 Hz (and the control band analyses) overlooks the critical frequency range (~6-8 Hz), which other studies have suggested may serve as proxies for the adult alpha rhythm. This omission does not align with the hypotheses regarding the role of the alpha rhythm in visual information processing.

The introduction discusses both alpha rhythm and its significance in perceptual echo phenomena, and theta rhythm and its role in mnemonic function, but these remain as separate phenomena. While the paradigm aims to assess perceptual echo phenomena in infants, one would expect the hypothesis to relate to precursors of the alpha rhythm in infancy (slower frequencies, yet related to alpha, ~6 Hz; Stroganova et al., 1999). However, the authors hypothesize that theta rhythm (4 Hz) is a precursor of the alpha rhythm in infancy: "Given the prominence of the theta rhythm in infancy, we expected the presence of a 4 Hz theta response and resonant activity in the infant visual system upon periodic stimulation and broadband visual input, respectively."

Why did the authors not study the 6-9 Hz frequency range, which previous work suggests may serve as a proxy for alpha in infants? Currently, the analyses are restricted to the theta range (i.e., 4 Hz) and a control band (adult-classical alpha range [8-14 Hz]), but [8-14 Hz] is not adapted to the infant population. At this age, prior work has reported ~6 Hz as the age-adapted range corresponding to alpha. It would be more appropriate to investigate this range. I can see some trace of this in Figure 2a, but perhaps this is weaker compared to the 4 Hz stimulation due to the cross-modal nature of the paradigm.

Stroganova, T. A., Orekhova, E. V., & Posikera, I. N. (1999). EEG alpha rhythm in infants. Clinical Neurophysiology, 110(6), 997-1012.

In the adult results, we also see similar ("two types of") responses: the main response at 8 Hz, which to me is the upper band of the theta rhythm (related to cross-modal learning), and traces around 10 Hz, which are more in line with perceptual echo mechanisms. The cited literature in adults (VanRullen & Macdonald, 2012), on which the authors base their framework and analysis, indicates a response at 10 Hz (not 8 Hz). This supports the idea that the 8 Hz response observed in this work might be related to the cross-modal presentation of stimuli. The authors could evaluate this more easily through a control group of adults with an unimodal (visual-only) presentation of stimuli.

(3) Methodological Approach and Clarity:

The methodological approach is not sufficiently detailed, which is crucial for reproducibility and wider contribution, especially given the difficulties in studying infants. Key points requiring clarification include preprocessing, choice of electrode clusters, and statistical details.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigate high-frequency oscillations (HFOs) in the prefrontal cortex during REM sleep. They identify a specific pattern where these HFOs occur in "chains" that are phase-locked to theta oscillations, primarily during the "phasic" periods of REM. The study contrasts these events with isolated HFOs and NREM ripples, suggesting a unique role for these chains in coordinating activity between the prefrontal cortex and the hippocampus. Most notably, the authors report that a specific subset of hippocampal cells-those that co-fire with the prefrontal cortex during these HFOs-increase their firing rates over the course of sleep, suggesting a potential mechanism for selective memory consolidation.

Strengths:

The study addresses an under-explored area of sleep physiology: the fine-grained temporal coordination between the cortex and hippocampus during REM sleep. The identification of HFO "chains" and their association with higher theta power provides an interesting framework for understanding how the brain might organize information transfer outside of NREM sleep. The observation that specific hippocampal populations show differential firing rate changes based on their participation in these HFO events is a striking finding that warrants further investigation.

Weaknesses:

The primary weakness of the study lies in the lack of a clear distinction between global brain states and the specific events being analyzed. Because the authors compare HFOs across different sleep stages (NREM, tonic REM, and phasic REM) without sufficient controls, it is difficult to determine if the observed differences are intrinsic to the HFOs themselves or simply a reflection of the different physiological states in which they occur.

Furthermore, the evidence for "structured reactivation" is not yet convincing. The temporal alignment of these reactivation events appears inconsistent, with peaks occurring well before the HFO itself, and the analysis does not sufficiently control for pre-existing cellular assembly strengths. Additionally, some of the sleep architecture presented appears atypical, such as very short REM bouts and direct NREM-to-REM transitions that bypass standard progression, raising questions about the consistency of the sleep detection across animals. Finally, the study does not account for potential confounds like baseline firing rates when interpreting the behavior of "high-cofiring" neurons, which may simply be the most active cells in the population.

Reviewer #2 (Public review):

Summary:

The work of Harada and collaborators fills an important gap in our knowledge of neuronal identities in the adult hagfish brain. There is essentially no modern, cell-type-level characterisation of neuronal identity in the hagfish brain yet. Existing data are limited to classical neuroanatomy (e.g. Nieuwenhuys) and sparse transmitter/gene-expression studies, mostly in embryos (e.g. work from the Kuratani lab). This study reveals a very broad peculiar pattern of dopaminergic identities and a strikingly unusual pattern of serotonergic transmission, with serotonergic cell bodies present in the telencephalon, which is uncommon for vertebrates and contrasts with previous reports (e.g., Kadota, 1991).

Strengths:

The three-dimensional reconstruction of the brain, including the ventricular system, is novel and very useful. Most of the neurotransmitter identity patterns presented here have not been previously described, and those that were published earlier, such as the serotonergic system (e.g. Kadota, Nieuwenhuys, Wicht), are old and would clearly benefit from re-evaluation using more modern approaches.

Weaknesses:

Neurotransmitter identities are highly relevant for interpreting the possible presence of LGE/MGE territories in hagfish (e.g. GABAergic patterns), for characterising the raphe nuclei (e.g. serotonergic system), and for refining our understanding of the central prosencephalic complex in relation to other vertebrate brain architectures. However, the authors do not address these points and overlook recent evidence from the amphioxus brain that could help interpret their results in an evolutionary context. Overall, the results are insufficiently discussed in relation to the current state of the art.

The study would clearly benefit from complementary gene expression profiling to place these neurotransmitter patterns within a broader framework of brain partitions, to enable more direct comparisons with other vertebrates, and, importantly, to interpret them in relation to the prosomeric model. Furthermore, the work lacks appropriate controls for the in situ hybridization experiments; Datx2 does not show any expression, so there is currently no evidence that this probe is functional. Including such controls would also strengthen the overall description of the dopaminergic system, especially given that the expression patterns of the different genes analysed appear very diffuse and somewhat random.

Reviewer #2 (Public review):

Summary and strengths:

The authors present a description of their online tool to estimate real-world performance of predictive models. The authors bring together different calculations to make better-informed implementation choices. It is a very nice tool to go from effect sizes to base rates to decision curve analysis. The paper describes the background and use of the tool with examples and seems like an extended version of their online how-to. The methods themselves are not new, but I think the tool will be valuable for researchers from different fields. Tools already exist for the conversion of effect sizes (my current favorite is https://www.escal.site/), but I haven't seen measurement noise being incorporated previously. The main benefit is the evaluation of performance under different real-world scenarios. Code is available on GitHub, and the manuscript is well-written.

Weaknesses:

While comprehensive explanation and examples are important for correct use of the tool, I don't really see the added value above their online how-to guide, as the software itself has already been published (Karvelis, P. and Diaconescu, A. O. (2025b). E2p simulator: An interactive tool for estimating real world predictive utility of research findings. Journal of Open Source Software, 10(114):8334.)

Reviewer #3 (Public review):

Xiaoyu Wu and colleagues examined a potential role in sleep of a Drosophila ribosomal RNA methyltransferase, mettl5. Based on sleep defects reported in CRISPR generated mutants, the authors performed both RNA-seq and Ribo-seq analyses of head tissue from mutants and compared to control animals collected at the same time point. A major conclusion was that the mutant showed altered expression of circadian clock genes, and that the altered expression of the period gene in particular accounted for the sleep defect reported in the mettl5 mutant. In this revision, the authors have added a more thorough analysis of clock gene expression and show that PER protein levels are increased relative to wild type animals a specific times of day, indicating increased stability of the protein. Given that PER inhibits its own transcription, the per RNA is low in the mutants. The revised manuscript included efforts toward a more detailed understanding of how clock gene expression was altered in the mutants, as well as other clarification of sleep phenotypes.

Comments on revisions:

All critiques have been addressed by the authors; the manuscript is much improved from its original submission. Thank you.

Reviewer #2 (Public review):

Summary:

This study investigates the role of MATR3 in oocyte development and folliculogenesis using conditional knockout mouse models together with in vitro follicle culture and molecular analyses. The authors aim to determine whether MATR3 regulates oocyte maturation and follicle development and to explore potential mechanisms linking MATR3 function to transcriptional and epigenetic regulation in growing oocytes.

Strengths:

A major strength of the work is the use of a conditional knockout mouse model combined with complementary in vitro follicle culture approaches, which together provide a useful framework for examining gene function during oocyte development. The study also attempts to integrate cellular phenotypes with molecular analyses of transcriptional activity and epigenetic markers.

Weaknesses:

Several weaknesses limit the strength of the conclusions. These include insufficient validation of key experimental manipulations (such as the efficiency of MATR3 knockdown in siRNA experiments), limited quantification or statistical analysis for some datasets, inconsistencies between the text and presented data in certain figures, and incomplete methodological descriptions that make it difficult to fully evaluate reproducibility.

Reviewer #2 (Public review):

In this manuscript, the authors dissect how Gβγ potentiates PLCβ3 signaling in cells. Using engineered crosslinking to stabilize a Gβγ-PLCβ3 complex, single particle cryo-EM, and cell-based functional assays, they identify and map multiple putative Gβγ interaction surfaces on PLCβ3, including a previously unrecognized binding mode. Structure-guided mutagenesis supports the functional relevance of these interactions and suggests that Gβγ potentiation is not primarily mediated by PLCβ3 membrane recruitment, but instead enhances PLCβ3 activity after the lipase is already at the membrane.

Previous reconstitution work on the membrane surface (Falzone & MacKinnon, 2023) proposed a recruitment/partitioning-centric model in which Gβγ increases PLCβ3 output largely by elevating its membrane surface concentration, whereas Gαq primarily increases catalytic turnover; under those reconstitution conditions, the two inputs can combine approximately multiplicatively. In receptor-driven cellular signaling, however, PLCβ3 is robustly recruited to the plasma membrane upon Gαq activation, which raises the question of whether Gβγ contributes mainly through additional recruitment or through a post-recruitment mechanism once PLCβ3 is already at the membrane.

This manuscript helps address that gap by using membrane-anchored PLCβ3 and complementary cellular readouts to separate "getting PLCβ3 to the membrane" from "boosting activity once PLCβ3 is already there." Their results argue that, in cells, membrane recruitment is largely dominated by Gαq·GTP, while Gβγ can further potentiate PIP2 hydrolysis after membrane association, consistent with a modulatory role at the membrane rather than primary recruitment.

Overall, the work provides a structural and mechanistic framework for Gβγ-PLCβ3 cooperation and helps clarify the basis of Gq pathway amplification. The manuscript is generally strong, but some issues need to be addressed.

Major comments:

(1) BMOE/BM(PEG)2 crosslinking may enforce a non-native docking geometry, potentially compromising the physiological relevance and precision of the Gβγ-PLCβ3 interface as described. Although a >50% 1:1 crosslinked complex is formed and remains active, the solution maps show lower local resolution for Gβγ, consistent with a dynamic, potentially heterogeneous, interface. One interface is captured via a single engineered cysteine pair (PLCβ3 E60C-Gβ C271), which could potentially bias the pose. It would be helpful if the authors could provide additional orthogonal support (e.g., alternative crosslinked sites) and bolster the clarification of its uniqueness and relevance.

(2) In the crosslinked structure, the authors report that GβD228 interacts with PLCβ3 R199 and K183. In Figure 2A, R199 appears closer to Gβ D228 than K183, yet only K183 is functionally tested. Testing R199 (e.g., R199E/R199A) would strengthen the structure-guided validation of this interface.

(3) The mutagenesis strategy appears inconsistent across figures/assays, which makes it difficult to interpret phenotypes and directly link the functional data to the proposed interfaces. For example, in Figure 2E, we see R185L but R215E, while residue L40 is mutated to Gly in the IP accumulation assays but to Glu/Lys (L40E/K) in the BRET assays (Figures 3B/3D/3F). The authors should (i) clearly justify the rationale for each substitution (conservative vs charge-reversal, interface disruption, etc.) and (ii), where possible, test the same mutants across assays (or provide evidence that alternative substitutions yield consistent conclusions).

Reviewer #2 (Public review):

Summary:

In this manuscript, Samuel Wagner and colleagues describe an elegant mechanism to prevent promiscuous assembly of a core virulence type III secretion system protein, SctS. Starting from a bioinformatic standpoint, they demonstrate that synteny is highly conserved, and sctT occurs immediately downstream of sctS. Secretion is greatly reduced when sctT is removed or scrambled from its genomic context, and sctT expression is accordingly reduced (sctS synteny is also important, though less so). The distance between sctS and sctT is crucial. An elegant series of genetic experiments leads the authors to pinpoint a stem loop structure that occludes the Shine-Dalgarno sequence of sctT. This property is independent of the actual gene preceding sctT. In sum, this means that SctS is already expressed before SctT is expressed, preventing SctT from forming cytotoxic homooligomers.

Strengths:

The manuscript is very well-written, easy to follow, and describes a substantial amount of genetic detective work to identify the underlying mechanism. I have only a number of textual suggestions, mainly for the Introduction text, which I believe could be revised for a flagellar and broader audience.

Weaknesses:

Major concern:

While the work is rigorous and substantial, I am unsure as to whether its findings will appeal beyond a niche audience.

Minor points:

(1) Line 117: The number here seems to be very small. RefSeq has ~200,000 genomes. My guess is that at least 100,000 of these will be bacterial. Many (most?) bacteria have flagella, and some unflagellated strains have injectisomes, meaning I would have guessed that the authors would have ~50,000 genomes with SctRSTU. This estimate is error-prone, but not by too much. Can the authors explain the discrepancy between my estimate and their figure of almost two orders of magnitude? (SctRSTU/FliPQGFlhB should also be easy to pick up by sequence searches, so I don't think this is due to false negatives).

(2) Discussion: I would appreciate some discussion of how species that do not conserve the synteny of sctS and sctT prevent problems of sctT oligomerisation? It doesn't need to be evidence-based at this stage, but I'm sure the authors have thought about this, and the Discussion is an appropriate place to share their speculations.

Reviewer #2 (Public review):

Summary:

The current work investigates the evolution of sideward locomotion in Brachyura in light of a single evolutionary origin. To this end, the authors first analysed the mode of locomotion in 50 crab species and observed mutually exclusive presence of sideways vs. forward movement. The phylogenetic analysis confirmed that there is indeed a single evolutionary origin for sideways movement, which was sometimes followed by several reversions to forward locomotion. This way, authors demonstrate how locomotor movement modes shape evolutionary diversification in animals by showing that species richness is much higher in side-ways-moving crabs than in the nearest groups. This is an interesting work that integrates behavioural analysis and phylogenetic relations, capitalising largely on crabs. I have a few suggestions and questions.

Firstly, I think the paper spends too much time on a straightforward analysis of the mode of locomotion. I was also wondering whether the phylogenetic analysis could be simply achieved by maximising an objective function in which the modes of movement are inversely coded for two putative groups, with all values calculated at all possible nodes.

Unfortunately, I find that the authors did not sufficiently discuss differences in the ecological niches of species with forward vs. sideways locomotion modes (including challenges of locomotion and substrate).

Likewise, what are the anatomic correlates of forward vs. sideways locomotion? For instance, how are the advantages assumed for sideways movement associated with a flattened body? Is it possible that the mode of motion is secondary to flattened/narrow body structure, which basically limits the distance between legs and thus makes the forward movement difficult - under this logic, the mode of movement would be a secondary phenomenon to body shape traits. How can one differentiate between this alternative and the one that puts the mode of movement in the centre of the story? On a related note, how do different modes of movement relate to the ability to fit into tight spaces - how does it relate to differences in leg joints?

Is it possible that the sideways movement maximises the scanned visual field per unit time/displacement, which may be beneficial for mostly forward-moving predators?

It is really difficult to decipher the information contained in the nodes (circles) in the printed black-and-white version of the manuscript.

Briefly, although I find the study interesting, the presented complexity may not be necessary given the endpoints; it can be achieved much more simply. Furthermore, the degree to which the conceptual analysis of different modes of locomotion was exercised was limited. The general approach may serve as a good model for the evolutionary analysis of other traits. The demonstration of traceability of the relations in question is a major contribution of the work.

Strengths:

The research question and the novel combination of different data types.

Weaknesses:

The complexity of the methods used, along with a limited discussion of the potential dynamics that may underlie the evolution of the sideways movement mode.

运行一次 452 个任务的评测，GPT-5.4 花费 1110 美元，Claude Opus 4.6 花费 1055 美元——每个任务平均约 2.3 美元。而 Gemini 3 Flash 只需要 596 美元，实现了 27.7% 的成绩（vs 顶级模型的 33.3%）。这个性价比数据对 AI 选型决策极为关键：如果业务场景可以接受 27% 而非 33% 的成功率，Gemini 3 Flash 能节省近一半成本。在金融服务的大规模部署中，这个差异将被放大数千倍。

Reviewer #2 (Public review):

Summary:

The goal of this study was to investigate the degree to which low-level stimulus features (i.e., grating orientation) are processed in V1 when stimuli are not consciously perceived under conditions of continuous flash suppression (CFS). The authors measured the activity of a population of V1 neurons at single neuron resolution in awake fixating monkeys while they viewed dichoptic stimuli that consisted of an oriented grating presented to one eye and a noise stimulus to the other eye. Under such conditions, the mask stimulus can prevent conscious perception of the grating stimulus. By measuring the activity of neurons (with Ca2+ imaging) that preferred one or the other eye, the authors tested the degree of orientation processing that occurs during CFS.

Strengths:

The greatest strength of this study is the spatial resolution of the measurement and the ability to quantify stimulus representations during CSF in populations of neurons preferring the eye stimulated by either the grating or the mask. There have been a number of prominent fMRI studies of CFS, but all of them have had the limitation of pooling responses across neurons preferring either eye, effectively measuring the summed response across ocular dominance columns. The ability to isolate separate populations offers an exciting opportunity to study the precise neural mechanisms that give rise to CFS, and potentially provide insights into nonconscious stimulus processing.

Weaknesses:

(The authors have now included a computational model in the second revision.)

Reviewer #2 (Public review):

Summary:

Stanojcic et al. investigate the origins of DNA replication in the unicellular parasite Trypanosoma brucei. They perform two experiments, stranded SNS-seq and DNA molecular combing. Further, they integrate various publicly available datasets, such as G4-seq and DRIP-seq, into their extensive analysis. Using this data, they elucidate the structure of origins of replications. In particular, they find various properties located at or around origins, such as polynucleotide stretches, G-quadruplex structures, regions of low and high nucleosome occupancy, R-loops, and that origins are mostly present in intergenic regions. Combining their population-level SNS-seq and their single-molecule DNA molecular combing data, they elucidate the total number of origins as well as the number of origins active in a single cell.

Between the initial submission and this revision, the raised major concerns have not been resolved, and no additional validation has been provided.

Strengths:

(1) A very strong part of this manuscript is that the authors integrate several other datasets and investigate a large number of properties around origins of replication. Data analysis clearly shows the enrichment of various properties at the origins, and the manuscript is concluded with a very well-presented model that clearly explains the authors' understanding and interpretation of the data.

(2) The DNA combing experiment is an excellent orthogonal approach to the SNS-seq data. The authors used the different properties of the two experiments (one giving location information, one giving single-molecule information) well to extract information and contrast the experiments.

(3) The discussion is exemplary, as the authors openly discuss the strengths and weaknesses of the approaches used. Further, the discussion serves its purpose of putting the results in both an evolutionary and a trypanosome-focused context.

Weaknesses:

I have major concerns about the origin of replication sites determined from the SNS-seq data. As a caveat, I want to state that, before reading this manuscript, SNS-seq was unknown to me; hence, some of my concerns might be misplaced.

(1) There are substantial discrepancies between the origins identified here and those reported in previous studies. Given that the other studies precede this manuscript, it is the authors' duty to investigate these differences. A conclusion should be reached on why the results are different, e.g., by orthogonally validating origins absent in the previous studies.

(2) I am concerned that up to 96% percent of all SNS-seq peaks are filtered away. If there is so much noise in the data, how can one be sure that the peaks that remain are real? Upon request, the authors have performed a control, where randomly placed peaks were run through the same filtering process. Only approximately twice as many experimental peaks passed filtering compared to random peaks. While the authors emphasize reproducibility between replicates, technical artifacts from the protocol would also be reproducible. Moreover, in other SNS-seq studies, for example, Pratto et al. Cell 2021, Fig. 1B, + and − strand peaks always appear closely paired. This pattern contrasts strongly with Fig. 2A in this manuscript.

Further, I have some minor concerns that do not affect the main conclusions of the manuscript:

- Fig 2C: The regions shown in the heatmap have different sizes, and I presume that the regions are ordered by size on the y-axis? If so, does the cone-shaped pattern, which is origin-less for genic regions and origin-enriched for intergenic regions, arise from the size of the regions? (I.e., for each genic region, the region itself is origin-less and the flanking intergenic regions contain origins.) If this is the case, then the peaks/valleys, centered exactly on the center of the regions on the mean frequency plots, arise from the different sizes of the analyzed regions, not from the fact that origins are mostly found at the center of intergenic regions. This data would be better presented with all regions stretched to the same size. This has not been addressed in the revision.

- Line 123, "and the average length of origins was found to be approximately 150 bp.": To determine origins, the authors filter away overlapping peaks and peaks that are too far from each other. Both restrict the minimal and maximal length of origins that can be observed, and this, in turn, affects the average length. This has not been addressed in the revision.

Are claims well substantiated?:<br /> The identification of origins via SNS-seq appears to be incompletely supported to me.<br /> All downstream analyses depend on the reliability of origin identification.

Impact:<br /> This study has the potential to be valuable for two fields: In research focused on T. brucei as a disease agent, where essential processes that function differently than in mammals are excellent drug targets. Further, this study would impact basic research analyzing DNA replication over the evolutionary tree, where T. brucei can be used as an early-divergent eucaryotic model organism.

Reviewer #2 (Public review):

Summary:

The authors propose that leftover heparin plasma can serve as a source for cfDNA extraction, which could then be used for downstream genomic analyses such as methylation profiling, CNV detection, metagenomics, and fragmentomics. While the study is potentially of interest, several major limitations reduce its impact; for example, the study does not adequately address key methodological concerns, particularly cfDNA degradation, sequencing depth limitations, statistical rigor, and the breadth of relevant applications.

Strengths:

The paper provides a cheap method to extract cfDNA, which has broad application if the method is solid.

Weaknesses:

(1) The introduction lacks a sufficient review of prior work. The authors do not adequately summarize existing studies on cfDNA extraction, particularly those comparing heparin plasma and EDTA plasma. This omission weakens the rationale for their study and overlooks important context.

(2) The evaluation of cfDNA degradation from heparin plasma is incomplete. The authors did not compare cfDNA integrity with that extracted from EDTA plasma under realistic sample handling conditions. Their analysis (lines 90-93) focuses only on immediate extraction, which is not representative of clinical workflows where delays are common. This is in direct conflict with findings from Barra et al. (2025, LabMed), who showed that cfDNA from heparin plasma is substantially more degraded than that from EDTA plasma. A systematic comparison of cfDNA yields and fragment sizes under delayed extraction conditions would be necessary to validate the feasibility of their proposed approach.

(3) The comparison of methylation profiles suffers from the same limitation. The authors do not account for cfDNA degradation and the resulting reduced input material, which in turn affects sequencing depth and data quality. As shown by Barra et al., quantifying cfDNA yield and displaying these data in a figure would strengthen the analysis. Moreover, the statistical method applied is inappropriate: the authors use Pearson correlation when Spearman correlation would be more robust to outliers and thus more suitable for methylation and other genomic comparisons.

(4) The CNV analysis also raises concerns. With low-coverage WGS (~5X) from heparin-derived cfDNA, only large CNVs (>100 kb) are reliably detectable. The authors used a 500 kb bin size for CNV calling, but they did not acknowledge this as a limitation. Evaluating CNV detection at multiple bin sizes (e.g., 1 kb, 10 kb, 50 kb, 100 kb, 250 kb) would provide a more complete picture. In addition, Figure 3 presents CNV results from only one sample, which risks bias. Similar bias would exist for illustrations of CNVs from other samples in the supplementary figures provided by the authors. Again, Spearman correlation should be applied in Figure 3c, where clear outliers are visible.

(5) It is important to point out that depth-based CNV calling is just one of the CNV calling methods. Other CNV calling software using SNVs, pair-reads, split-reads, and coverage depth for calling CNV, such as the software Conserting, would be severely affected by the low-quality WGS data. The authors need to evaluate at least two different software with specific algorithms for CNV calling based on current WGS data.

(6) The authors omit an important application of cfDNA: somatic mutation detection. Degraded cfDNA and reduced sequencing depth could substantially impact SNV calling accuracy in terms of both recall and precision. Assessing this aspect with their current dataset would provide a more comprehensive evaluation of heparin plasma-derived cfDNA for genomic analyses.

Comments on revisions:

As suggested previously, the Pearson correlation analysis tends to be overstated; please replace it with Spearman correlation in the whole manuscript. Currently, the authors include both of them in the abstract, method, results, and graphics, all of which are required to be updated to only use Spearman correlation results.

I don't have other concerns about the manuscript.

Reviewer #2 (Public review):

Summary:

This manuscript reports the high-resolution cryo-EM structures of the endogenous TolC-YbjP-AcrABZ complex and a TolC-YbjP subcomplex from E. coli, identifying a novel accessory subunit. This work is an impressive effort that provides valuable structural insights into this native complex.

Strengths:

(1) The study successfully determines the structure of the complete, endogenously purified complex, marking a significant achievement.<br /> (2) The identification of a previously unknown accessory subunit is an important finding.<br /> (3) The use of cryo-EM to resolve the complex, including potential post-translational modifications such as N-palmitoyl and S-diacylglycerol, is a notable highlight.

Weaknesses:

(1) Clarity and Interpretation: Several points need clarification. Additionally, the description of the sample preparation method, which is a key strength, is currently misplaced and should be introduced earlier.<br /> (2) Data Presentation: The manuscript would benefit significantly from improved figures.<br /> (3) Supporting Evidence: The inclusion of the protein purification profile as a supplementary figure is essential. Furthermore, a discussion comparing the endogenous AcrB structure to those obtained in other systems (e.g., liposomes) and commenting on observed lipid densities would strengthen the overall analysis.

Comments on revisions:

In the revision, all my concerns have been addressed.

Reviewer #2 (Public review):

Summary:

The authors are investigating cerebellar-mediated motor behaviors in a large sample of adults, including 30 individuals over the age of 80 (a great strength of this work). They employed a large battery of motor tasks that are tied to cerebellar function, in addition to a cognitive task and motor tasks that are more general. They also evaluated cerebellar structure. Across their behavioral metrics, they found that even with cerebellar degeneration, cerebellar-mediated motor behavior remained intact relative to young adults. However, this was not the case for measures not directly tied to cerebellar function. The authors suggest that these functions are preserved and speak to the resiliency and redundancy of function in the cerebellum. They also speculate that cerebellar circuits may be especially good for preserving function in the face of structural change. The tasks are described very well, and their implementation is also well-done with consideration for rigor in the data collection and processing. The inclusion of Bayesian estimates is also particularly useful, given the theoretically important lack of age differences reported. This work is methodologically rigorous with respect to the behavior, and certainly thought-provoking.

Strengths:

The methodological rigor, inclusion of Bayesian statistics, and the larger sample of individuals over the age of 80 in particular are all great strengths of this work. Further, as noted in the text, the fact that all participants completed the full testing battery is of great benefit. Please note, upon my second review the strengths remain. This is a really wonderful investigation and amazingly comprehensive from a behavioral perspective given the numerous tasks and domains that were considered.

Weaknesses:

The suggestion of cerebellar reserve, given that at the group level there is a lack of difference for cerebellar specific behavioral component,s could be more robustly tested. That is, the authors suggest that this is a reserve given that volume of cerebellar gray matter is smaller in the two older groups, though behavior is preserved. This implies volume and behavior are seemingly dissociated. However, there is seemingly a great deal of behavioral variability within each group and likewise with respect to cerebellar volume. Is poorer behavior associated with smaller volume? If so, this would suggest still that volume and behavior are linked; but, rather than being age that is critical it is volume. On the flip side, a lack of associations between behavior and volume would be quite compelling with respect to reserve. More generally, as explicated in the recommendations, there are analyses that could be conducted that, in my opinio,n would more robustly support their arguments given the data that they have available.

The authors have done wonderful work to address the comments from the initial feedback/reviews. While I may ultimately disagree with the approach of including the imaging data in another manuscript, that is at the same time, a reasonable decision. This, however, does not change the impression that the paper would be stronger with the inclusion of the volumetric imaging data. I can understand why it may be published separately - it would be a very long paper to include both. At the same time the assertions made here, which are largely nicely supported by the preprint, would ultimately strengthen this work. The behavior certainly stands on its own as an excellent and needed investigation; together, both pieces make for a truly excellent contribution to the literature.

Reviewer #2 (Public review):

Summary:

The authors assemble a variety of information from biochemical experiments on oligomeric and higher-order assembly of the spore coat protein SafA, which functions as a hub in spore coat development. Together, the data indicate a robust process of assembly, guided initially by an organized process of disulfide bond formation and ultimately leading to cross-linking by the enzyme Tgl. Interestingly, neither process is strictly necessary for the formation of highly assembled oligomeric forms of SafA, but instead, these processes are mutually supportive in creating a strong, intercrosslinked assembly. Given this lead-up, it is somewhat disappointing to find that the cross-linking defective SafA mutants do not exhibit any obvious defects in sporulation in vivo, and one is left with the conclusion that this stage of spore coat assembly is accomplished by multiple independent co-occurring activities. The information is sufficient to support a detailed model for SafA assembly, which is significant in that it helps to explain the process of building a critically important hub-scaffold for spore coat development.

Strengths:

The main body of experiments supports a detailed model for the assembly of SafA monomers into spore coat superstructures. This is interesting because it shows how a protein can be used as both a scaffold and a hub in contributing to the assembly of a super-resilient biological material.

Weaknesses:

(1) The weak sporulation phenotype of the crosslinking mutants diminishes the significance of the mechanism that is described.

(2) The narrative flow of the originally submitted manuscript could be improved by removing some unnecessary and confusing figures on peripheral subjects and rearranging some of the latter figures to arrive at a conclusion that focuses more on SafA assembly.

(3) The original manuscript appears to have a labeling error in the supplementary figures, but a correctly labeled version of the figures would not support one of the manuscript's claims.

Reviewer #2 (Public review):

Frangos et al present a set of studies aiming to determine mechanisms underlying initiation and tumour progression. Overall, this work provides some useful datasets, further establishing mitochondrial dysfunction during the cellular transformation process.

A key strength is the coordinated analysis of transcriptomics and proteomics from tumour samples derived from a Neu-dependent mouse model for breast cancer. This analysis provides rigorous datasets that show robust patterns, including down-regulation across many components of mitochondrial OXPHOS that were generally consistent at both the mRNA and protein level. Parallel analysis of corresponding tumour samples thereby clearly shows the opposite trend of increased mitochondrial function, which is unexpected. As such, this work further establishes altered mitochondrial phenotypes in tumour contexts and further illustrates that mitochondrial function is not necessarily always tightly correlated with mitochondrial gene expression patterns.

Several key weaknesses remain. It remains unclear how increased mitochondrial function is being sustained despite wide decreases in mRNA and protein levels of OXPHOS components. In terms of mechanism, the study confirmed that pharmacologic EGFR inhibition decreases OXPHOS in a EGFR-dependent breast cancer line. However, it remains unclear if the cell culture system recapitulates other key observations of the tumour model (namely decreased expression with increased function).

Therefore, the mechanistic basis of increased mitochondrial function in light of decreased mitochondrial content remains speculative, as does the role of these changes for tumour initiation or progression.

Comments on revisions:

We agree with the overall findings of the study and appreciate that the claims in text and title have been appropriately toned down.

As additional suggestions eg for presentation, many of the graphics/labels are still too small to be useful. It would be interesting to see if this cell line is similar to the tumours in terms of all the phenotypes. The lapatinib experiment was good. I wonder how quick this drug affects the mitochondria. Also it would be interesting to see if these cells have higher OXPHOS than other non-transformed breast epithelial cells.

The WB on oxphos components is good with ab110413 but this looks like many subunits are detected so this should be made clear.

Reviewer #2 (Public review):

Summary:

The authors generated a zebrafish mutant of the pdgfrb gene. The presented analyses and data confirm previous studies demonstrating that Pdgfrb signaling is necessary for mural cell development in zebrafish. In addition, the data support previously published studies in zebrafish showing that mural cell deficiency leads to hemorrhages later in life. The authors presented quantified data on vessel density and branching, assessed tracer extravasation, and investigated the vasculature of adult mice using electron microscopy.

Strengths:

The strength of this article is that it provides independent confirmation of the important role of Pdgfrb signaling for the development of mural cells in the zebrafish brain. In addition, it confirms previous literature on zebrafish that provides evidence that, in the absence of pericytes/VSMC, hemorrhages appear (Wang et al, 2014, PMID: 24306108 and Ando et al 2021, PMID: 3431092)".

The Reviewing Editor has carefully reviewed the revised manuscript and is fully satisfied with the authors' revisions.

Reviewer #4 (Public review):

Summary:

In this study, He et al. investigate the mechanisms underlying Kupffer cell (KC) loss during metabolic stress. It has long been observed that embryonically derived KCs decline in obesity and liver disease, a loss that is compensated by monocyte recruitment, although the underlying mechanisms remain unclear. The authors propose that metabolic reprogramming, particularly excessive glycolysis, drives KC death. Using an original murine genetic model to modulate glycolysis, they further demonstrate that enhanced glycolytic activity exacerbates KC damage.

Strengths:

Overall, the study is extremely clearly presented, with a convincing and simple message destined to a vast audience.

Weaknesses:

This manuscript has already undergone one round of revisions in which I was not involved. The authors have tried to address several points raised by the previous reviewers, notably regarding the unexpectedly high level of TUNEL staining observed in KCs. However, I share these concerns expressed by the three reviewers that the reported levels remain difficult to reconcile with the biology. A TUNEL positivity rate of ~60% at week 16 of the HFHC diet would imply massive KC death, which should have led to a near-complete depletion of the KC population, something that is not observed. While I agree that the KC compartment is clearly affected under this dietary challenge, I would strongly encourage the authors to carefully rule out potential technical biases that could account for this implausibly high rate of cell death.

Considering the new in-vivo experiment with 2-DG, it is definitely convincing and clearly adds some value to the full study.

So the full story deserves publication.

Reviewer #2 (Public review):

Summary:

In the present manuscript, Cordeiro et al. show that α-mangostin, a xanthone obtained from the fruit of the Garcinia mangostana tree, behaves as an agonist of the BK channels. The authors arrive at this conclusion by examining the effects of mangostin on macroscopic and single-channel currents elicited by BK channels formed by the α subunit and α + β1 subunits, as well as αβ1 channels coexpressed with voltage-dependent Ca2+ (CaV1,2) channels. The single-channel experiments show that α-mangostin produces a robust increase in the probability of opening without affecting the single-channel conductance. The authors contend that α-mangostin activation of the BK channel is state-independent, and molecular docking and mutagenesis suggest that α-mangostin binds to a site in the internal cavity. Importantly, α-mangostin (10 μM) alleviates noradrenaline-induced contracture. Mangostin is ineffective if the contracted muscles are pretreated with the BK toxin iberiotoxin.

In this revised version of the manuscript by Cordeiro et al., the authors have adequately answered my previous concerns. However, as I stated in my comments, without determining the probability of opening across a wide range of voltages, any conclusion about the drug's mechanism of action can be questioned. For example, the statement in Discussion line 481: "The higher shift observed in 1 μM Cai 2+ may reflect the steep Cai2+-dependence of the closed-open equilibrium (Cui, Cox and Aldrich, 1997) and the allosteric coupling of voltage and Cai2+ signals (Horrigan and Aldrich, 2002; Magleby, 2003; Clay, 2017), which are effective in this concentration range, which may lead to a higher apparent activation when voltage activation is facilitated by Cai 2+ (Sun and Horrigan, 2022)." has no support in the data and is not predicted by the allosteric model. In order to have a larger shift induced by the drug in the presence of Ca2+, you need either to alter the Ca2+ binding or the allosteric coupling factor C.<br /> Please note that in the manuscript, there are several problems with the English in this sentence.

Minor

In Figure 1E, BKa should read BKalpha.

Reviewer #2 (Public review):

Summary:

In this manuscript, "Cryo-EM structure of the bicarbonate receptor GPR30," the authors aimed to enrich our understanding of the role of GPR30 in pH homeostasis by combining structural analysis with a receptor function assay. This work is a natural development and extension of their previous work on Nature Communications (PMID: 38413581). In the current body of work, they solved the cryo-EM structure of the human GPR30-G-protein (mini-Gsqi) complex in the presence of bicarbonate ions at 3.15 Å resolution. From the atomic model built based on this map, they observed the overall canonical architecture of class A GPCR and also identified 3 extracellular pockets created by ECLs (Pockets A-C). Based on the polarity, location, size, and charge of each pocket, the authors hypothesized that pocket A is a good candidate for the bicarbonate binding site. To identify the bicarbonate binding site, the authors performed an exhaustive mutant analysis of the hydrophilic residues in Pocket A and analyzed receptor reactivity via calcium assay. In addition, the human GPR30-G-protein complex model also enabled the authors to elucidate the G-protein coupling mechanism of this special class A GPCR, which plays a crucial role in pH homeostasis.

Strengths:

As a continuation of their recent Nature Communications publication, the authors used cryo-EM coupled with mutagenesis and functional studies to elucidate bicarbonate-GPR30 interaction. This work provided atomic-resolution structural observations for the receptor in complex with G-protein, allowing us to explore its mechanism of action, and will further facilitate drug development targeting GPR30. There were 3 extracellular pockets created by ECLs (Pockets A-C). The authors were able to filter out 2 of them and hypothesized that pocket A was a good candidate for the bicarbonate binding site based on the polarity, location, and charge of each pocket. From there, the authors identified the key residues on GPR30 for its interaction with the substrate, bicarbonate. Together with their previous work, they mapped out amino acids that are critical for receptor reactivity.

Weaknesses:

When we see a reduction of a GPCR-mediated downstream signaling, several factors could potentially contribute to this observation: 1) a reduced total expression of this receptor due to the mutation (transcription and translation issue); 2) a reduced surface expression of this receptor due to the mutation (trafficking issue); and 3) a dysfunctional receptor that doesn't signal due to the mutation.

Altogether, the wide range of surface expression across the different cell lines, combined with the different receptor function readouts, makes the cell functional data only partially support their structural observations.

Reviewer #2 (Public review):

Summary:

Hurst et al. developed a new Tol2-based transgenesis system, ImPaqT, an Immunological toolkit for PaqCl-based Golden Gate Assembly of Tol2 Transgenes, to facilitate the production of transgenic zebrafish lines. This Golden Gate assembly-based approach relies on only a short 4-base-pair overhang sequence in the final construct, and the insertion construct and backbone vector can be assembled in a single-tube reaction using PaqCl and a ligase. This approach can also be expandable by introducing new overhang sequences while maintaining compatibility with existing ImPaqT constructs, allowing users to add fragments as needed.

The generation of several transgenic zebrafish lines for immunological studies demonstrates the feasibility of the ImPaqT in vivo. Lineage tracing of macrophages via LPS injection demonstrates the approach's functionality and validates its use in vivo.

Comments on revisions:

The authors have addressed all my concerns.

Reviewer #2 (Public review):

The authors aim to investigate the ability of evolution to create strong transcription factor binding sites (TFBSs) de novo in E. coli. They focus on three global transcriptional regulators: CRP, Fis, and IHF, using a massively parallel reporter assay to evaluate the regulatory effects of over 30,000 TFBS variants. By analyzing the resulting genotype-phenotype landscapes, they explore the ruggedness, accessibility, and evolutionary dynamics of regulatory landscapes, providing insights into the evolutionary feasibility of strong gene regulation. Their experiments show that de novo adaptive evolution of new gene regulation is feasible. It is also subject to a blend of chance, historical contingency, and evolutionary biases that favor some peaks and evolutionary paths.

(1) Strengths of the methods and results:

The authors successfully employed a well-designed sort-seq assay combined with high-throughput sequencing to map regulatory landscapes. The experimental design ensures reliable measurement of regulation strengths. Their system accounts for gene expression noise and normalizes measurements using appropriate controls.

Comprehensive Landscape Mapping:<br /> The study examines ~30,000 TFBS variants per transcription factor, providing statistically robust and thorough maps of the regulatory landscapes for CRP, Fis, and IHF. The landscapes are rigorously analyzed for ruggedness (e.g., number of peaks) and epistasis, revealing parallels with theoretical uncorrelated random landscapes.

Evolutionary Dynamics Simulations:<br /> Through simulations of adaptive walks under varying population dynamics, the authors demonstrate that high peaks in regulatory landscapes are accessible despite ruggedness. They identify key evolutionary phenomena, such as contingency (multiple paths to peaks) and biases toward specific evolutionary outcomes.

Biological Relevance and Novelty:<br /> The author's work is novel in focusing on global regulators, which differ from previously studied local regulators (e.g., TetR). They provide compelling evidence that rugged landscapes are navigable, facilitating de novo evolution of regulatory interactions. The comparison of landscapes for CRP, Fis, and IHF underscores shared topographical features, suggesting general principles of global transcriptional regulation in bacteria.

(2) Weaknesses of the methods and results:

Undersampling of Genotype Space:<br /> Approximately 40% of the theoretical TFBS genotype space remains uncharacterized after quality filtering. The authors now discuss this limitation more explicitly and provide analyses suggesting that undersampling does not strongly bias their conclusions at the landscape level. Nevertheless, predictive modeling approaches could further extend these landscapes in future work.

Simplified Regulatory Architecture:<br /> The study considers a minimal system consisting of a single TFBS upstream of a reporter gene. While this simplification allows clean interpretation and high-throughput measurement, natural promoters often involve combinatorial regulation and chromosomal context effects that may alter landscape topography.

Lack of Experimental Evolution Validation:<br /> The evolutionary conclusions are based on simulations rather than direct experimental evolution. The authors provide a reasonable justification for this choice and frame their conclusions at the statistical level rather than for specific trajectories, but experimental validation would be a valuable future extension.

Impact on the Field:<br /> This study advances our understanding of adaptive landscapes in gene regulation and offers a critical step toward deciphering how global regulators evolve de novo binding sites. The findings provide foundational insights for synthetic biology, evolutionary genetics, and systems biology by highlighting the evolutionary accessibility of strong regulation in bacteria.

Utility of Methods and Data:<br /> The sort-seq approach, combined with landscape analysis, provides a robust framework that can be extended to other transcription factors and systems. If made publicly available, the study's data and code would be valuable for researchers modeling transcriptional regulation or studying evolutionary dynamics.

Additional Context:<br /> The study builds on a growing body of work exploring regulatory evolution. For instance, recent studies on local regulators like TetR and AraC have revealed high ruggedness and epistasis in TFBS landscapes. This study distinguishes itself by focusing on global regulators, which are more complex biologically and more influential in bacterial gene networks. The observed evolutionary contingency aligns with findings in other biological systems, such as protein evolution and RNA folding landscapes, underscoring the generality of these evolutionary principles.

Conclusion:<br /> The authors successfully mapped the genotype-phenotype landscapes for three global regulators and simulated evolutionary dynamics to assess the feasibility of strong TFBS evolution. They convincingly demonstrate that ruggedness and epistasis, while prominent, do not preclude the evolution of strong regulation. Their results support the notion that gene regulation evolves through a blend of chance, contingency, and evolutionary biases.

This paper makes a significant contribution to the understanding of regulatory evolution in bacteria. While minor limitations exist, the authors' methods are robust, and their findings are well-supported. The work will likely be of broad interest to researchers in molecular evolution, synthetic biology, and gene regulation.

Reviewer #2 (Public review):

This study uses mass spectrometry to quantify how LPS + IL-4 modify the mouse B cell proteome as naïve cells undergo blastogenesis and enter the cell cycle. This analysis revealed changes in key proteins involved in amino acid transport and cholesterol biosynthesis. Genetic and pharmacological experiments indicated important roles for these metabolic processes in B cell proliferation.

This work provides new information about the regulation of TI B cell responses by changes in cell metabolism and also a comprehensive mass spectrometry dataset which will be an important general resource for future studies. The experiments are thorough and carefully carried out. The majority of conclusions are backed up by data that is shown to be highly significant statistically. The comprehensive mass spectrometry dataset will be an important general resource for future studies.

After revision, the study now includes new data showing that the up regulation of amino acid uptake and cholesterol metabolism is not restricted to LPS + IL-4 (TLR4 + IL4R) stimulation but is also observed after stimulation of TLR7, TLR9, CD40 and the BCR. This increases the impact of this work and shows that this metabolic rewiring is a common feature of B cell activation. The inclusion of inhibitor data showing important roles for MTOR and ERK/p38a MAP kinases in the metabolic changes identified and provides preliminary insights into the mechanisms involved.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors examine vocal communication during same-sex dyadic interactions in mice, comparing periods of physical separation (with limited sensory access) to direct social contact. They report that separation dramatically alters the vocal repertoire, shifting it away from canonical ultrasonic vocalizations (USVs) toward low-frequency vocalizations (LFVs) and broadband "noisy" calls. While LFVs and noisy calls have been described previously, largely in aversive contexts, this study provides a detailed, systematic characterization of these vocalizations during social interactions, thereby extending prior work.

The authors explore several experimental manipulations and analyses, including divider hole size, strain and sex differences, anxiolytic drug treatment, and correlations with spatial proximity, to infer potential functions of these call types. Although the dataset is rich, the results are largely descriptive, and many conclusions remain tentative. Several experimental variables are not fully controlled, and in some cases, the interpretation exceeds what the data can clearly support. Nonetheless, with improved experimental framing, additional analyses of existing data, and a clearer discussion of limitations, this work has the potential to make a valuable contribution by broadening the field's focus beyond USVs to understand a wider vocal repertoire relevant to social context.

Strengths:

Much work on mouse vocal communication focuses almost exclusively on USVs. This manuscript convincingly demonstrates that non-USV vocalizations (LFVs and noisy calls) are prominent and systematically modulated by social context, highlighting an underappreciated dimension of mouse communication. Furthermore, the authors employ several experimental manipulations, including sensory access, strain, sex, and pharmacological treatment, to assess changes in vocalization repertoire. This provides a valuable resource for the field and reveals robust context dependence of vocalization. The discussion is thoughtful and integrative, particularly in its consideration of potential communicative roles of LFVs and noisy calls and their relationship to sensory constraints and signal propagation, although these ideas will require further experimental validation.

Weaknesses:

There are several concerns regarding experimental design and data interpretation that could be addressed to strengthen the manuscript.

(1) The terminology used for vocalization types is confusing and needs better clarification. The authors refer to Grimsley et al. (2016) multiple times, yet they use the same names for their vocalizations while applying different definitions. This makes it very difficult to compare the two papers. Since this study and Grimsley et al. use different mouse strains (FVB vs CBA), a direct comparison of absolute frequencies may also not be appropriate. Please explicitly clarify the definitions of the call types (e.g., frequency range, voiced vs. USV) and explain how they relate to those in the previous study earlier in the manuscript.

(2) In the initial experiment, mice always experience separation first (15 minutes), followed by unification (5 minutes), using novel same-sex dyads. Multiple factors besides physical contact could influence vocalization across this sequence, including habituation to the arena, reduced anxiety over time, or increasing familiarity with the partner despite physical separation. It is unclear whether the authors have tested the reverse order (unification first, followed by separation). If not, this limitation should be explicitly acknowledged. In addition, examining whether vocalizations or behaviors change over the course of the 15-minute separation period, for example, by comparing early vs late phases, could help disentangle effects of habituation from those of physical separation per se.

(3) The conclusion that separation-induced LFVs are unlikely to be anxiety-driven may overinterpret the buspirone experiment (Figure 8). Vehicle injections themselves produced large changes in call rate and call-type distribution, raising concerns about stress or arousal induced by the injection procedure. Comparisons between buspirone-treated animals and untreated animals are therefore problematic, as these groups differ in their experimental histories, including the number of exposures. The manuscript would benefit from independent measures confirming the anxiolytic efficacy of buspirone compared to vehicle injection in this paradigm, such as behavioral readouts of anxiety. In addition, the experimental design requires a clearer description. It is not always clear whether the same dyads were tested twice, or how social familiarity, contextual familiarity, and habituation to injections were handled. Male data comparing first and second exposures should also be included as supplementary figures to allow direct comparison with the excluded female dataset.

(4) The idea that noisy calls function to attract conspecific attention is intriguing. However, in Figure 5, all call types, including LFVs and USVs, are most likely to occur when mice are already in close proximity during separation, which seems inconsistent with a long-distance signaling role. Analyses of the temporal relationship between vocalizations and behavior would strengthen this claim. For example, it would be informative to test whether bouts of noisy calls precede approach behavior or a reduction in inter-animal distance. Examining whether calls occur before, during, or after orientation toward the partner could further clarify whether these vocalizations actively modulate social behavior.

(5) The effects of divider hole size on vocal repertoire are striking but difficult to interpret. Unexpectedly, small holes and no holes yield similar call distributions, whereas large holes produce a markedly different profile dominated by LFVs, which also differs from free interactions. If large holes allow greater tactile or close-range interaction, the reduction in USVs and MFV is counterintuitive. Incorporating behavioral metrics such as distance, orientation, or specific interaction types alongside call classification would greatly aid interpretation and help link vocal output to interaction quality rather than divider type alone.

(6) Throughout the study, vocalizations are pooled across both animals in the dyad. Because the arena is neutral rather than a home cage, either animal could be initiating vocalization. Assigning calls to individuals, where possible, using spatial or acoustic cues, would substantially strengthen functional interpretations. Even limited analyses, e.g., identifying which animal vocalizes first or whether calls precede approach by the partner, could provide important insight into the communicative role of different call types.

Reviewer #2 (Public review):

Summary:

The authors aimed to determine Molidustat targets and the potential utility of these findings. They clearly demonstrate that Molidustat interferes with GSTP1 and some other proteins on top of PHD2. They also demonstrate that PHD2 deletion is not sufficient to recapitulate Molidustat effects in cells and proteomes. Finally, they demonstrate synthetic lethality in organoids for Molidustat and APC deletion.

Strengths:

The data on Molidustat proteomes, GSTP1 binding, inhibition and metabolic health of organoids is really clear. All biochemical, docking and omic data are really strong. The potential impact of these findings could be the use of Molidustat in APC null tumours and awareness of potential off-target effects.

Weaknesses:

A main but minor weakness is that Molidustat also inhibits other PHDs, although these are less expressed. PHD1 has been shown to control the cell cycle and be expressed in the colon, where it is needed for viability. Although this does not explain the lack of effect of other PHD inhibitors, it does warrant some discussion. The use of MTT is not very good to detect viability when it measures metabolism; this also needs to be discussed and perhaps supplemented with colony or cell number measurements.

Reviewer #3 (Public review):

In this paper, the authors revealed that Molidustat can induce a dose-dependent increase in Caspase-3/7 activity in the HT29 cell line, which is an APC-mutant colorectal cancer cell line. More importantly, they found that targeting PHD2 alone cannot cause cell death. By using thermal proteome profiling (TPP) and orthogonal chemical proteomic competition assays, they determined GTSP1 as a previously undiscovered off-target of Molidustat. They also revealed that combined PHD2 and GSTP1 loss leads to an increase in intracellular ROS and apoptosis. Moreover, they evaluated the effects of Molidustat in colonic organoids and showed that Molidustat has a high selectivity for colonic organoids with activated WNT signaling and/or KRAS pathway alterations, and this effect is not reproduced by hydroxylase inhibition alone, providing a new potential approach to targeting both PHD2 and GTSP1 for the treatment of APC-mutant CRC.

Specific comments:

(1) What is the possible molecular mechanism of dual GSTP1/PHD2 loss, inducing cell death?

(2) Can the authors mutate the binding site of Molidustat on GTSP1 to verify the in silico docking results?

(3) Evidence for Molidustat inhibiting PHD2 activity or stabilising HIF-1α should be provided.

Reviewer #3 (Public review):

This study concerns how observers (human participants) detect changes in the statistics of their environment, termed regime shifts. To make this concrete, a series of 10 balls are drawn from an urn that contains mainly red or mainly blue balls. If there is a regime shift, the urn is changed over (from mainly red to mainly blue) at some point in the 10 trials. Participants report their belief that there has been a regime shift as a % probability. Their judgement should (mathematically) depend on the prior probability of a regime shift (which is set at one of three levels) and the strength of evidence (also one of three levels, operationalized as the proportion of red balls in the mostly-blue urn and vice versa). Participants are directly instructed of the prior probability of regime shift and proportion of red balls, which are presented on-screen as numerical probabilities. The task therefore differs from most previous work on this question in that probabilities are instructed rather than learned by observation, and beliefs are reported as numerical probabilities rather than being inferred from participants' choice behaviour (as in many bandit tasks, such as Behrens 2007 Nature Neurosci).

The key behavioural finding is that participants over-estimate the prior probability of regime change when it is low, and under estimate it when it is high; and participants over-estimate the strength of evidence when it is low and under-estimate it when it is high. In other words participants make much less distinction between the different generative environments than an optimal observer would. This is termed 'system neglect'. A neuroeconomic-style mathematical model is presented and fit to data.

Functional MRI results how that strength of evidence for a regime shift (roughly, the surprise associated with a blue ball from an apparently red urn) is associated with activity in the frontal-parietal orienting network. Meanwhile at time-points where the probability of a regime shift is high, there is activity in another network including vmPFC. Both networks show individual differences effects, such that people who were more sensitive to strength of evidence and prior probability show more activity in the frontal-parietal and vmPFC-linked networks respectively.

Strengths

(1) The study provides a different task for looking at change-detection and how this depends on estimates of environmental volatility and sensory evidence strength, in which participants are directly and precisely informed of the environmental volatility and sensory evidence strength rather than inferring them through observation as in most previous studies

(2) Participants directly provide belief estimates as probabilities rather than experimenters inferring them from choice behaviour as in most previous studies

(3) The results are consistent with well-established findings that surprising sensory events activate the frontal-parietal orienting network whilst updating of beliefs about the word ('regime shift') activates vmPFC.

Weaknesses

(1) The use of numerical probabilities (both to describe the environments to participants, and for participants to report their beliefs) may be problematic because people are notoriously bad at interpreting probabilities presented in this way, and show poor ability to reason with this information (see Kahneman's classic work on probabilistic reasoning, and how it can be improved by using natural frequencies). Therefore the fact that, in the present study, people do not fully use this information, or use it inaccurately, may reflect the mode of information delivery.

In the response to this comment the authors have pointed out their own previous work showing that system neglect can occur even when numerical probabilities are not used. This is reassuring but there remains a large body of classic work showing that observers do struggle with conditional probabilities of the type presented in the task,

(2) Although a very precise model of 'system neglect' is presented, many other models could fit the data.

For example, you would get similar effects due to attraction of parameter estimates towards a global mean - essentially application of a hyper-prior in which the parameters applied by each participant in each block are attracted towards the experiment-wise mean values of these parameters. For example, the prior probability of regime shift ground-truth values [0.01, 0.05, 0.10] are mapped to subjective values of [0.037, 0.052, 0.069]; this would occur if observers apply a hyper-prior that the probability of regime shift is about 0.05 (the average value over all blocks). This 'attraction to the mean' is a well-established phenomenon and cannot be ruled out with the current data (I suppose you could rule it out by comparing to another dataset in which the mean ground-truth value was different).

More generally, any model in which participants don't fully use the numerical information they were given would produce apparent 'system neglect'. Four qualitatively different example reasons are: 1. Some individual participants completely ignored the probability values given. 2. Participants did not ignore the probability values given, but combined them with a hyperprior as above. 3. Participants had a reporting bias where their reported beliefs that a regime-change had occurred tend to be shifted towards 50% (rather than reporting 'confident' values such 5% or 95%). 4. Participants underweighted probability outliers resulting in underweighting of evidence in the 'high signal diagnosticity' environment (10.1016/j.neuron.2014.01.020 )

In summary I agree that any model that fits the data would have to capture the idea that participants don't differentiate between the different environments as much as they should, but I think there are a number of qualitatively different reasons why they might do this - of which the above are only examples.

Reviewer #2 (Public review):

Summary

Using mouse embryos early in development, this excellent paper from Prudhomme et al. shows that Vinculin's recruitment to adherens junctions during mammalian cranial neural tube closure is essential for maintaining junctional integrity in response to increased tension during this process. Previous work had shown that during neural tube elevation, planar polarity of Myosin II and mechanical forces in the tissue are increased. Additionally, mouse embryos lacking Vinculin were known to display neural tube closure failure, and mutations in human Vinculin had been associated with increased risk of neural tube defects, but the mechanism remained unclear. Here, the authors utilize a high-throughput embryonic stem cell (ESC)-based pipeline to generate Vinculin-depleted embryos, complemented by a conditional mutant lacking Vinculin in the embryonic lineages, to investigate this question. The authors show that Vinculin is not required for force generation, but Vinculin is recruited to cell-cell junctions in a tension-dependent manner and is needed to transmit actomyosin-mediated tension to junctions - particularly tricellular and higher-order multicellular junctions - so that apical constriction can happen during neural fold elevation. Furthermore, they find that Vinculin is required to maintain adhesion during high force events (e.g., rosette resolution and cell division) during neural tube closure. The research builds on previous studies about Vinculin's role in mechanotransduction at cell-cell junctions carried out in cultured epithelial cells, zebrafish cardiomyocytes, or early Xenopus embryos, and investigates how physiological forces required for mouse neural tube closure challenge junction integrity and the important role that Vinculin plays in maintenance of junction integrity and translation of mechanical forces into changes in tissue structure during this process.

Strengths:

This study stands out for its sophisticated use of laser ablation and live imaging in neurulating mouse embryos, enabling quantification of junctional tension, Vinculin recruitment to multicellular junctions, and assessment of junction integrity during neural tube elevation. The authors' use of both ESC-derived Vinculin mutant embryos complemented by a second conditional mutant of Vinculin convincingly demonstrates that their findings are specific to the loss of Vinculin. Additionally, the authors demonstrated proof-of-principle for their ESC-based pipeline with a Shroom3 mutant known to be important for neural tube closure. The Zallen lab's application of the genetically engineered ESC-derived mouse embryo pipeline to efficiently generate larger numbers of mutant mouse embryos exhibiting neural tube closure defects (compared with traditional genetic crossing strategies) that can be utilized for live imaging and mechanical perturbations like laser ablation will be valuable for future work in the field. The authors show that Vinculin depletion disrupts tricellular and multicellular junctions. Notably, over 75% of higher-order (5+) vertices in Vinculin mutant embryos display gaps, but interestingly, about one third of 5+ cell junctions in Control embryos also display gaps, indicating that transient vertex remodeling events are needed for normal neural tube closure. Overall, this is a well-written paper that places the authors' findings within the context of prior literature; their beautiful data that is robustly analyzed and clear figure presentation will make the authors' exciting findings accessible to readers.

Weaknesses:

The criteria for selection of junctions targeted by laser ablation, including specifics of location, Myosin II intensity, and initial junction length, should be more clearly described in the Methods, especially given the use of different reporter strains (MyoIIB-GFP vs. GFP-Plekha7) across figures, which may influence junction selection for laser ablation. Analysis of Myosin II in Vinculin mutant embryos would benefit from staining for active Myosin II (pMRLC), and further examination of actomyosin organization at different stages of neural fold elevation in controls vs. Vinculin mutants would be informative. Although the authors note that ZO-1 gaps are limited to a subset of vertices where adherens junction gaps are detected, the increased frequency of tight junction gaps in Vinculin mutants could have functional significance that should be noted. Finally, inclusion of schematics to detail how the adherens and tight junction gaps were defined and measured at cell vertices, as well as how cell division completion was defined, would improve transparency and strengthen readers' understanding of how the data were quantified.

Reviewer #2 (Public review):

Summary:

Liang et al. explore an unusual observation of membrane discontinuities in dividing C. elegans embryonic cells. This report is the first to demonstrate that, instead of the classical invagination of membranes during cytokinesis, cells in the early embryos of C. elegans exhibit separation of sister membranes that extend independently. TEM images of high-pressure-frozen samples provide strong evidence for the presence of Membrane Openings (MOs) in cells at various stages of the cell cycle, predominantly during mitosis. High-resolution images (x 30,000) clearly show the wrinkled plasma membrane and smooth MOs.<br /> The electron microscopy data are supported by the live cell imaging of strains with fluorescently tagged membrane markers. This study opens up the possibility of tracking MOs at other stages of C. elegans development, and also asks if it might be a common phenomenon in other species that exhibit rapid embryonic growth and divisions.

Strengths:

(1) Thorough verification of Membrane Openings (MO) by several methods:

(a) 4 independent sample batches.

(b) Examined historical collections.

(c) Analysed embryos at different stages of development. The absence of MOs in later stages (comma) serves as a negative control and gives confidence that MOs are genuine and not technical artifacts.

(2) Live cell imaging of strain with fluorescently labelled membranes provides real-time dynamics of membrane rupture.

(3) After observing the membrane rupture, the next obvious question is - what prevents the cytosol from leaking out? The EM images showing PBL and PEL - extracellular matrix serving as barriers for the cytosol are convincing.

Weakness:

(1) The association of membrane discontinuities with cell division is not convincing, as there are 159 cells out of 425 showing MOs, but it is not mentioned clearly how many of these are undergoing cell division. Also, it's not clear whether the 20 dividing cells analysed for MOs are a part of the 159 cells or a separate dataset. A graphical representation of the number of samples and observed frequencies would be helpful to understand the data collection workflow.

(2) In Figures 3A and 3B, the resolution of the images is not enough to verify 3A as classical membrane invagination and 3B as detached sister membranes.

(3) Figure 6 lacks controls. How does the classical invagination look in this strain? Also, adding nuclear dye would be informative, in order to correlate the nuclear division with membrane rupture, as claimed.

Reviewer #2 (Public review):

Summary:

The study of Pilipenko et al evaluated the role of alpha phase in a visual perception paradigm using the framework of signal detection theory and reverse correlation. Their findings suggest that phase-related modulations in perception are mediated by a reduction in internal noise and a moderate increase in tuning to relevant features of the stimuli in specific phases of the alpha cycle. Interestingly, the alpha phase did not affect the criterion. Criterion was related to modulations in alpha power, in agreement with previous research.

Strengths:

The experiment was carefully designed, and the analytical pipeline is original and suited to answer the research question. The authors frame the research question very well and propose several models that account for the possible mechanisms by which the alpha phase can modulate perception. This study can be very valuable for the ongoing discussion about the role of alpha activity in perception.

Conclusion:

This study addresses an important and timely question and proposes an original and well-thought-out analytical framework to investigate the role of alpha phase in visual perception. While the experimental design and theoretical motivation are strong, the very limited sample size substantially constrains the strength of the conclusions that can be drawn at the group level.

Bibliography:

Button, K., Ioannidis, J., Mokrysz, C. et al. Power failure: why small sample size undermines the reliability of neuroscience. Nat Rev Neurosci 14, 365-376 (2013). https://doi.org/10.1038/nrn3475

Tamar R Makin, Jean-Jacques Orban de Xivry (2019) Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript eLife 8:e48175 https://doi.org/10.7554/eLife.48175

Reviewer #3 (Public review):

In the manuscript titled "Heat Shock Factor Regulation of Antimicrobial Peptides Expression Suggests a Conserved Defense Mechanism Induced by Febrile Temperature in Arthropods", the authors investigate the role of heat shock factor 1 (HSF1) in regulating antimicrobial peptides (AMPs) in response to viral infections, particularly focusing on febrile temperatures. Using shrimp (Litopenaeus vannamei) and Drosophila S2 cells as models, this study shows that HSF1 induces the expression of AMPs, which in turn inhibit viral replication, offering insights into how febrile temperatures enhance immune responses. The study demonstrates that HSF1 binds to heat shock elements (HSE) in AMPs, suggesting a conserved antiviral defense mechanism in arthropods. The findings are informative for understanding innate immunity against viral infections, particularly in aquaculture. However the logical flow of the paper can be improved.

Comments on revisions:

Some aspects of the initial study design, regarding the selection of representative candidate genes and the logical flow, raised concerns. However, these issues have been addressed in the revised manuscript through additional validations and clarifications. Most of my comments and concerns were sufficiently addressed in the revised manuscript. The results support the authors' conclusion that HSF1-dependent regulation of AMP expression contributes to antiviral defense under febrile conditions.

Reviewer #2 (Public review):

In 'Developmental constraints mediate the reversal of temperature effects on the autumn phenology of European beech after the summer solstice', Rebindaine and co-authors report on two experiments on Fagus sylvatica where they manipulated temperatures of saplings between day and night and at different times of year. I think the experiments are interesting, but note that the treatments are extreme compared to natural conditions. Further, given that much of the experiment happened outside, I am not sure how much we can generalize from one year for each experiment, especially when conducted on one population of one species.

Reviewer #2 (Public review):

Summary:

This article addresses a very pertinent question - what are the computational mechanisms underlying risky behaviour in patients having attempted suicide. In particular, it is impressive how the authors find a broad behavioral effect whose mechanisms they can then explain and refine through computational modeling. This work is important because currently, beyond previous suicide attempts, there has been a lack of predictive measures. This study is the first step towards that: understanding the cognition on a group level. Before then being able to include it in future predictive studies (based on the cross-sectional data, this study by itself cannot assess the predictive validity of the measure).

Strengths:

- Large sample size<br /> - Replication of their own findings<br /> - Well-controlled task with measures of behaviour and mood + precise and well-validated computational modeling

Questions, based on revised manuscript and replies to other reviewers:

(1) Replies to reviewers in general: Bayes Factors have been added, it would be good to also use common verbal terms to describe them (e.g. 'anecdotal', 'moderate' etc). For example, my reading of table S8 would be that for gambling rate there is only anecdotal evidence that it does not relate to PSWQ, BDI, and moderate evidence it does not relate to TAI.

(2) Reply to reviewer 1 Q2 (Predicting STB):<br /> For the regression predicting suicidal ideation, it seems to me that what you did was a regression STB ~ gambling behaviour + approach + mood? Could you clarify? I had expected as a test of whether the task can predict STB risk something slightly different - a cross-validation (LOO or maybe 5-fold in the large sample): STB ~ gambling behaviour + approach [parameter from model] + mood [parameter from model]; and then computing in the left out participants: predicted STB. Then checking correlation between STB and predicted STB. This would allow testing whether the diverse task measures together predict STB (with the caveat, that it's cross-validated, rather than hold-out sample, unless you could train on one sample (in lab) and test on the other (online).

(3) Reply to reviewer 2 Q1 (parameter recovery): I'm looking at S3, it seems to still show only the scatter plots and not the correlation matrices, which are now added as text notes. Can you actually show these matrices? An off-diagonal correlation of 0.63 appears quite high. I think it needs to be discussed exactly which parameters those are, and whether that impacts the interpretation of the results.

(4) Reply to reviewer 3 Q3 (mood model): I would have imagined that the response would involve changing the mood equations (equation 8 main text) to include a term for whether the participant gambled or not, independent of the gamble value.

Reviewer #2 (Public review):

Summary:

In this study the authors compared the transcriptomes of the various different types of hair cells contained in the sensory epithelia of the cochlea and vestibular organs of the mouse inner ear. The analysis of their transcriptomic data lead to novel insights into the potential function of the kinocilium.

Strengths:

The novel findings for the kinocilium gene expression along with the demonstration that some kinocilia demonstrate rhythmic beating as would be seen for known motile cilia is fascinating. It is possible that perhaps the kinocilium known to play a very important role in the orientation of the stereocilia, may have a gene expression pattern that is more like a primary cilium early in development and later in mature hair cells more like a motile cilium. Since the kinocilium is retained in vestibular hair cells it makes sense that it is playing a different role in these mature cells than its role in the cochlea.

Another major strength of this study which cannot be overstated is that for the transcriptome analysis they are using mature mice. To date there is a lot of data from many labs for embryonic and neonatal hair cells but very little transcriptomic data on the mature hair cells. They do a nice job in presenting the differences in marker gene expression between the 4 hair cell types. This information is very useful to those labs studying regeneration or generation of hair cells from ES cell cultures. One of the biggest questions these labs confront is what type of hair cell develop in these systems. The more markers available the better. These data will also allow researchers in the field to compare developing hair cells with mature hair cell to see what genes are only required during development and not in later functioning hair cells.

Comments on revision:

I am satisfied with the revision, the authors made an effort to incorporate the changes requested.

Reviewer #2 (Public review):

Summary:

The question of how caloric and taste information interact and consolidate remains both active and highly relevant to human health and cognition. The authors of this work sought to understand how nutrient sensing of glucose modulates sweet sensation. They found that glucose intake activates hugin signaling to AstA neurons to suppress feeding, which contributes to our mechanistic understanding of nutrient sensation. They did this by leveraging the genetic tools of Drosophila to carry out nuanced experimental manipulations, and confirmed the conservation of their main mechanism in a mammalian model. This work builds on previous studies examining sugar taste and caloric sensing, enhancing the resolution of our understanding.

Strengths:

Fully discovering neural circuits that connect body state with perception remains central to understanding homeostasis and behavior. This study expands our understanding of sugar sensing, providing mechanistic evidence for a hugin/AstA circuit that is responsive to sugar intake and suppresses feeding. In addition to effectively leveraging the genetic tools of Drosophila, this study further extends their findings into a mammalian model with the discovery that NMU neural signaling is also responsive to sugar intake.

Weaknesses:

The effect of Glut1 knockdown on PER in hugin neurons is modest in both fed and starved flies, suggesting that glucose intake through Glut1 may only be part of the mechanism. The authors address this in their discussion.

Reviewer #3 (Public review):

The DNA and RNA binding protein TDP-43 has been pathologically implicated in a number of neurodegenerative diseases including ALS, FTD, and AD. Normally residing in the nucleus, in TDP-43 proteinopathies, TDP-43 mislocalizes to the cytoplasm where it is found in cytoplasmic aggregates. It is thought that both loss of nuclear function and cytoplasmic gain of toxic function are contributors to disease pathogenesis in TDP-43 proteinopathies. Recent studies have demonstrated that depletion of nuclear TDP-43 leads to loss of its nuclear function characterized by changes in gene expression and splicing of target mRNAs. However, to date, most readouts of TDP-43 loss of function events are dependent upon PCR based assays for single mRNA targets. Thus, reliable and robust assays for detection of global changes in TDP-43 splicing events are lacking. In this manuscript, Xie, Merjane, Bergmann and colleagues describe a biosensor that reports on TDP-43 splicing function in real time. Overall, this is a well-described unique resource that would be of high interest and utility to a number of researchers validated in multiple cell types as a sensitive readout of TDP-43 loss of function. Future studies validating the utility of this biosensor in models of TDP-43 loss of function (e.g. disease iPSNs) that do not rely on TDP-43 knockdown will be of further interest.

Reviewer #2 (Public review):

Summary:

The paper proposes a workflow to accelerate EM connectomics by combining multi-scale imaging with image processing and analysis (image alignment, registration, neuron tracing, automated segmentation and synapse prediction, proof-reading) to derive a brain region connectome. The paper argues and (partially) demonstrates that this approach facilitates comparative connectomics.

The data acquisition pipeline uses a well-established sample preparation protocol, uCT guided acquisition, and SBEM imaging at cellular and synaptic resolution.

Data processing and analysis combine existing state-of-the-art components and focus on the alignment and complementary analysis of the two SBEM resolution levels. The paper applies the workflow to the central complex of six different insects and performs some preliminary analysis based on this (which is acceptable for a resource/tool).

Disclaimer for the rest of the review: I am an expert in image analysis and segmentation, so I have mainly focused on these aspects as I am not qualified to analyze the details of image acquisition.

Strengths:

The paper addresses an important problem and promises an acceleration and democratization of comparable connectomics. The time savings of the imaging approach are well-motivated and derived. The methods used for image alignment, segmentation, synapse detection, and proofreading are state-of-the-art.

Weaknesses:

I see two major weaknesses in the paper:

(1) The paper introduces the (approximate) equivalence of the projectome and connectome in the insect brain very prominently in the introduction and uses this as a central motivation for the multi-resolution image acquisition protocol. But - to me - it is unclear how this principle is really used in the analysis presented in the last results and if this assumption is evaluated at all. Specifically, Figure 4 a shows the anatomical neuron reconstructions (from cellular resolution SBEM), d-g show connectome-level analysis from the synaptic resolution data. The only link I can see between the two is that the neural processes in the synapse-resolution data can be mapped to the neurons from the cellular resolution data, thanks to the image alignment. This is certainly important, BUT it is only tangentially related to the projectome vs. connectome claim from the introduction. This claim implies that a tentative connectome is derived from projectome-level data (e.g. by assuming a uniform probability of synapse-formation given surface or distance between projections) that is then validated by the "true" connectome data from synaptic resolution. Instead, what is actually solved - to my understanding - is mapping the local connectome to the projectome. While related, these are different things and the current framing of the paper and the quite brief description of the section on comparative connectomics (also no corresponding Methods section) make this claim inadequately supported.

(2) Reporting on segmentation and proofreading is purely qualitative. Given that this is claimed as a core contribution of the paper (e.g. statement in line 497 and following), I would expect substantially more reporting and evaluation of this claim:<br /> a) Report the actual time needed for proofreading the segmentations in CAVE. I could not find any numbers on this.<br /> b) Report the initial segmentation quality of the model: How many errors does it make? Note: There is a brief mention of VoI-based quantification in Methods (around line 1060), but the results are not reported.

What should be done: Report the error rates (with an accurate measure such as skeleton VoI) independently for all 6 volumes. Given that the authors have the proofread versions, this is feasible. Only then can the claims be made here be evaluated. Note that the F1-score of synapse prediction is quantified. This is a good starting point, but could also be extended to further species in order to assess the actual transferability. Furthermore, none of the data from the study seems to be available. The training data of the network has to be made available. If possible, high-resolution data should be proofread too.

Further points:

(1) Why isn't reconstruction at the cellular level addressed with ML? This is surely possible and should be easier than the full connectome analysis. Similar to before, the actual times needed for tracing with CATMAID are not reported; the manuscript only states that this can be done in minutes for a neuron, but it's unclear if this is the best or average case. It would help to have quantitative numbers to assess whether automation would bring any benefits.

(2) Finally, regarding the underlying software. I did not try this myself due to time constraints, but did check the repositories. They seem to be in an ok state with some documentation in a README. However, given the central role of the software contribution, I would expect a centralized doc page that explains how to use the different parts of the software, including a full example with sample data. Without this, application by other labs - a central claim - will be difficult.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors test the hypothesis that whole-brain functional magnetic resonance imaging in behaving mice, coupled with reinforcement-learning modeling, can dissociate neural substrates of initial cue-reward acquisition versus contingency reversal, and potentially reveal underappreciated contributors to cognitive flexibility. Using a head-fixed go/no-go odor discrimination task with subsequent rule reversal in a subset of mice, they model trial-by-trial state-action values with a model-free Q-learning algorithm (hierarchical Bayesian fit) and use the model-derived decision variable as a parametric regressor in whole-brain analyses. They report that acquisition-related signals prominently involve ventral and dorsal striatal regions, whereas reversal learning additionally recruits the periaqueductal gray (negative correlation with the decision variable) and shows an apparent double dissociation between nucleus accumbens and periaqueductal gray responses for hit versus correct-rejection outcomes during reversal.

Strengths:

(1) The reversal manipulation is implemented without explicit punishment, targeting suppression of previously rewarded actions under reward omission - an underexplored regime for midbrain contributions beyond canonical threat/pain framing.

(2) The manuscript provides a credible MR-compatible olfactory/licking platform with synchronized sniff/lick/valve/reward timing and high-field imaging, supporting feasibility and broader utility for mesoscale systems neuroscience in rodents.

(3) Trial-by-trial value estimates from a Q-learning variant are fit via hierarchical Bayesian inference and explicitly integrated into subject-level general linear models with a mouse hemodynamic response function, which is appropriate for leveraging within-subject dynamics in small-N rodent fMRI.

(4) The decision-variable maps during acquisition recover expected basal ganglia involvement (including nucleus accumbens and dorsal striatum), providing face validity; the reversal-stage map yields an interpretable set of cortical/striatal/pallidal regions plus periaqueductal gray/hippocampus.

(5) The finite impulse response analysis stratified by behavioral outcomes (hit, false alarm, correct rejection, miss) adds interpretability beyond the model regressor alone, and the reported crossover interaction between nucleus accumbens and periaqueductal gray is potentially impactful if robust.

Weaknesses:

(1) The core claim regarding selective periaqueductal gray engagement rests on a subset of n = 6 mice for reversal. With permutation-based whole-brain inference and very small cluster sizes, the robustness of the periaqueductal gray effect to reasonable analytic perturbations is not yet convincing. I would suggest providing leave-one-animal-out analyses for the periaqueductal gray cluster/ROI effects and reporting how often the key findings survive.

(2) The authors note that due to temporal resolution and hemodynamics, they cannot separate stimulus, choice, and feedback and therefore model "whole trials." This limitation creates ambiguity about whether periaqueductal gray signals reflect value updating, action inhibition (no-lick), reward omission, autonomic arousal, or motor preparation/withholding, especially given the strong hit versus correct-rejection opponency. I would suggest adding targeted analyses that disambiguate "withholding" from "reversal-related updating".

(3) ROIs are defined from the whole-brain decision-variable maps and then interrogated by outcome types; the manuscript acknowledges non-independence. This can inflate apparent dissociations. It would be better if the authors define ROIs independently (anatomical periaqueductal gray/nucleus accumbens masks, or split-half ROI definition with held-out data) and repeat the key ROI conclusions.

(4) The reversal group is a subset of the acquisition cohort and also experiences a different task phase structure and additional sessions; the paper attempts to address exposure differences descriptively. I would suggest that the authors formally test whether periaqueductal gray effects are explained by session count, time-in-scanner, or learning rate differences (e.g., include these as covariates, or match sessions more strictly).

(5) The platform records sniffing and licking, but the imaging models described include motion, global, and ventricle regressors and do not clearly include trialwise lick/sniff covariates. Given the periaqueductal gray's known autonomic and defensive coordination roles, physiological state confounding is a major concern. Could the authors incorporate sniff and lick metrics (and their derivatives) as nuisance regressors and show whether the periaqueductal gray effects persist?

Reviewer #2 (Public review):

Summary:

The JAK-STAT pathway (JSP) exhibits cell-type-specific functional heterogeneity in breast cancer. This study investigates the JSP in breast cancer and its response to anti-PD‑1 immunotherapy. JSP displays distinct cell‑type heterogeneity: it promotes malignant phenotypes and immunosuppression in tumor cells, while enhancing cytotoxicity and reducing exhaustion in T cells. Elevated JSP expression correlates with improved immunotherapy responses, especially in triple‑negative breast cancer. These findings highlight the paradoxical roles of JSP, indicating that broad inhibition may compromise anti‑tumor immunity.

Strengths:

The major strengths of this study include the comprehensive characterization of JSP heterogeneity across epithelial, tumor, and T cells in breast cancer. The identification of JSP and STAT4 as predictive biomarkers for immunotherapy response, particularly in triple‑negative breast cancer, provides clinically relevant insights for patient stratification.

Weaknesses:

The findings rely heavily on public dataset analyses.

Reviewer #2 (Public review):

Summary:

Bhattacharya and colleagues here use cell culture, single-cell RNA and ATACseq sequencing of such in vitro cultures and of ex vivo isolated B-lineage cells to infer an ontogeny for extra-germinal centre B cell differentiation. The manuscript presents a useful potential ontogeny for plasma cells, wherein in vitro cultured naïve human B cells enter a CD30+ intermediate state before moving in subsequent days through a CD44v9+ state before ultimately obtaining a 'mature' antibody-secreting plasma cell phenotype. Ex vivo isolated germinal centre B cells obtain the plasma cell state without expressing CD30 in their development. Phenotype analysis of tonsillar B-lineage cells supports the same phenotype conversion in vivo, although the intermediate cell population was smaller in vivo. The link to CD44v9 expression on developing plasma cells is inferred to be for extra-GC (T-independent) responses, but the data presented leave this equivocal, and the functional importance of developing via a CD30+CD44v9+ intermediate is not investigated.

Strengths:

The article presents a solid potential ontogeny for PC development, wherein some differentiating B cells acquire a CD30+ state, transition through a CD44v9+CD30+ state, then downmodulate CD30 before obtaining canonical CD38+ 'PC' status. A strength is the integration of in vitro cultured B cell results with tonsillar B-lineage cell data sets, and careful flow cytometry of the in vitro cultures over several days to infer lineage. The data provide reasonable support for the concept. CD30+ cells are shown to develop readily from naïve B cells in culture, but uncommonly from GC B cell cultures. A nice piece of data is Figure 6B, which shows reasonably strong correlative changes in phenotype through the assumed ontogeny, and this fits with the expected trajectory of maturation.

Weaknesses:

The most important weakness throughout is the non-absolute nature of the relationship. An example is seen in that the sorted ex vivo GC B cells also give rise to the 'extra-GC' phenotype of plasma cell, suggesting that while the profile is enriched, it is not absolute. There is a further weakness, as while cultures are run for several days, division-associated shifts in PC phenotype are not mapped; such would greatly strengthen the weight of the argument, and show conditional shifts in phenotype associated with division, an uncontrolled parameter in the mix. For example, for the MEF2C A388 inhibition experiments, it would be strong evidence of the pathway/process contributing if a by-division peak increase in CD30+ population was demonstrated in the early days of culture.

There are some basic sort experiments performed (e.g. 3C-3F), which show that the CD30+ cells do give rise to PC preferentially, but what is missing is the step-wise phenotype shifts in these sorted populations, which should support the trajectory shown in Figure 3B and (the in vitro equivalent of) 6B. It would emphatically support the trajectory to show the cellular phenotypes on the PC with sorting based on CD30, CD44v9, CD27, and CD20 expression, and following outcome phenotypes 24-48 hours later, if the inferred maturation trajectory is true.

There are also specific weaknesses with the bioinformatics, in that, while the analyses are likely appropriate, unpresented data is necessarily used to shape the argument. For example, Figure 1C shows bubble plots for two plasma cell sets, yet, of archetypal PC-expressed genes, only IRF4 is demonstrated to confirm they are true PC, and the gene is not universally expressed in cells in the clusters. For this figure, it would help to expand the bubble plot to show J-CHAIN, XBP-1, CIITA and PRDM1 or other appropriate PC demarcating molecules. Similarly, in Fig 2B, more evidence of a bifurcation in state is needed than that CD44v9 distinguishes PC1 from PC2 clusters-this is the stated conclusion, but 2A depicts that 50% of PC1 relatively weakly express CD44, while <25% of PC2 express it. Demonstrating additional molecules or genes distinguishing the clusters would improve veracity. Figure 2F shows clonal lineages, but it would be helpful to see somatic hypermutation burdens and learn if they differ between the demarcated subsets. I also find the pseudotime analyses of limited value, as some of the branches follow trajectories that are unrealistic biologically, so less weight should be placed on the pathways to which they do or do not point (i.e., the notion that GC B cells do or do not give rise to particular PC subsets).

Statistically, some of the experiments are single wells from single donors, so there is a low level of confidence and no reproducibility demonstrated for some aspects of the study, which is a weakness.

Paradoxical to the argument that it is the TI response process being modelled, it is presented that CpG stimulation, plus proxy T cell help (CD40L), drives the CD30+ phenotype best with the addition of the GC-associated cytokine IL-21. This should be carefully considered and discussed.

Overall, in addition to presenting more contextual information from the bioinformatics, the best way to solidify the data set, in my vie,w would be to revisit the hypothesis with two additional experimental approaches: (1) to incorporate division tracing into the ontogeny studies and (2) to perform lineage tracing on sort-purified populations at different stages of the maturation process.

cephalexin, dicloxacillin, penicillin VK, amoxicillin/clavulanate, or clindamycin (for penicillin-allergic patients). [1-2] These beta-lactam antibiotics provide excellent coverage against streptococci and methicillin-susceptible S. aureus (MSSA

endovascular stenting emerging as first-line therapy for rapid symptom relief, while definitive treatment targets the underlying cause

Reviewer #2 (Public review):

Summary:

In this study, the authors took advantage of a semi-intact ex vivo somatosensory preparation that includes hindlimb skin to characterize the response of projection neurons in the dorsal horn of the spinal cord to peripheral stimulation, including cold thermal stimuli. The main aim was to characterize the connectivity between peripheral afferents expressing the cold sensing receptor TRPM8 and a set of genetically tagged neurons of the anterolateral system (ALS). These ALS neurons expressed high levels of the calcium binding protein calbindin 1.

In addition, combining different viral tracing methods, the authors could identify the anatomical targets of this specific subset of projection neurons within the brainstem and diencephalon.

Strengths:

The use of a relatively new (seldom used previously) transgenic line to label TRPM8-expressing afferents, combined with the genetic characterization of a previously identified subset of projections neurons add specificity to the characterization. The transgenic line appears to capture well the subpopulation of Trpm8-expressing neurons.

In addition, the use of electron microscopy techniques makes the interpretation of the structural contacts more compelling

The writing is clear and the presentation of findings follows a logical flow.

Overall, this study provides solid, novel information about the brain circuits involved in cold thermosensation.

Weaknesses:

In the characterization of recorded neurons in close contact or in the absence of this contact with TRPM8 afferents, the number of recordedd neurons is relatively low. In addition, the strength of thermal stimuli is not very well controlled, preventing a more precise characterization of the connectivity.

The authors acknowledge that, technically, this is a very difficult preparation with very low yield as far as obtaining successful recordings. Moreover, the tissue needs to be maintained at room temperature which is obviously not ideal when characterizing cold thermoreceptors due to the unavoidable effects of low temperature on cold-activated receptors.

Reviewer #2 (Public review):

Summary:

In this study, the authors took advantage of a semi-intact ex vivo somatosensory preparation that includes hindlimb skin to characterize the response of projection neurons in the dorsal horn of the spinal cord to peripheral stimulation, including cold thermal stimuli. The main aim was to characterize the connectivity between peripheral afferents expressing the cold-sensing receptor TRPM8 and a set of genetically tagged neurons of the anterolateral system (ALS). These ALS neurons expressed high levels of the calcium-binding protein calbindin 1.

In addition, combining different viral tracing methods, the authors could identify the anatomical targets of this specific subset of projection neurons within the brainstem and diencephalon.

Strengths:

The use of a relatively new (seldom used previously) transgenic line to label TRPM8-expressing afferents, combined with the genetic characterization of a previously identified subset of projection neurons, adds a specificity to the characterization. The transgenic line appears to capture well the subpopulation of Trpm8-expressing neurons

In addition, the use of electron microscopy techniques makes the interpretation of the structural contacts more compelling.

The writing is clear, and the presentation of findings follows a logical flow.

Overall, this study provides solid, novel information about the brain circuits involved in cold thermosensation.

Weaknesses:

In the characterization of recorded neurons in close contact or in the absence of this contact with TRPM8 afferents, the number of recorded neurons is relatively low. In addition, the strength of thermal stimuli is not very well controlled, preventing a more precise characterization of the connectivity.

The authors could provide some sense of the effort needed to record from the 6 cold-activated neurons described. How many preparations were needed, etc?

Reviewer #2 (Public review):

Summary:

In this study, the authors have demonstrated, through a comprehensive approach combining electrophysiology, chemogenetics, fiber photometry, RNA interference, and multiple behavioral tasks, the necessity of projections from CCK+ CAMKIIergic neurons in the hippocampal CA3 region to the CA1 region for regulating spatial memory in mice. Specifically, authors have shown that CA3-CCK CAMKIIergic neurons are selectively activated by novel locations during a spatial memory task. Furthermore, authors have identified the CA3-CA1 pathway as crucial for this spatial working memory function, thereby suggesting a pivotal role for CA3 excitatory CCK neurons in influencing CA1 LTP. The data presented appear to be well-organized and comprehensive.

Strengths:

(1) This work combined various methods to validate the excitatory CCK neurons in the CA3 area; these data are convincing and solid.

(2) This study demonstrated that the CA3-CCK CAMKIIergic neurons are involved in the spatial memory tasks; these are interesting findings, which suggest that these neurons are important targets for manipulating the memory-related diseases.

(3) This manuscript also measured the endogenous CCK from the CA3-CCK CAMKIIergic neurons; this means that CCK can be released under certain conditions.

Weaknesses:

In summary, this work can be formally accepted after the revision. For the limitations of the revision, the distinct neural effects of cholecystokinin (CCK) receptors (CCK-1R, CCK-2R, and CCK-3R) on hippocampal function have not been fully elucidated. Recent studies indicate that CCK-2R can modulate hippocampal activity at CA3-Schaffer collateral synapses; however, the roles of CCK-1R and CCK-3R in hippocampal function remain poorly characterized, with limited experimental evidence supporting their involvement. Overall, this study provides an interesting and novel perspective on the role of excitatory CCK signaling in hippocampus-dependent navigation learning.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the response of the amphibian respiratory rhythm generator under varying excitability conditions. They use pharmacological agents to increase and/ or decrease synaptic excitability and demonstrate the resilience of buccal rhythms under different conditions. They employ these results to formulate their primary thesis, that there is no obligatory locus of the buccal respiratory rhythm in the frog, and that their respiratory rhythmogenic mechanisms should be considered diffuse and anatomically distributed across a larger brainstem region.

Strengths:

This manuscript is well written, with a sufficiently large number of experiments, for which the authors should be congratulated.

Weaknesses:

The presented results don't support the authors' main conclusions, and the interpretation of the data is heavily biased toward their hypothesis. This impregnates an unsubstantiated narrative in the Abstract, Introduction, and Discussion of this manuscript, which must be reexamined with the following points in consideration:

(1) The authors seem to confuse degeneracy with redundancy. For instance, at line 54, they state, "These findings support the broader hypothesis that respiratory rhythm-generating circuits can switch to being diffuse and redundant, with discrete oscillators quickly drowning in a sea of excitations."

Redundancy means having the same component repeated multiple times to buffer the failure of any single component, whereas degeneracy means different functional components that compensate for one another under perturbations (Goaillard and Marder, ARN 2021)

Since the premotor-lung units get converted to buccal units under high excitability, this suggests a degenerate mechanism for respiratory rhythm generation- rather than a redundant mechanism, where there should be multiple buccal units that get recruited under different excitability conditions.

(2) Line 83, "but the essential requirement for a discrete, rudimentary buccal oscillator is also lost".

This statement is not supported by the data presented in this study. How does the expansion of the buccal unit imply that the essential requirement for discreteness is lost? Under increased excitability, does the burst/rhythm initiation zone also expand? Or does it still remain centered around the location of buccal units under physiological conditions? Increased excitability can lead to recruitment of a larger area, without a change in the location of the rhythmogenic kernel.

(3) Line 86, "... oscillators should be viewed as promiscuous flexible functional entities that expand or contract...".

Oscillators can be regarded as promiscuous only if, under physiological conditions, they switch positions. Under high excitability, only the flexibility argument holds, which has been established in mammals before (e.g., CA Del Negro, K Kam, JA Hayes, JL Feldman, The Journal of physiology 587 (6), 1217-1231; CA Del Negro, C Morgado-Valle, JL Feldman,Neuron 34 (5), 821-830; NA Baertsch, LJ Severs, TM Anderson, JM Ramirez, Proceedings of the National Academy of Sciences 116 (15), 7493-7502; NA Baertsch, HC Baertsch, JM Ramirez Nature communications 9 (1), 843).

Results:

(4) Interpretation of data in Figure 6.

How does the Buccal activity and L2 Power stroke change with 60nm AMPA (in CN5)? Does the increase in the Buccal neurons and decrease in power stroke neurons also reflect in the CN5 activity? Also see comments on Figure 9 data below.

(5) Interpretation of data in Figure 7.

Here, classifying buccal neurons solely by spiking may obscure the fact that the 'silent' neurons under baseline conditions were part of the rhythmic network but could not spike due to subthreshold inputs. 60 nM AMPA increased their firing in response to previously subthreshold synchronous inputs during the buccal burst. Intracellular recordings are required to negate this possibility and establish that the neuronal classification is robust.

(6) Interpretation of data in Figure 8.

"Lung units can transform into buccal units under excitation".<br /> CN5 buccal and lung bursts need to be compared before and after AMPA injection. From Figure 8 A-D, it is apparent that the example Unit2's activity increases during the buccal bursts, after AMPA injection. However, they are also present in buccal burst pre-AMPA, albeit with less frequency.

It is striking that the pre-AMPA epoch (panel A) is less than half of the post-AMPA epoch. This would, in itself, lead to a biased estimate of lung units that are active under the baseline condition during the buccal bursts.

Figure 8G, meta-analysis of lung units spiking during the baseline buccal bursts is warranted to interpret the main claim of this figure. Similarly, analysis of spiking per lung burst for the post-AMPA condition is essential for comparing the lung unit's contribution under high excitability.

(7) Interpretation of data in Figure 9

"Buccal area loses importance under increased excitation."

This interpretation is not fully supported by the data presented in this manuscript. Under 60 nm AMPA, does the ratio of lung burst to buccal burst change in CN5? This analysis is crucial for determining whether the lung units are indeed converted into buccal bursts at the expense of lung activity or whether their appearance during buccal bursts is incidental due to increased excitability. In the baseline, there are 4-5 buccal bursts per lung burst, whereas under high excitability, there are 2-3 buccal bursts per lung burst (Figure 9 A-B). This seems inconsistent with the conclusion that increased excitability converts lung units into buccal units (Figures 6 &7).

Could the authors comment on the connectivity between the lung and the buccal units? Results in Figure 9A-B indicate that lung units may receive an efference copy of buccal units, and under high excitability, their spikes may generate negative feedback onto the buccal units, terminating their bursts. This could explain the decrease in the buccal-to-lung burst in high-AMPA conditions. This type of circuit interaction resembles the mammalian breathing CPG, in which the parafacial/RTN (which controls the abdominal muscles) and preBötC (which controls the diaphragm) interact and cross-inhibit each other.

(8) Line 382.

"Buccal-like bursting produced from two independent slices".

The two "independent" slices have portions of the same anatomical kernel, the buccal rhythm generator. This experiment is like the sandwich slice preparation of preBötC (Del Negro Lab), in which two thinner slices exhibit rhythmic activity. Thus, the two slices are not independent; they are anatomically adjacent and functionally overlapping.

Reviewer #2 (Public review):

Summary:

This study by Choucri and Treiber aims to directly address a recent critique regarding the role of transposable elements (TEs) in diversifying the neural transcriptome of Drosophila. The authors seek to demonstrate that TEs are not merely genomic "noise" but are frequently and reliably "exonized" into brain-specific mRNA. By introducing an upgraded computational pipeline, TEChim, and conducting precise experimental validations, the authors set out to show that TE-mediated splicing represents a genuine biological phenomenon that expands the molecular repertoire of the nervous system.

Strengths:

The study's primary strength lies in its rigorous technical "forensic" analysis of previous failed replication attempts. The authors convincingly demonstrate that the lack of signal in the opposing study stemmed from a fundamental methodological mismatch: the software used by the critics (TIDAL) is logically incapable of detecting splice sites located within TE sequences. Importantly, the authors complement this computational clarification with definitive experimental evidence through an effective "experimental rescue." By employing correctly designed primers and matching the genetic backgrounds of the fly strains, thereby accounting for genomic polymorphisms, they successfully validated all seven loci that were previously reported as undetectable. This dual-pronged strategy, addressing both algorithmic bias and experimental design, establishes a more robust technical benchmark for the detection and validation of TE-derived exons in neural tissues.

Weaknesses:

While the technical rebuttal is highly convincing, the scope of the study remains primarily defensive. As a response to a prior critique, the work focuses on establishing the existence and detectability of chimeric TE-derived transcripts rather than exploring their broader functional consequences. As a result, there is limited new insight into how these TE-modified isoforms influence neural circuit function or organismal behavior. In addition, the detection and validation of these events remain technically demanding, requiring deep sequencing and specialized bioinformatic expertise, which may limit broader adoption by laboratories without dedicated computational resources.

Reviewer #3 (Public review):

Xu, Cao and colleagues aimed to overcome the obstacles of high-resolution imaging of intact liver tissue. They report successful modification of the existing CUBIC protocol into Liver-CUBIC, a high-resolution multiplex 3D imaging method that integrates multicolor metallic compound nanoparticle (MCNP) perfusion with optimized liver tissue clearing, significantly reducing clearing time and enabling simultaneous 3D visualization of the portal vein, hepatic artery, bile ducts, and central vein spatial networks in the mouse liver. Using this novel platform, the researchers describe a previously unrecognized perivascular structure they termed Periportal Lamellar Complex (PLC), regularly distributed along the adult liver portal veins.<br /> Using available scRNAseq data, the authors assessed the CD34⁺Sca-1⁺ cells' expression profile, highlighting mRNA presence of genes linked to neurodevelopment, bile acid transport, and hematopoietic niche potential. Different aspects of this analysis were then addressed by protein staining of selected marker proteins in the mouse liver tissue. Next, the authors addressed how the PLC and biliary system react to CCL4-induced liver fibrosis, implying PLC dynamically extends, acting as a scaffold that guides the migration and expansion of terminal bile ducts and sympathetic nerve fibers into the hepatic parenchyma upon injury.

The work clearly demonstrates the usefulness of the Liver-CUBIC technique and the improvement of both resolution and complexity of the information, gained by simultaneous visualization of multiple vascular and biliary systems of the liver. The identification of PLC and the interpretation of its function represent an intriguing set of observations that will surely attract the attention of liver biologists as well as hepatologists. The importance of the CD34+/Sca1+ endothelial cell population and claims based on transcriptomic re-analysis require future assessment by functional experimental approaches to decipher the functional molecules involved in PLC formation, maintenance, and the involvement in injury response before establishing their role in biliary, arterial, and neural liver systems.

Strengths:

The authors clearly demonstrate an improved technique tailored to the visualization of the liver vasulo-biliary architecture in unprecedented resolution.<br /> This work proposes a new morphological feature of adult liver facilitating interaction between the portal vein, hepatic arteries, biliary tree, and intrahepatic innervation, centered at previously underappreciated protrusions of the portal veins - PLCs.

Weaknesses:

The importance of CD34+Sca1+ endothelial cell sub-population for PLC formation and function was not tested and warrants further validation.

Reviewer #2 (Public review):

In this study, the authors investigate how increasing cognitive demand shapes activity patterns in the dorsal dentate gyrus (DG). Using a touchscreen-based TUNL task combined with TRAP/c-Fos tagging, birth-dating of adult-born granule cells (abDGCs), and chemogenetic inhibition, they show that higher task demand increases mature granule cell (mGC) recruitment and enhances suprapyramidal (SB) versus infrapyramidal (IB) blade bias. Functionally, mGC inhibition reduces overall activity and impairs performance without disrupting blade bias, whereas inhibition of {less than or equal to}7-week-old abDGCs increases mGC activity, abolishes blade bias, and impairs discrimination under high-demand conditions. These findings suggest that effective pattern separation depends not only on overall DG activity levels but also on the spatial organization of recruited ensembles.

The integration of touchscreen TUNL with temporally controlled activity tagging and birth-dated cohorts is technically strong. Quantification of SB-IB bias and radial/apical distributions adds anatomical precision beyond bulk activity measures. The comparison between mGC and abDGC inhibition is conceptually compelling and supports dissociable functional roles. Overall, the data convincingly demonstrate that increasing cognitive demand amplifies blade-biased DG recruitment and that mGCs and abDGCs differentially contribute to both behavioral performance and network organization.

However, how abDGCs are integrated into the mGC network under high cognitive demand remains unresolved. Additional experiments are needed to clarify how abDGCs shape spatial recruitment patterns and whether they directly inhibit or indirectly regulate mGC activity to maintain high performance.

Furthermore, the authors frame "high cognitive demand" as a multidimensional construct encompassing broad behavioral challenge. It would strengthen the work to delineate how local abDGC-mGC circuit interactions regulate specific task components in real time. This will require higher temporal resolution approaches, as TRAP and c-Fos labeling integrate activity over prolonged windows and primarily reflect sustained engagement rather than moment-to-moment computations.<br /> The central conclusion that dentate function depends on coordinated spatial recruitment rather than total activity magnitude is supported by the data, although mechanistic interpretations should be tempered given methodological limitations.<br /> Overall, this work advances models of adult neurogenesis by emphasizing a critical-period modulatory role of abDGCs in organizing DG network activity during high-demand discrimination. The combined behavioral and circuit-level framework is likely to be influential in the field.

Reviewer #2 (Public review):

Summary:

The authors present a new method, Unbend, for measuring motion in cryo-EM images, with a particular emphasis on more challenging in situ samples such as lamella and whole cells (that can be more prone to overall motion and/or variability in motion across a field of view). Building on their previous approach of full-frame alignment (Unblur), they now perform full-frame alignment followed by patch alignment, and then use these outputs to generate a 3D model of the motion. This model allows them to estimate a continuous, per-pixel shift field for each movie frame that aims to better describe complex motions and so ultimately generate improved motion-corrected micrographs. Performance of Unbend is evaluated using the 2D template matching (2DTM) method developed previously by the lab, and results are compared to using full-frame correction alone and to the leading local motion correction methods. Several different in situ samples are used for evaluation covering a broad range that will be of interest to the rapidly growing in situ cryo-EM community.

Strengths:

The method appears an elegant way of describing complex motions in cryo-EM samples and the authors present sound data that Unbend generally improves SNR of aligned micrographs as well as increases detection of particles matching the 60S ribosome template when compared to using full-frame correction alone and since review to the leading local motion correction methods. The authors also give interesting insights into how different areas of a lamella behave with respect to motion by using Unbend on a montage dataset collected previously by the group. There is growing interest in imaging larger areas of in situ samples at high resolution and these insights contribute valuable knowledge. Additionally, the availability of data collected in this study through the EMPIAR repository will be much appreciated by the field.

Weaknesses:

A major weakness was comparing this method to full-frame approaches only but this has since been addressed by the authors during review and Unbend is compared to MotionCor2, 3, CryoSPARC and Warp. The improvements here are smaller, generally it seems to perform on par with the above methods, but there are significant gains for certain samples (e.g. the M. pneumoniae sample). A comment from this reviewer about using an adaptive approach to decide if/when to proceed to the full Unbend pipeline, over full-frame alone, has been addressed by the authors.

Reviewer #2 (Public review):

Summary:

Yamashiro et al. investigated how transient absence of visual input (i.e. darkness) impacts tactile neural encoding in the rat primary somatosensory cortex (S1). They recorded local field potentials (LFPs) using a 32-channel array implanted in forelimb and hindlimb primary somatosensory cortex while rats walked on smooth or rough textures under illuminated and dark conditions. Employing a convolutional neural network (CNN), they successfully decoded both texture and lighting conditions from the LFPs. The authors conclude that the subtle differences in LFP patterns underlie tactile representation surface roughness and become more distinct in darkness, suggesting a rapid cross-modal reorganization of the neural code for this sensory feature.

Strengths:

• The manuscript addresses a valuable question regarding how sensory cortices dynamically adapt to changes in sensory context.<br /> • The use of machine learning (CNNs) enables the analysis to go beyond conventional amplitude-based metrics, potentially uncovering subtle but meaningful effects.<br /> • The authors have substantially improved the manuscript with clearer figures, additional statistical analyses (including permutation tests and cross-validation), and greater methodological transparency.

Weaknesses:

• The new analyses (grand-average LFPs, correlation maps, wavelet decompositions, attribution-score correlations) improve transparency but do not yet clarify which specific neural features the CNN exploits, leaving the central interpretability question unresolved.<br /> • A plausible alternative explanation for the increased discriminability in darkness remains insufficiently ruled out: visually driven activity in the light condition (e.g., ambient illumination changes or self-motion-induced visual input) could contaminate S1 LFPs and account for the effect without reflecting a true neural representational change.<br /> • Behavioural and order controls have been improved but remain somewhat limited in sample size.

Overall assessment:

The revised manuscript is clearer, more transparent, and technically strengthened. However, the true nature of the signal changes underlying the observed differences in discriminability remains unclear, limiting the scientific strength of the conclusions. The possibility that visual interference contributes to the observed effects remains a plausible and untested alternative interpretation. Additional experiments or analyses quantifying visually evoked activity in S1 would be required to confirm the claim of genuine reorganization of neural representation depending on the illumination condition.

Reviewer #3 (Public review):

The paper identifies effects of gonadal hormones within hormone-responsive GABAergic neurons in the MPOA. Although it is not surprising that hormones have effects on neurons that express hormone receptors, the current paper adds insights with higher cellular and spatial resolution than previous work and focuses on adolescence period. The paper also identifies a major role for Esr1-dependent mechanisms on behavior using an intersectional genetic strategy to ablate Esr1 in GABAergic or glutamatergic neurons in the MPOA.

The authors have thoughtfully addressed the reviews, in particular by focusing quantitative analyses on Vgat+Esr1+ clusters and adding important technical and conceptual considerations in the limitations section.

I have one remaining minor concern. I appreciate that the text now defines "transcriptional maturation". However, the term seems inappropriate when describing the "minimal transcriptional changes" in Vgat+hormone RLow clusters, which implies that they are transcriptionally immature. Do the authors mean to imply that transcriptional maturation is observed in Vgat+Esr1+ clusters but not Vgat+hormone RLow clusters? The authors also use the term "hormone-dependent transcriptional dynamics", which I think is more appropriate. For example, hormone-dependent transcriptional dynamics are observed in Vgat+Esr1+ clusters but not Vgat+hormone RLow clusters.

Reviewer #2 (Public review):

In this manuscript, the authors test growth, behavior, and gene expression in pairs of clownfish as they establish social dominance hierarchies, examining patterns of gene expression in these pairs after dominance has been established. The authors show solid evidence that emerging dominant clownfish show increased growth, aggression, and food consumption compared to their submissive or solitary counterparts, eventually adopting distinct gene expression profiles.

Major Comments:

(1) The Introduction is comprehensive, but it could be condensed. Likewise, the discussion could be condensed. There is considerable redundancy between the methods, the results, and the legend in Figure 1. The authors should consolidate and remove the redundancy.

(2) For Figure 3, the authors are showing PC2 and PC3; why is PC1 not shown? There is so much overlap between the three groups in PC2 vs PC3; it seems unlikely that researchers could conclusively identify any individual as belonging to a group based on the expression profile. The ovals shown do not capture all the points within each of the groups, and particularly the grey S oval seems misaligned with the datapoints shown.

(3) The authors indicate that the 15 replicates exhibiting the greatest size difference between P1 and P2 were selected for gene profiling. Does this mean that each of the P1 and P2 were pairs with each other? Have the authors tried examining the gene expression patterns in a paired manner? E.g., for the pairs that showed the greatest size differences, do they also show the greatest differences in gene expression? Do the P1s show the most extreme differences from P2s that also show the most extreme P2 differences? Perhaps lines on Figure 3A connecting datapoints from the P1 and P2 pairs would be informative.

(4) For the specific target pathways that are up- and downregulated in the different backgrounds, I recommend that the authors include boxplots (or heatmaps) showing the actual expression values for these targets. Figure 6 shows a heatmap for appetite-related genes, and it would be great to see a similar graph for the metabolism and glycolysis genes; it would also be informative to see similar graphs for hormonal and sexual maturation pathways as well.

(5) Particularly given that there is a relatively small number of genes enriched in the different rank conditions, I did not understand the need to do the WGCNA module analysis. I thought that an analysis of GO terms across the dataset would have been more meaningful than the GO term analysis shown in Figure 4, which considers only genes assigned to the "brown WGCNA module". This should be simplified or clarified.

(6) The authors say that they have identified coordinated changes in behaviors and the "underlying gene expression, leading to the emergence" of social roles. This is a little bit misleading, since the gene expression analysis occurred well after the behavioral and phenotypic differences emerged. Presumably, the hormonal and genetic shifts that actually caused the behavioral and phenotypic difference occurred during the weeks during which the experiment was underway, and earlier capture of the transcriptome would presumably reveal different patterns, and ones that would be considered more causative. The authors acknowledge this in 434-435, but it could be emphasized further.

(7) The authors have measured a number of differences between the different dominance classes of fish. All these differences were measured relative to the other classes, but in my view, the Solitary group was the closest to a baseline control. So I'm not sure that it is fair to say that "P2 and S individuals showed consistent downregulation of these genes and pathways" (line 401). I encourage the authors to emphasize the differences in gene expression from the "perspective" of the P1 individuals compared to the baseline of P2 and S individuals. Line 474 says that "P2 fish showed significant upregulation" of a number of pathways. It should be very clear what that is compared to (compared to P1, presumably?)

(8) Along the same lines, the authors say in line 514 that subordinates and solitaries strategically downregulate their growth. I'm not convinced that this is the case: I would consider this growth trajectory to be the default and the baseline. I would interpret that under certain social conditions, a P1 dominant pattern of growth, behavior, and gene expression is allowed to emerge.

Reviewer #2 (Public review):

Summary:

The authors use a combination of genomics, genome conformation assays, and CRISPR-mediated deletion to study the transcriptional regulation of the SOX2 gene in human neural stem cells (hNSCs).

Strengths:

The authors show that two distal elements, located ~550kb downstream of the SOX2 gene, are important for SOX2 transcription in hNSC. They investigate both the deletion of these elements in established hNSCs and in hNSCs generated by differentiation of human pluripotent stem cells, suggesting these elements are important in both the establishment and maintenance of SOX2 expression in hNSCs.

Weaknesses:

Homologous elements have been studied in the mouse genome and have conserved function in mouse NSCs, yet these findings are not mentioned. Inclusion of biological replicates for the scRNA-seq and replicate CRISPR-deleted clones would strengthen the study.

Reviewer #2 (Public review):

Summary:

Silva and co-workers exploit their previously established methods of analyzing release events at single parallel fiber to molecular layer interneuron synapses. They observed synaptic depression at low transmission frequencies (< 5 Hz), which rapidly recovers during high-frequency transmission. Analysis of the time course of low-frequency depression revealed an initial rapid and a slow linearly increasing time course. Strikingly, the initial depression occurred even in the absence of preceding release, arguing against vesicle depletion as the underlying mechanism.

Strengths:

The main strength of the study is the careful demonstration of an interesting synaptic phenomenon challenging the classical vesicle-centered interpretation of synaptic depression.

Weaknesses:

No major weaknesses were identified by this reviewer.

The finding of release-independent synaptic depression is important and would have widespread implications. Therefore, some more analyses to increase the confidence in these findings could be performed.

My concern is whether rundown could explain the findings. If the rate of failures in s1 increases and at the same time the amplitude decreases during the experiments, an apparent depression in s2 could arise. The Supplementary Figure 5A addresses run-down, but the figure is not easy to understand, and, as far as I understood, it does not address the question of whether the release-independent depression could be caused by a rundown. To address this, the analysis of Figure 5 could be repeated by investigating the failure rate and amplitude separately or by analyzing the 1st and 2nd half of the recordings separately.

Reviewer #2 (Public review):

Summary:

Prior work identified TMEM30B (knockout mice) as well as ATP8B1 (human genetics and mouse model), ATP8A2 (knockout mice), and ATP811A (human genetics) as relevant for hearing. The authors also reasoned that, given the recent discovery of TMC1 and TMC2's dual function as mechanotransduction channels of the inner ear and as lipid scramblases, a counterpart flippase should be in the sensory hair-cell stereocilia bundle where mechanotransduction happens. They use CRISPR/CAS to modify the endogenous mouse genes and add an HA tag at the N-terminus of the ATP8B1, ATP8A1, ATP8A2, and ATP11A proteins. Their experiments with these mice unambiguously localized ATP8B1 at the base of outer hair cell stereocilia bundles. Knockout of ATP8B1 results in loss of outer hair cells, deficient auditory function (ABR), and degeneration of outer hair cell stereocilia bundles. Similarly, hair cells from genetically modified mice with endogenous HA-tagged TMEM30B proteins show localization of this protein to outer hair cell stereocilia bundles. TMEM30B knock-out mice phenocopy the ATP8B1 knock-out model. Interestingly, the authors show that annexing V staining precedes hair cell loss in ATP8B1 and TMEM30B knockout mice and that proper localization of these proteins is lost in mice that lack CIB2, a protein essential for hair cell mechanotransduction.

Strengths:

(1) Use of knock-in HA-tagged proteins, rather than antibody staining, to unambiguously localize ATP8B1 and TMEM30B.

(2) Systematic characterization of auditory function (ABR), hair cell loss, and hair-cell stereocilia bundle morphology.

(3) Advances our understanding of the role played by lipid homeostasis in auditory function.

(4) Reports on mouse models that will be helpful to further understand the mechanistic role played by ATP8B1 and TMEM30B in normal hearing and hereditary deafness.

Weaknesses:

(1) Are the HA tags causing any functional issues? Function and localization of tagged proteins can sometimes be compromised. It would be good to know, for each knock-in model (TMEM30B, ATP8B1, ATP8A1, ATP8A2, and ATP11A ), whether the HA-tagged protein is causing any issues with the mice and particularly with hearing (ABRs). Are these mice normal? Can they hear? These data are missing.

(2) Following on the point above, is it possible that ATP8B1-HA is well localized, but localization for the other three flippases (ATP8A1-HA, ATP8A2-HA, and ATP11A-HA) is compromised by the tag? Is this potential mislocalization causing any functional phenotypes? (ABRs of point 1). I find it surprising that there are flippases only in outer hair cells, and only formed by ATP8B1. A possible explanation is that the tag is interfering with trafficking. If so, there should be a phenotype (ABRs), although this might be masked by redundancy among these flippases or caused by systemic issues (admittedly difficult to sort out). Given that this manuscript will likely become foundational, and that there is evidence that at least two of the other flippases are involved in hearing loss, it would be good to provide more information about the mice and HA-tagged proteins in the other knock-ins (ATP8A1-HA, ATP8A2-HA, and ATP11A-HA). Depending on the data available for the knock-ins, the authors may want to discuss these scenarios and soften the statement indicating that inner-hair cells may lack flippase activity altogether.

(3) Expression of ATP8B1 at P0 (Figure 1D), when there should not be protein in outer hair cells yet, seems high. Does this mean that other cells in the cochlea also express ATP8B1? Is this a concern?

(4) Fluorescence scales in Figure 6 B and D and Figure 7 B and D are very different. So are the values for WT. One would expect that the WT would be similar in all cases (at least within the same compartments), given that the methods section indicates that "All images were collected using identical acquisition parameters, including zoom and laser power, across genotypes". If WT shows such variability, how can we compare?

Reviewer #3 (Public review):

Summary:

This study addresses a long-standing question in visual neuroscience concerning how the human visual system balances stability and plasticity when sensory input is altered from early in life. Using achromatopsia as a model of lifelong cone deprivation, the authors examine whether early visual cortex undergoes retinotopic reorganization to compensate for the absence of foveal cone input, or whether canonical retinotopic organization is largely preserved. By combining fMRI-based population receptive field (pRF) mapping with connective field (CF) modelling, the authors characterize changes across multiple hierarchical stages of visual processing.

The main findings indicate that primary visual cortex (V1) shows no systematic remapping of the foveal projection zone, whereas extrastriate cortex, particularly V3, exhibits altered patterns of sampling from V1. The authors interpret these results as evidence for hierarchical adaptation, whereby downstream readout mechanisms adjust to make more efficient use of degraded rod-mediated input while preserving early-stage retinotopic organization.

Strengths:

A major strength of this work is the use of silent substitution to generate rod-selective stimuli. This approach enables a principled comparison between achromats and typically sighted controls by isolating rod-driven responses in both groups. In doing so, the study overcomes a key limitation of prior work, where differences in cortical organization could often be confounded by differences in photoreceptor class rather than reflecting neural reorganization per se. The inclusion of a rod-driven baseline in controls provides an important reference for distinguishing long-term adaptation from transient or stimulus-driven effects.

Another notable strength is the integration of CF modelling alongside conventional pRF mapping. While pRF analyses alone suggest enlarged receptive fields in V1, consistent with reduced spatial resolution, the CF analysis offers a more mechanistic account by revealing changes in how V3 samples information from the V1 surface. This multi-level modelling approach moves beyond descriptive accounts of cortical map structure and provides a framework for interpreting how downstream areas may adjust their integration strategies under conditions of altered input.

Weaknesses:

Although the study is methodologically strong, the central claims regarding stability and compensatory plasticity require clearer conceptual framing and stronger empirical support. Stability is primarily defined as the absence of large-scale retinotopic remapping in V1, yet the presence of significantly enlarged V1 pRFs indicates substantial tuning-level plasticity at the input stage; distinguishing topographic stability from functional reorganization would therefore strengthen the interpretation. Moreover, the proposed compensatory mechanism raises a signal-processing concern, as reduced downstream sampling (smaller CFs in V3) cannot restore spatial information lost due to coarse upstream representations, and may instead limit integration. The mechanistic link between altered CF properties and normalization of extrastriate pRFs is not directly tested, as group differences are not shown to covary across individuals or visual field locations. Finally, the interpretation of these changes as compensatory implies functional benefit, yet no behavioral or performance measures are provided to establish that the observed reorganization preserves or enhances visual function, leaving open whether these effects reflect adaptive optimization or passive downstream consequences of altered input.

Reviewer #2 (Public review):

The paper proposes a hierarchically layer approach to larval locomotion, chemotaxis and learning. The model consists of a basic locomotor layer with two coupled oscillators, one for crawls and one for turns. The intermediate layer modulates the frequency and amplitude of tunings to enables chemotaxis. The higher layer, integrates a spiking neural network model of the Mushroom Body to modify the door valence in response to experience as during learning.

The model is compared to experimental data with a good degree of agreement. This modular framework provides a valuable advance for modeling larva behavior.

Strengths:

A novel multilayer level model that reflects current thinking of the neuronal organisation of motor control. The model is very useful to investigate the neuronal architecture of central pattern generators<br /> and higher order motor control circuits that could be linked to larval connectome data.

Weaknesses:

All the limitations of the model are discussed and therefore the paper perfectly fits its purpose.

Reviewer #2 (Public review):

This study investigates the visual information that is used for the recognition of faces. This is an important question in vision research and is critical for social interactions more generally. The authors ask whether our ability to recognise faces, across different viewpoints, varies as a function of the orientation information available in the image. Consistent with previous findings from this group and others, they find that horizontally filtered faces were recognised better than vertically filtered faces. Next, they probe the mechanism underlying this pattern of data by designing two model observers. The first was optimised for faces at a specific viewpoint (view-selective). The second was generalised across viewpoints (view-tolerant). In contrast to the human data, the view-specific model shows that the information that is useful for identity judgements varies according to viewpoint. For example, frontal face identities are again optimally discriminated with horizontal orientation information, but profiles are optimally discriminated with more vertical orientation information. These findings show human face recognition is biased toward horizontal orientation information, even though this may be suboptimal for the recognition of profile views of the face.

One issue in the design of this study was the lowering of the signal-to-noise ratio in the view-selective observer. This decision was taken to avoid ceiling effects. However, it is not clear how this affects the similarity with the human observers.

Another issue is the decision to normalise image energy across orientations and viewpoints. I can see the logic in wanting to control for these effects, but this does reflect natural variation in image properties. So, again, I wonder what the results would look like without this step.

Despite the bias toward horizontal orientations in human observers, there were some differences in the orientation preference at each viewpoint. For example, frontal faces were biased to horizontal (90 deg) but other viewpoints had biases that were slightly off horizontal (e.g. right profile: 80 deg, left profile: 100 deg). This does seem to show that differences in statistical information at different viewpoints (more horizontal information for frontal and more vertical information for profile) do influence human perception. It would be good to reflect on this nuance in the data.

Comments on revisions:

I am happy with the response and changes to the comments in my review. The key findings from this study are: (1) that there is bias toward the use of horizontal information across all viewpoints for face recognition in humans using an old-new recognition task. (2) In contrast, the optimal information for matching faces varies as a function of viewpoint. The view-selective model shows horizontal information is dominant for frontal views and vertical information is dominant for profile views.

The data from the view-tolerant model is less easy to interpret as it doesn't fit with any theoretically plausible model of face recognition. It might be a useful model for a face matching task in which participants had to match unfamiliar faces across viewpoints. This might be a possible extension of the current work.

Nonetheless, I still think this is an interesting contribution to the literature.

Reviewer #2 (Public review):

Summary:

This article is a useful compendium of advice for MD/PhD students (and research-focused MD students) to consider when it is time to decide on a clinical field for residency training. The authors are a distinguished group of physician-scientists and program directors who are drawing on published data and their own experience as mentors to provide advice and resources to students about to make what can be a career-defining choice. It makes an effective argument for considering important differences between clinical fields in their ability to sustain research integration, provide mentorship, meet lifestyle expectations, and foster a long-term career as a research-focused physician-scientist.

Strengths:

(1) A lot has been written about physician-scientists as an endangered species. Given the important role that physician-scientists can play if they engage in research that is informed by experience in patient care, not nearly enough has been written about the choices that students make during training that can keep them on track or throw them off.

(2) The article provides not only general advice, but specific information in the 2 tables that can help trainees to weigh their priorities and consider their options.

(3) Among the best advice is to weigh clinical demands, maintenance of procedural skills, recognition of the impact of research time on salary, and the impact of high salaries on the tension between research effort and clinical effort in clinical departments, which is where most physician-scientists in academia are employed.

Areas for potential improvement:

(1) Some of the most useful pieces of advice are scattered through the text when they might be more impactful if focused. For example, what are the 4 or 5 most essential factors that someone in an MD/PhD or an MD program should weigh when they are deciding between clinical disciplines? There are also published data on the experience of past graduates in achieving a research-focused career in each clinical discipline. How should that data be applied by trainees? What are the factors that should be weighed in deciding where to work as a research-focused physician once training has been completed?

(2) Some clinical fields at academic institutions have proved to be much more hospitable to careers as research-focused physicians than others. Published data highlight the challenges. I believe the authors have tried very hard to present a balanced perspective, but in the process, they have, I believe, missed an opportunity to guide trainees and make them aware of what they should look for to avoid making a decision that may prove incompatible with their long-term goals.

(3) An issue that hasn't been raised: Where will be the jobs for physician-scientists who have an MD {plus minus} PhD and want to do research and discovery? How many openings will there be for physician-scientists in academia 5-10 years from now? In industry? How are recent events in Washington affecting the continuation of those jobs? Unfortunately, I am not aware of labor statistics for physician-scientists, but perhaps the authors can find them.

(4) Additional questions that can be raised and addressed in the article: Should one of the "smart choices" in the article's title be where you do the residency, and not just which residency you do? How important is it to be at a successful, research-intensive medical center/university, both during and after residency and fellowship training? If being in an institution where there are numerous very successful physician-scientists and scientists improves the likelihood of being able to sustain a physician-scientist career, how should graduating students improve their chances of being at one of those institutions?

(5) In every clinical discipline, there are departments that value physician-scientists more than other departments and invest accordingly. What advice would the authors give to help graduating students identify those departments?

Reviewer #2 (Public review):

I think this paper is an excellent and timely contribution. It clearly shows that learning overlapping relationships in a disjoint training schedule (where the overlaps are not encountered close together in time) appears to aid the formation of an integrated associative memory structure (a cognitive map) and supports generalisation. I believe the methods are sound and the results are clear. I only have a couple of methodological questions that may not warrant any changes to the paper (or only very minor changes/additions):

(1) The mixed effects models did not include random slopes for the within-subject factors ("spatial manipulation" and "block"), and so the corresponding fixed effect inferences may be unsafe. Having said that, it is likely that including these slopes may not be warranted given their contribution to the model's fit. I recommend that the authors check this.

(2) The mixed effects models for accuracy appear to model average performance across trials rather than using a generalised linear model with a (e.g.) logit link function and the binomial distribution to characterise performance. I think this is a little sub-optimal, as the latter is often more sensitive. Nonetheless, it is not in any way wrong; the results are clear enough as is, and there may be a good reason to avoid a non-linear link function, which can alter the interpretation of effects close to the ceiling and floor.

I think the introduction and/or discussion would benefit from contrasting their results with Berens & Bird (2022, PLOS Comp Bio). In this paper, it is shown that blocking the training of discriminations in a linear hierarchy (what we call progressive training) substantially benefited transitive inference performance. This seems at odds with the author's finding that "participants struggle to integrate information across rows and columns, i.e. across groups of transitions that were trained separately in time".

I would really like to know what the authors think about this discrepancy (or, indeed, whether they think there is one at all). Is it possibly because "progressive" learning is some combination of "grouping", "blocking" and "chaining" (where there is a structured overlap between adjacently trained relationships)? Or is it something else, e.g., that there is a fundamental difference between learning associations and discriminations (personally, I lean on this explanation)?

Relevant to this, the authors note that their "findings do contradict recent reports from the category learning literature, where blocking seems to help learning and generalisation (Dekker et al., 2022; Flesch et al., 2018; Noh et al., 2016). It may be that where the goal is not to learn a complex knowledge structure - like a map - but simply to compress exemplars by mapping them onto a smaller number of labels - the benefits of blocking emerge." However, the benefit of progressive (blocked) training in my own work was observed in a task that required learning a complex/relational structure in the form of a transitive hierarchy, which theoretical accounts suggest depends on learning map-like representations (Whittington et al., 2020).

Reviewer #2 (Public review):

Summary:

The manuscript by Puno and colleagues investigates the impact of hypoxia on cortical interneuron migration and downstream signaling pathways. They establish two models to test hypoxia, cortical forebrain assembloids and primary human fetal brain tissue. Both of these models provide a robust assay for interneuron migration. In addition, they find that ADM signaling mediates the migration deficits and rescue using exogenous ADM. The findings are novel and very interesting to the neurodevelopmental field, revealing new insights into how cortical interneurons migrate and as well, establishing exciting models for future studies.The authors use sufficient iPSC lines including both XX and XY, so analysis is robust. In addition, the RNAseq data with re-oxygenation is a nice control to see what genes are changed specifically due to hypoxia. Further, the overall level of valiation of the sequencing data and involvement of ADM signaling is convincing, including the validation of ADM at the protein level. Overall this is a very nice manuscript. I have a few comments and suggestions for the authors.

Strengths/Weaknesses:

(1) Can they comment on the possibility of inflammatory response pathways being activated by hypoxia - has this been shown before? While not the focus of the manuscript, it would be discussed in the Discussion as an interesting finding and potential involvement of other cells in the Hypoxic response.

(2) Can they comment on the mechanism at play here with respect to ADM and binding to RAMP2 receptors - is this a potential autocrine loop, or is the source of ADM from other cell types besides inhibitory neurons? Given the scRNA-seq data, what cell-to-cell mechanisms can be at play? Since different cells express ADM, there could be different mechanisms at place in ventral vs dorsal areas.

(3) For data from Figure 6 - while the ELISA assays are informative to determine which pathways (PKA, AKT, ERK) are active, there is no positive control to indicate these assays are "working" - therefore, if possible, western blot analysis from assembloid tissue could be used (perhaps using the same lysates from Fig 3) as an alternative to validate changes at the protein level (however, this might prove difficult); further to this, is P-CREB activated at the protein level using WB?

(4) Can the authors comment further on the mechanism and what biological pathways and potential events are downstream of ADM binding to RAMP2 in inhibitory neurons? What functional impact would this have linked to the CREB pathway proposed? While the link to GABA receptors is proposed, CREB has many targets beyond this.

(5) Does hypoxia cause any changes to inhibitory neurogenesis (earlier stages than migration?) - this might always be known but was not discussed.

(6) In the Discussion section - it might be worth detailing to the readers what the functional impact of delayed/reduced migration of inhibitory neurons into the cortex might results in, in terms of functional consequences for neural circuit development

Comments on revisions:

The authors have addressed my comments thoroughly. I have no further comments or suggestions

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors report that Nora virus, a natural Drosophila pathogen that also persistently infects many laboratory fly stocks, infects intestinal stem cells (ISCs), leading to a shorter life span and increased sensitivity to intestinal infection with the Pseudomonas bacterium. Nora virus infection was associated with an increased proliferation of ISC and disrupted gut barrier function. Genetically, the authors show that increased ISC division in Nora virus and Pseudomonas coinfected flies is driven by signaling through the JAK-STAT pathway and apoptosis.

Accordingly, blocking apoptosis and JAK-STAT signaling reduces viral load, suggesting that in this context the JAK-STAT pathway is proviral in contrast to other previous observations in systemically infected flies. This work adds to the findings of another recent paper showing that another persistent fruit fly virus, Drosophila A virus, also increases ISC proliferation and decreases gut barrier function. Intestinal viruses should therefore be considered confounders in studies of fly intestinal physiology.

Strengths:

Overall, the data are convincing and robust, starting with two wildtype fly stocks (Ore-R strain) that differ in their Nora virus infection status, followed by experiments in which cleared stocks are reinfected with a purified Nora virus stock preparation. The conclusions of the paper will be of interest to scientists working on insect physiology, virology, and immunology, but should also serve as a warning for scientists that use the fly as a model to study gut physiology.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

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

Comments on revision:

The authors state that OPT is a new software tool and have posted example code on GitHub. However, the Jupyter notebook does not display any figures or workflows that would allow the process to be replicated. Please provide documentation and code that can reproduce the results/figures presented in the paper.

Reviewer #2 (Public review):

Summary:

This paper examines resting-state electroencephalography (EEG), the electrophysiological underpinnings of the temporal integration window in perception, and its modulation by priors (serial dependence) as measured through the perceptual fusion point of two continuous alternating stimuli. The study also includes a measure of perceptual confidence. Separating periodic from aperiodic EEG activity, the results show that the faster the individual alpha-frequency at rest and the steeper the aperiodic slope (previously linked to higher sampling/ lower noise), the lower the perceptual fusion point (corresponding to narrower integration windows), with independent contributions of the period and aperiodic activity to the integration window. The data also reveal that the point of fusion depends on prior history, and that the strength of this effect depends on individual alpha frequency and aperiodic slope: the lower the individual alpha frequency and the aperiodic slope, the stronger the serial dependence, with the two contributions being again independent. Higher alpha frequency also led to higher confidence. The results are interpreted to suggest that speed of alpha oscillations and aperiodic slope of the power spectrum (presumably reflecting rate/fidelity of visual sampling and the level of background noise) jointly shape the perceptual measure under study: high rate/ fidelity and low noise promote temporal precision in integration, while lower rate/fidelity and higher noise lead to a higher reliance on prior history. It is concluded that it is the interaction between two EEG features that shapes temporal integration and hence perceptual fusion.

Strengths:

The strength lies in the use of a continuous visual stream of two alternating stimuli whose timing shapes fusion or separation of the two stimulus precepts, avoiding some of the pitfalls of previous fusion probes through discrete (not continuous) stimulus pairs (missed detection of one stimulus of the pair may be misinterpreted as fusion). The results seem robust (based on n=83 participants), the results are interesting, and the interpretations are sound.

Weaknesses:

The main weakness lies in the reliance on resting state EEG for correlation with the behavioural measures. This captures trait-based relationships, but does miss out on the brain activity dynamics within/across trials, which could be used for a direct readout of evidence accumulation to a decision, for capturing spontaneous fluctuations of the processes under study, etc. Also, in terms of resting state EEG, both eyes-closed (EC) and eyes-open (EO) data have been recorded, but their links to perceptual fusion point/ confidence seem somewhat inconsistent across the results. This is a bit confusing. Are the EO and EC signals in any way related/ correlated, and if not, what are they supposed to represent? Would an analysis of these EEG measures during task performance (e.g., in a pre-stimulus = baseline time window) provide more consistent results? These points could be resolved by additional analyses and/or more elaborate discussions.

Reviewer #2 (Public review):

Summary:

Using a public dataset of retinotopic mapping and resting-state data, the authors find that the default mode network has voxels that respond (positively or negatively) to visual stimulation at specific retinotopic positions, and that resting-state activity in these voxels is correlated with activity in more traditional sensory voxels with the same visual-location preference. The retinotopic specificity is bidirectional, such that high activity in default mode voxels drives activity only in voxels with matching receptive fields in sensory cortex, and vice versa. These findings are at odds with traditional views of the default mode network as having abstract (non-retinotopic) representations and competing (rather than cooperating) with external sensory representations.

Strengths:

This study continues an intriguing line of research about how default mode regions interact with the sensory cortex. Demonstrating that there are structured interactions between these regions at rest, and that these interactions are in fact organized according to retinotopic location (as opposed to traditional views of representational format in the default mode network), provides a new framework for thinking about large-scale internal and external brain networks. The authors make use of a well-powered public dataset that allows for precise estimates of pRFs and individual-specific resting-state networks, and develop a number of interesting analyses that characterize the relationships between DN and dATN voxels. The findings are exciting and could have a major impact on future studies in cognitive neuroimaging.

The authors mention that these findings could shed light on internal/external interactions such as "anticipatory saccades or memory-guided attention," which is true, though I would argue that constructing DN representations of external stimuli is in fact even more fundamental than these specific cases (e.g., see Barnett and Bellana, 2025, "Situation models and the default mode network"). The "highways" identified in this study could play a vital role in real-world perceptual processes that are constantly translating external input into internal mental models.

Weaknesses:

(1) The criterion used for defining voxels as retinotopic seems very liberal. The authors show that only 5% of voxels have R^2>0.14 in a null analysis, and therefore define voxels with R^2>0.14 as retinotopic. Although all the networks in 1C show voxel distributions that differ from the null, the number of false positives above R^2>0.14 seems problematic, especially for the DN positive pRFs (red distribution) and to a lesser extent the DN negative pRFs (blue distribution). From visual inspection of the plot, the false discovery rate (fraction of voxels labeled as retinotopic that are false positives) looks like it would be greater than 50% for the DN-positive pRFs. The authors do show that the positive pRF voxels have above-chance consistency across runs, again providing evidence that there are true positive voxels in this set, but perhaps a stricter criterion (such as having consistent negative fits across runs) would provide more targeted identification of the DN voxels with true retinotopic sensitivity.

(2) The claim that "opponency at rest between the DN and dATN appears to be driven by the subset of DN voxels with negative retinotopic tuning" is not well supported. The fraction of DN voxels with negative pRFs is small: 9.42% of DN voxels have pRFs, and 58.77% are negative, so about 6% of DN voxels have negative pRFs. The fact that any DN voxels have negative pRFs is notable, but the authors do not provide evidence that these 6% are driving the overall behavior of the DN. They do show (e.g., in Figure 2B) that negative and positive pRFs have opposing influences, but the overall correlation with dATN does not look similar to the negative pRF connectivity. I'm also unsure whether "opponency" is a reasonable description for two networks that are "independent (i.e., not correlated)" in this analysis.

(3) The event-triggered analysis is effective at testing the bidirectional relationship between DN and dATN, with high activity in either network triggering a response in the other network. However, it would be helpful to show more validation that these "events" are meaningful windows of time to study. First, is 13 TRs a typical length of time that activity is elevated during one of these events? Second, the top-down and bottom-up terminology is perhaps too loaded and not well-justified; if the negative pRFs in the DN reflect a meaningful coding system, then couldn't low (rather than high) activity indicate a top-down event?

(4) The framing of this paper relative to the authors' past week, such as Steel et al. 2024 ("A retinotopic code structures the interaction between perception and memory systems"), could be improved. The existence of negative pRFs in the DN and a functional relationship between these pRFs and the sensory pRFs have already been described in prior work. My understanding of the primary novelty here is that this paper examines resting-state data, showing that there are widespread spontaneous interactions between broad internal and external networks, but this distinction is not made explicit in the Introduction.

(5) The definition of the default mode (DN) in this study aligns with past research, but the definition of the dorsal attention network (dATN) seems at odds with standard terminology. For example, the authors cite Fox et al. 2006, which depicts the dATN as including regions such as IPS, FEF, SMA, and MT+. Here, however, the "dATN" seems to be primarily lateral and ventral visual cortex (e.g., Figure S5). The exact location of these sensory pRFs is not critical to the authors' claims, but this labeling seems incorrect, and the motivation for defining/selecting the sensory network in this way is not described.

Reviewer #2 (Public review):

Summary:

The manuscript by Garcia-Alcala et al. reports an interesting paradox: the cost of gene expression slows the population-average growth rate, whereas at the single-cell level, expression levels from these genes positively correlate with the growth rate. The effect is observed in the expression of flagellar genes and a gene under a synthetic promoter in E. coli. The findings are explained by the inheritance of growth factors, including ribosomes, during asymmetric division.

Strengths:

(1) The manuscript adds strength to an emerging body of literature showing that the population-level bacterial growth laws do not match correlations based on single-cell data. The evidence presented here is more striking than in previous works (such as Pavlou et al., Nat. Commun. 2025), as the trends in population-level data and single-cell data are reversed.

(2) A relatively simple model correctly explains the trends in the data.

Weaknesses:

(1) It is not clear whether flagellar proteins are expressed proportionally to the reporter signal. Furthermore, it is questionable if E. coli bacteria in the mother machine channels are flagellated. If they are, they could potentially swim out of the channels, which is not the case when they do not carry the MotA E98K mutation. The authors should provide some evidence that E. coli expresses the actual filament proteins in the channels.

(2) It is unclear what fraction of the total proteome mVenus represents in different measurements. Some quantification is needed (for example, using the Coomassie staining). Using f_U as high as 14.4% in simulations is questionable.

(3) The data from the MC4100 strain does not directly match the trends of MG1655. The justification for filtering out the low-frequency components of MC4100 is not particularly convincing. It appears unlikely that ribosomes or other growth factors partition significantly differently in the MC4100 strain than in the MG1655 strain. Further discussion and a plot similar to Figure 1 (Left) for this strain are warranted.

(4) The model needs to be described in more detail. A closed set of equations that has been simulated must be presented, along with all values of the model parameters and their sources. The authors should consider depositing their code on GitHub or another publicly accessible repository.

Reviewer #2 (Public review):

McGaughey & Gold trained rhesus macaque monkeys to perform a motion-direction discrimination task in which a behaviorally irrelevant adapting stimulus with either fast or slow direction alternations preceded a variable-duration test stimulus, while simultaneously recording single-unit activity in area MT and pupil diameter. They report that adaptation to the more rapidly changing stimulus was associated with reduced behavioral sensitivity, attenuated test-evoked MT responses, and larger pupil-linked arousal signals. The authors interpret these behavioral changes as evidence for a more "leaky" evidence-accumulation process, and argue that this apparent leak is implemented in part through context-dependent sensory adaptation in MT and in part through arousal-related mechanisms. More broadly, they conclude that flexible evidence accumulation in dynamic environments arises from distributed adjustments across sensory encoding and neuromodulatory systems rather than solely from changes within a downstream accumulator. If correct, this interpretation has significant implications not only for our understanding of the neural mechanisms of perceptual decision-making but also for broader theories concerning the functional role of sensory adaptation.

The conclusions of the paper are mostly well supported by the data. Evidence for robust adaptation-induced changes in sensory encoding, behavior, and pupil dynamics is convincing, but further clarification and refinement are needed to establish a clear mechanistic link between these effects and decision-making processes.

Aspects of the behavioral analysis would benefit from a tighter connection between theoretical claims about evidence accumulation and the empirical features of the psychometric functions. For example, the rightward shifts observed across adapting conditions are interpreted as consistent with a reset of accumulation on switch trials, but similar patterns could also arise from failures to detect the test stimulus on a subset of trials, leading responses to default to the final adaptor direction. Likewise, changes in psychometric slope and asymptote are attributed to differences in evidence accumulation without explicit modelling or consideration of alternative explanations. Clarifying how specific features of the psychometric functions map onto distinct components of the decision process will strengthen the link between the theoretical framework and the behavioral data.

A slight concern is the lack of a consistent analytical approach for relating behavioral changes to neural and pupil-linked measures. Different sections of the manuscript rely on different behavioral metrics-such as differences in accuracy within a selected stimulus-duration range (e.g., Figure 5C) or psychometric slope differences (Figure 6C) - without clear justification for these choices. The analytical approach likewise varies between simple correlational analyses (Figure 5C, Figure 6C), pseudo-experimental group comparisons (Figures 5D, E), and the inclusion of neural or pupil terms in the behavioral psychometric regression model (Figure 7B). While each metric and approach may be defensible in isolation, adopting a more consistent framework will help convince readers that the reported effects are robust and not contingent on the selective choice of metric or analysis.

Reviewer #2 (Public review):

Summary:

In natural visual behavior, such as when one is looking for a face in the crowd, the eyes are moved from site to site, seeking possible matching targets. This involves attention both to the current view at the center of vision (the foveal location) as well as to upcoming views via attention to targets in the periphery. While it has been established that attention generally enhances neuronal response (compared to simple visual activation) at the attended spatial location, this study provides solid evidence that attention during active visual search leads to neuronal response enhancement only when the eye moves towards targets that exhibit the desired feature and category. This study thus moves the field towards understanding the neural encoding of active vision.

This study examines the neuronal basis of feature-selective attention during active, freely behaving visual search. Traditional electrophysiological studies on visual attention in monkeys commonly used an eye fixation with a covert attention paradigm, but have not sufficiently addressed the roles of both foveal and peripheral attention in play during natural looking behavior. Here, the authors present a novel paradigm in which, during eye-movement mediated search, neuronal receptive fields are recorded in multiple cortical areas (sensory V4, temporal, and prefrontal areas). In this manner, as the eye foveates, items in the array fall into foveal or non-foveal recorded sites. Thus, the experimental paradigm is elegant, offering the opportunity to make multiple types of comparisons: target/distractor, towards/away from fovea, and areal. Specifically, following a category cue (face, house, hand, flower), freely initiated saccades are made to locate a categorically matching 'target' in an array of distractors. Feature attention is assessed by comparing eye saccades made to targets vs to distractors. Spatial attention is assessed by comparing saccades made 'towards' vs 'away' from targets. Statistics are rigorous and nicely designed. The detailed association of simultaneously obtained eye movement sequences and neural parameters is well done. These are valuable data that will contribute to our understanding of attentional modulation in visual search.

Strengths:

The significance of these findings is fundamental. Decades of attention research in vision have been based on the paradigm of visual fixation and covert peripheral attention. However, increasingly, the field has moved towards understanding how the visual system works during active vision. Here, the authors use an active visual search paradigm and record from multiple areas (V4, IT, PFC). They find enhancement of attention both in the foveal and peripheral locations, and, furthermore, a high degree of feature and categorical specificity. This provides valuable data for the concept of a foveal-peripheral attentional window in natural vision. The controls (comparisons of neuronal response during looks to targets vs distractors, and looks towards and away from the target) and statistical rigor make these findings quite compelling.

Weaknesses:

While the study is generally quite strong, there are a few weaknesses to be addressed.

(1) Little rationale is provided for recording in the selected areas, V4, IT, and PFC. Given the respective roles in sensory, object recognition, and goal-directed behavior, some rationale for this design should be offered, and commonalities/distinctions between these areas should be discussed.

(2) Given the reliance of all analyses on saccadic behavior (towards target/distractor, towards/away from target), additional description and summaries of eye movement behavior during single trials and across trials should be provided.

(3) The dependency of findings on top-down (categorical & feature-specific) task design should be discussed.

Reviewer #2 (Public review):

Summary:

Spike sorting, that is, assigning events detected in extracellular electrophysiology data to the firing of individual neurons, is an inherently difficult computational problem involving multiple steps. The difficulty arises from low signal-to-noise, instability in signal due to the relative motion of the tissue and recording sites, and large volumes of data. Experimental ground truth data - where the correct assignment of spikes is known - is not available in large enough quantities to test algorithms. This paper describes a tool for creating fully synthetic ground truth data and benchmarking the individual steps of spike sorting to dissect the impact of signal-to-noise, firing rate, and motion correction on each step. This information is used to construct an optimized algorithm for sorting the ground truth data. One result of particular interest is the dominant role of motion correction in degrading accuracy. Another important technical result is that motion correction via interpolation of the voltage traces yields similar accuracy to interpolation of the spike templates.

Strengths:

The paper clearly shows the benefits of analyzing the complex process of spike sorting step by step. While this analysis has also been done in papers presenting spike sorters (for example, reference [32]), the tools presented here allow users and developers to do similar studies for their own work. This toolset will be very useful to many labs, especially those working in less studied brain areas or model systems, cases where the tuning of standard spike sorting tools is not a good match to the data.

Weaknesses:

The model ground truth data used in the paper does not need to be a perfect match to experimental data to provide useful benchmarking. However, as with all measurements of spike sorting accuracy, extrapolation to experimental data can be complicated. Users of these tools will need to assess how well the simulated data matches their recordings.

Reviewer #2 (Public review):

Summary:

In this study, Yang et al. develop a real-time system for automatic face detection and identification of multiple unrestrained common marmosets in a home cage setting.

Strengths:

The study aims to address an unmet need in behavioral neuroscience: the ability to non-invasively identify animals is crucial to the automated and rigorous study of neural behaviors; this is especially true for common marmosets, which are rapidly becoming a model system of choice for the study of complex social cognition. By using a YOLOv8 backbone, the study achieve human level performance, both in terms of precision and recall of the trained models.

Weaknesses:

The robustness of the system is not clear from the limited datasets presented. The use of color-coded beads undercuts the study's premise that the system achieves truly non-invasive tracking. Although the system achieves good performance in face detection, it does not perform as well for classification using faces alone (especially when the faces are similar, as in twin animals). Here, too, the color-coded beads play a key role in identity discrimination. The stated goals of the study and the actual results presented are therefore at odds.

Reviewer #2 (Public review):

Summary:

The authors used an LFA-1 αI-Fc fusion protein to pull down potential ligands and LC-MS/MS, leading to the selection of PfGBP-130 as a potential membrane protein on the surface of infected cells. PfGBP-130 antibodies were raised and used to support the surface localization. This putative ligand interacted strongly with LFA-1 (Kd = 15 nM). A presumed PfGBP-130 ectodomain interacts with monocytes and NK cells but not cells that lack LFA-1. PfGBP-130 antibodies also interfered with NK cell-mediated infected cell killing; the effect, although statistically significant, is modest. The authors propose that NK cells recognize infected cells via LFA-1 interaction with PfGBP-130 exposed on the host cell and that this interaction is critical to initiation of NK cell activation and killing of infected cells.

Major points:

(1) PfGBP-130 is proposed to be a membrane protein based on a single predicted transmembrane domain. Figures 2b and 3a show ribbon schematics with this TM domain at residues 51-68, in agreement with TM prediction algorithms such as TMHMM 2.0 and Phobius. However, this predicted TM is upstream of the PEXEL motif (residues 84-88, sequence RILAE), a conserved sequence for parasite protein export to host cytosol that is proteolytically processed at its 4th residue. Thus, residues 1-87 are removed from PfGBP-130 prior to export, yielding a mature protein without predicted TMs. Prior studies have determined that the mature PfGBP-130 lacks TMs and is retained as a soluble protein in host cell cytosol (PMID: 19055692, 35420481). Thus, the authors' model of PfGBP-130 as a surface-exposed membrane protein conflicts with both computational analysis of the mature protein and these prior reporter studies. An important simple experiment would be to evaluate PfGBP-130 membrane association in immunoblots using the authors' PfGBP-130 antibody after hypotonic lysis (PMID: 19055692) and after alkaline extraction (e.g. 100 mM NaCO3, pH 11 as frequently used, PMID: 33393463). If the prior studies and computational analyses are correct, the protein will be predominantly in the soluble and/or alkaline supernatant fractions.

(2) Many findings rely on the specificity of antibodies generated against PfGPB-130 or NK cell receptors. Although the authors have included key controls (use of isotype control antibodies, lack of anti-PfGBP-130 binding to uninfected cells), cross-reactivity between P. falciparum antigens is well-recognized and could significantly undermine the interpretation of experiments (PMID: 2654292 and 1730474 provide key examples of antigens recognized by antibodies raised against other proteins). For example, the surface localization in IFA experiments (Figure 2B(iii)) could reflect anti-PfGBP-130 binding to an unrelated parasite surface antigen, a possibility not addressed by any of the authors' controls. As another example, the iRBC lysate immunoblot using this antibody in Fig. 2B(iv) suggests a MW of 95 kDa, which corresponds to the unprocessed pre-protein before export; cleavage in the PEXEL motif yields a processed mature protein of 85 kDa, which should be readily resolved from the pre-protein in immunoblots (PMID: 19055692). A better immunoblot using immature infected cell stages might show both the pre-protein and the mature protein as a doublet band.

(3) PfGBP-130 is not essential for in vitro cultivation (PMID: 18614010 and MIS of 1.0 in the piggyBac mutagenesis screen as tabulated on plasmodb.org, indicating a highly dispensable gene). The authors should use the knockout line as a control in their IFA localization experiments to address antibody specificity. More fundamentally, their model predicts that NK cells should not recognize or kill infected cells from the knockout line when compared to their untransfected parent. Such results with the knockout line would compellingly support the authors' model without reliance on antibodies that may cross-react with other parasite antigens. PMID: 18614010 reported that the PfGBP-130 knockout exhibited increased membrane rigidity, suggesting an intracellular scaffolding protein rather than a surface localization and use as a ligand for LFA-1 interaction and NK cell-mediated killing.

(4) PfGBP-130 non-essentiality raises the question of why the gene would be retained if it triggers NK cell-mediated killing of infected cells in vivo. Presumably, this killing would pose strong selective pressure against retention of PfGBP-130. Some speculation is warranted to support the model.

Reviewer #2 (Public review):

In 'Developmental constraints mediate the summer solstice reversal of climate effects on European beech bud set [their original title]' Rebindaine and co-authors report on two experiments on Fagus sylvatica where they manipulated temperatures of saplings between day and night and at different times of year. I think the experiments are interesting, but I found the exact methods of them somewhat extreme compared to how the authors present them. Further, given that much of the experiment happened outside, I am not sure how much we can generalize from one year for each experiment, especially when conducted on one population of one species. I was also very concerned by the revisions.

I expand briefly on these concerns and a few others for readers of the paper (see `The below comments relate to my original review'). Subsequent edits to the paper addressed some of these by providing a new figure and moving around the methods. Further, I am at a loss about their hypothesis, when they write in their letter: "Importantly, the Solstice-as-Phenology-Switch hypothesis does not assume that the reversal is fixed to June 21." Why on earth reference the solstice if the authors do not mean to exactly reference the solstice?

The comments below relate to my original review with many of them still applying.

Methods: As I read the Results I was surprised the authors did not give more info on the methods here. For example, they refer to the 'effect of July cooling' but never say what the cooling was. Once I read the methods I feared they were burying this as the methods feel quite extreme given the framing of the paper. The paper is framed as explaining observational results of natural systems, but the treatments are not natural for any system in Europe of which I have worked in. For example a low of 2 deg C at night and 7 deg C during the day through end of May and then 7/13 deg C in July is extreme. I think these methods need to be clearly laid out for the reader so they can judge what to make of the experiment before they see the results.

I also think the control is confounded with growth chamber experience in Experiment 1. That is, the control plants never experience any time in a chamber, but all the treatments include significant time in a chamber. The authors mention how detrimental chamber time can be to saplings (indeed, they mention an aphid problem in experiment 2) so I think they need to be more upfront about this. The study is still very valuable, but -- again -- we may need to be more cautious in how much we infer from the results.

Also, I suggest the authors add a figure to explain their experiments as they are very hard to follow. Perhaps this could be added to Figure 1?

Finally, given how much the authors extrapolate to carbon and forests, I would have liked to see some metrics related to carbon assimilation, versus just information on timing.

Fagus sylvatica: Fagus sylvatica is an extremely important tree to European forests, but it also has outlier responses to photoperiod and other cues (and leafs out very late) so using just this species to then state 'our results likely are generalisable across temperate tree species' seems questionable at best.

Measuring end of season (EOS): It's well known that different parts of plants shut down at different times and each metric of end of season -- budset, end of radial expansion, leaf coloring etc. -- relate to different things. Thus I was surprised that the authors ignore all this complexity and seem to equate leaf coloring with budset (which can happen MONTHS before leaf coloring often) and with other metrics. The paper needs a much better connection to the physiology of end of season and a better explanation for the focus on budset. Relatedly, I was surprised the authors cite almost none of the literature on budset, which generally suggests is it is heavily controlled by photoperiod and population-level differences in photoperiod cues, meaning results may different with a different population of plants.

Somewhat minor comments:<br /> (1) How can a bud type -- which is apical or lateral -- be a random effect? The model needs to try to estimate a variance for each random effect so doing this for n=2 is quite odd to me. I think the authors should also report the results with bud type as fixed, or report the bud types separately.<br /> (2) I didn't fully see how the authors results support the Solstice as Switch hypothesis, since what timing mattered seemed to depend on the timing of treatment and was not clearly related to solstice. Could it be that these results suggest the Solstice as Switch hypothesis is actually not well supported (e.g., line 135) and instead suggest that the pattern of climate in the summer months affects end of season timing?

Reviewer #2 (Public review):

Summary:

Protein tyrosine kinases are subject to diverse regulatory mechanisms controlling their activity in normal situations. The authors previously identified SLAP (Src-like adaptor protein), a negative regulator of receptor tyrosine kinase (RTK) signaling, as a key suppressor of the cytoplasmic tyrosine kinase SRC in the normal colon and demonstrated that SLAP is downregulated in a majority of colorectal cancers (CRCs).

In this study, the authors further explored SLAP functions in mouse models using constitutive and inducible epithelial-specific Slap deletion (villin-CreERT2 model). They found that loss of SLAP augments colonic epithelial cell proliferation and that induction of tumorigenesis by the AOM/DSS protocol mimicking CRC leads to more aggressive tumors in the absence of SLAP. This effect is apparently cell-autonomous as growth of normal and tumoral colonic organoids is SLAP-dependent in in vitro settings. Finally, the authors define that, in colon, SLAP represses EphB2, an RTK lying upstream of SRC, and show that inhibitors of EphB2 can partially limit tumorigenic development in vitro.

Strengths:

The manuscript is clearly and concisely written, making it easy to follow. The data obtained in the mouse models are very convincing.

Weaknesses:

Direct evidence that EphB2 is activated/phosphorylated in the absence of SLAP is lacking, as conclusions are only based on results obtained with inhibitors. Some other issues have to be addressed before acceptance, in particular, the relevance of the findings in CRC patients.

Reviewer #2 (Public Review):

Summary:

In this work, Xiong and colleagues examine the relationship between the profile of the morphogen Shh and the resulting cell fate decisions in the zebrafish neural tube. For this, the authors combine high-resolution live imaging of an established Shh reporter with reporter lines for the different progenitor types arising in the forming neural tube. One of the key observations in this manuscript is that, while, on average, cells respond to differences in Shh activity to adopt distinct progenitor fates, at the single cell level there is strong heterogeneity between Shh response and fate choices. Further, the authors showed that this heterogeneity was particularly prominent for the pMN fate, with similar Shh response dynamics to those observed in neighboring LFP progenitors.

Strengths:

It is important to directly correlate Shh activity with the downstream TFs marking distinct progenitor types in vivo and with single cell resolution. This additional analysis is in line with previous observations from these authors, namely in Xiong, 2013. Further, the authors show that cells in different anterior-posterior positions within the neural tube show distinct levels of heterogeneity in their response to Shh, which is a very interesting observation and merits further investigation.

Weaknesses:

This is a convincing work, however, adding a few more analyses and clarifications would, in my view, strengthen the key finding of heterogeneity between Shh response and the resulting cell fate choices.

Reviewer #3 (Public review):

Summary:

This study addresses a central question in genome organization: whether the positions of chromosomal domain boundaries are functionally coupled to gene activity. The authors use high-throughput imaging to simultaneously measure distances between boundary markers and nascent RNA production in thousands of individual cells, enabling direct comparison of boundary positions and transcriptional status at single chromosomal copies. This approach is applied across multiple loci, genes, and cell types, and is combined with acute transcriptional perturbations and depletion of architectural proteins to test the relationship between chromosome structure and gene activity in both directions.<br /> This work makes a meaningful contribution by providing direct, single-cell evidence that domain boundary positions and gene activity are largely uncoupled in this system.

Strengths:

A major strength of the work is its single-cell, single-allele resolution, which overcomes the averaging inherent to population-based assays. The authors consistently find that boundary proximity is largely independent of transcriptional status: active and inactive alleles have similar boundary distances, transcriptional perturbations do not shift boundary distributions, and depletion of the boundary factor CTCF does not alter gene expression, whereas cohesin depletion affects both boundary organization and transcription. These conclusions are supported by large numbers of alleles, multiple loci and cell types, and internal controls that distinguish boundary-specific effects from broader chromatin influences. The study offers a robust, scalable imaging pipeline that will be valuable for future studies linking genome organization and transcription at single-cell resolution.

Weaknesses:

The study has important limitations that are acknowledged by the authors. Measurements are restricted to distances between flanking boundaries and do not capture internal domain architecture, sub-domain structure, or finer-scale regulatory contacts. Resolution is limited by probe size and imaging, potentially masking subtle positional changes, and only a small set of loci is examined, leaving open how broadly the uncoupling generalizes. Some perturbation effects, particularly for RAD21, may involve mechanisms beyond boundary disruption.

Reviewer #2 (Public review):

The goal of the study was to uncover the mechanisms mediating tactile-context-dependent locomotion modulation in C. elegans, which represents an interesting model of behavioral plasticity. Starting from a candidate genetic screen focusing on guanylate cyclase (GCY) mutants, the authors identified the AFD-specific gcy-18 gene as essential for tactile-context-dependent locomotion modulation. AFD has been primarily characterized as a thermosensory neuron. However, key thermosensory transduction genes and the sensory ending structure of AFD were shown here to be dispensable for tactile-context locomotion modulation. AFD actuates tactile-context locomotion modulation via the cell-autonomous actions of GCY-18 and the CNG-3 cyclic nucleotide-gated channel, and via AFD's connection with AIB interneurons through electrical synapses. At the circuit level, AIB also receive inputs from the mechanosensory neuron FLP, which was also shown to be relevant for tactile-context-dependent locomotion modulation.

For this study, the authors combined a very clever microfluidic-based behavioral assay with a large set of genetic manipulations to dissect the molecular and cellular pathways involved. Rescue experiments with single-copy transgenes are particularly convincing. The study is very clearly written, and the figures are nicely illustrated with diagrams that effectively convey the authors' interpretation. Overall, the convergence of behavioral assays, genetics, and circuit analysis provides convincing support for the proposed role of the AFD-AIB connection, potentially downstream of FLP via synapic and of other mechanosensory neurons via extra-synaptic communication.

The facts that AFD mediates tactile-context locomotion modulation, that this role relies on GCY-18, and on electrical synapses linking AFD to AIB are new, somewhat unexpected, and interesting. The study raises intriguing and addressable questions about the role of innexin-based cellular communication in a multimodal sensory-behavior microcircuit, including the direction and nature of the signal(s) transmitted through these electrical synapses. These questions remain difficult to address in most experimental systems. The compact and genetically tractable nervous system of C. elegans provides a powerful entry point for addressing them in the context of an intact in vivo circuit.

Reviewer #2 (Public review):

Summary:

In this paper, the authors set out to better understand the genetic mechanisms underlying thermal adaptation in insects. They experimentally evolved diamondback moth (Plutella xylostella) populations - a pest species with a wide distribution - under both hot (12h:12h 32{degree sign}C/27{degree sign}C) and cold (15{degree sign}C/10{degree sign}C) thermal conditions, and conducted phenotypic assays and metabolic and transcriptomic profiling to analyze how populations changed to deal with this thermal stress compared to the nonevolved ancestral population (constant 26{degree sign}C). Phenotypic assays showed that evolved hot populations had increased survival at high temperatures (42-43{degree sign}C) while evolved cold populations had lower freezing points compared to the ancestral population. When measured at the constant 26{degree sign}C conditions, metabolic and transcriptomic profiles of 3rd instar larvae from the evolved population were distinctive from the ancestral population, with a set of overlapping metabolic and transcriptomic pathways that were significantly differentially expressed in both hot and cold evolved populations compared to the ancestral. The authors narrowed down this set of candidate genes further by focusing on genes with high expression levels overall, whose expression profile was correlated with differentially expressed metabolites, and that contained mutants in both hot and cold strains. From this set, they chose the PxSODC gene for further functional validation, as it has previously been shown to be involved in the response of insects to abiotic stress with its antioxidative role in cellular defense. At the constant 26{degree sign}C, this gene showed lower expression across development in evolved strains compared to the ancestral population, while it showed similar expression patterns under thermal stress. Knockdown of PxSODC resulted in decreased survival rates at high temperatures and higher freezing points compared to the ancestral population. Based on this validation, the authors hypothesize that the non-synonymous mutation in the PxSODC gene that they found in the cold and hot evolved populations might alter the conformation of the PxSODC protein, increasing enzyme capacity. Their experimental evolution experiment furthermore indicates the capacity of the pest species, the diamondback moth, to adapt to a wide range of temperatures, providing insights into its capacity for global dispersal.

Strengths:

(1) The authors did a tremendous amount of work to characterize the mechanisms underlying thermal adaptation in the diamondback moth, artificially selecting populations for three years in the lab and characterizing how they evolved as a result at different biological levels: from phenotypes in different life stages, to larval metabolites and gene transcription, to functionally validating how one of the resulting gene candidates influences the capacity to deal with thermal stress.

(2) The paper identifies and provides further evidence for candidate genetic mechanisms that might be particularly important for thermal adaptation in insects, including lipid metabolism, oxidoreductase activity, and DNA methylation. It is furthermore interesting that the authors found similar mechanisms to be involved in both the adaptation to cold and hot environments. Their functional validation of some of the genes involved in these mechanisms is very useful to understand how these genes might be causally involved in insect thermal adaptation.

(3) The paper also has applied value: the diamondback moth is a pest species with a wide distribution, so understanding its adaptive capacity to different thermal environments is important for predicting the prevalence and potential further range expansion of this species under future climate change.

Weaknesses:

(1) The paper in its current form is hard to digest and would benefit from improved clarification of the storyline, as well as a tighter integration between the phenotypic, omics, and functional validation data. Currently, it is not always clear what the relevance is of all the reported results, nor why certain decisions were made, or how all the different methods the authors used fit together. For example, the authors functionally validated a second gene, PxDnmt1, but it is unclear why this particular gene was chosen, nor how it relates to their selection regimes when looking at the results obtained with the phenotyping and omics data collection. Seeing how much work the authors did, this makes the paper overwhelming and difficult to read.

(2) The authors at times stretch their results too far, as the ecological relevance of their study design and results is not clear, limiting the generalizability and value of the results for understanding species' adaptive potential under climate change. For example, the selection regimes used present the minimum and maximum known temperatures at which the species can survive and develop, but it is unclear how the temperatures relate to the natural environment of the source population, to what extent wild populations might experience these temperatures, and whether they would experience them at the extended duration used (12h at max/min temperature). Moreover, I wonder whether the comparisons made would identify the genes that matter under natural conditions, as unevolved populations were kept under constant conditions compared to 12h:12h temperature regimes for the evolved populations, and the metabolic and transcriptomic profiling was done under a constant favorable 26{degree sign}C rather than under thermal stress in a, as far as I can tell, randomly chosen life stage (larval stage).

(3) The paper in its current form does not adequately describe the statistical analyses underlying the results, nor do the authors share their code, making it very hard to judge whether the analyses used are appropriate and the results trustworthy. I have concerns about the inappropriate use of t-tests, the lack of correcting for confounding variables, and the need for multiple testing corrections.

Reviewer #2 (Public review):

In this study, the authors investigate the spatial organization of direction and orientation selectivity in the mouse superior colliculus (SC) and its retinal inputs. By combining two-photon imaging of retinal boutons and SC neurons with Neuropixels recordings, they assess whether tuning preferences form structured maps or are arranged in a salt-and-pepper fashion. They further compare SC tuning organization to previously described retinal geometric models to determine the extent to which collicular responses inherit retinal topography. The authors conclude that SC inherits a cardinally biased topographic scaffold from the retina, which progressively weakens with depth, and that strong global maps are absent.

A major strength of the study is the impressive combination of methodologies, including imaging of retinal boutons, imaging of SC neurons, and large-scale electrophysiological recordings across SC depth. The direct comparison to retinal geometric models is particularly valuable, as it situates the SC within a broader framework of retinotopic information transfer and advances our understanding of how retinal computations are transformed in downstream targets.

A limitation of the study, however, is that the imaging experiments sample only a relatively small and spatially homogeneous region of the visual field, whereas the electrophysiological recordings cover a different portion of SC. This separation makes it difficult to form a fully integrated, global picture of the spatial organization of direction and orientation selectivity across the entire collicular map.

Reviewer #2 (Public review):

This well-written manuscript proposes to use attractors in space and time (STA) as a mechanistic explanation for planning in the prefrontal cortex. The main conceptual hypothesis is that planning is implemented as attractor dynamics in a representation that encodes states at each time step jointly. Depending on inputs, the network relaxes to a trajectory that already contains future states that will be visited at each time step, rather than computing a scalar value at each point in time and space like other classical approaches from RL. The authors compare this approach to implementations such as TD learning and successor representation, and further show that trained recurrent neural networks on specific tasks involving planning develop structured subspaces resembling the ones postulated in STA.

The idea of treating attracting trajectories unfolding in time as the computational substrate for planning is very interesting and potentially important. The explicit construction of a state x time representational space and its implementation via recurrent dynamics are appealing and convincing in the idealized tasks considered. I found the manuscript to be refreshingly explicit regarding several of the assumptions and limitations of the models, for example, the fact that certain advantages can be viewed as properties of the state space itself and not necessarily of a fundamentally new planning mechanism.

Overall, the manuscript presents a cool attractor model that extends in time and explores its performance in a subset of illustrative tasks involving planning. My doubts concern mostly the interpretation and scope of the claims made in the manuscript. Here are a few comments where I detail my questions/concerns:

(1) The authors nicely discuss that much of the difference between STA and classical TD or SR agents is "in some sense a property of the state space rather than the decision making algorithm," and that TD and SR could in principle be implemented in a comparable space x time representation. This is fair, but it also suggests that the central contribution of the manuscript lies primarily in the representational factorization (state x time tiling) and its dynamical implementation via attractors, rather than in a fundamentally new planning algorithm or theory, mechanistic or not. I think theory should be distinguished from mechanism, and it would therefore help the reader to describe the conceptual advancement more as a novel mechanism or implementation than a novel (mechanistic) theory for decision/planning.

(2) Related to my previous point, I think it would be helpful to position STA more explicitly relative to computational/theoretical literature in which attractor networks encode temporally ordered patterns (so effectively including future times). For example, classical extensions of Hopfield networks with asymmetric connectivity implement retrieval of sequences and ordered transitions between patterns (Sompolinsky & Kanter, 1986). More recently, sequential attractors and limit-cycle dynamics have been constructed in structured recurrent networks by the Morrison group (Parmelee et al., 2021). These works do not implement an explicit discretized state x future-time tiling as in STA and do not specifically discuss the usage for planning. However, they do provide concrete precedents for attractor dynamics over temporally structured trajectories in terms of mechanism. It would be useful to discuss this literature and clarify a little what's new mechanistically in the view of the authors.

(3) A central claim of the manuscript is that space-time trajectories are attractors of the STA dynamics. The manuscript does provide empirical evidence consistent with attractor-like behavior. However, it is not explicitly shown whether trajectory representations persist in the absence of sustained external inputs. So it's not clear to me whether the trajectories should be interpreted as intrinsic attractors of the recurrent system, which can be selected by delivering transient inputs, or whether they must be stabilized by a specific continuous external drive. It would be useful if the author could clarify/discuss this point.

As far as I understand it, reward information is provided as input to specific populations encoding future time steps, and that's essential for rapid adaptation without rewiring connectivity. How such future-time-specific reward inputs would be generated and routed to distinct neural populations isn't entirely clear to me. Since this seems to be an essential component of the model, I think it would be important to discuss more deeply the source and plausibility of these reward signals related to different timesteps.

(4) The authors note that vanilla STA scales linearly with planning horizon, and discuss potentially hierarchical extensions for longer horizons. They acknowledge that learning abstractions remains an open challenge, yet the examples of planning in the manuscript are restricted to very short temporal horizons and limited branching complexity. It is not obvious to me in what cases the current implementation and interpretation of STA remains viable (for example, in terms of relaxation iterations) as the horizon and branching factor increase. Relatively simple planning can be managed by simpler, less costly models/algorithms, whereas complex planning is a lot harder to deal with, and it's something that a mechanistic "theory" should address. In the context of the claims of the paper in its present form, I think this is possibly the most important conceptual and practical limitation in the manuscript.

(5) The RNN analyses show that trained networks develop structured subspaces aligned with future time indices and exhibit perturbation behavior consistent with attractor-like dynamics. The manuscript also explicitly notes differences between the trained RNN and the handcrafted STA (e.g., long-range couplings between subspaces and differences in behavior of lower-value trajectories under perturbation), which I much appreciated. My doubt is on the specificity of this result, as trained RNNs on fixed-horizon tasks can develop latent dimensions correlated with temporal progress within a trial or time-to-goal. I think it would help the reader to clarify whether the results demonstrate that STA-like computations emerge in RNNs trained on planning tasks, or that RNNs generally develop some kind of structured spacetime representations when tasks involve future timesteps and some degree of flexibility in the decisions.

A few more minor points, mainly concerning clarity:

(1) The main dynamical equation combines a log-domain recurrent term, a floor operation, and a log-sum-exp normalization step, followed by exponentiation. The intuition/logic behind this specific formulation could be clarified for the reader. For example tt would be helpful to explain why the recurrent input appears inside a log, and also whether/how these operations relate to any multiplicative constraint.

(2) While the computational cost of successor representation in an expanded NT x NT representation is discussed, the corresponding scaling of STA in terms of number of units and connections (as a function, for example, of the planning horizon) isn't clear to me. Perhaps the authors could compare costs more explicitly.

(3) In the RNN analyses, structured subspaces aligned with future time indices are shown. I couldn't find a quantification of how much variance is captured by the subspaces, relative to other latent dimensions. Adding it would help get a feeling for the strength of the alignment.

Reviewer #2 (Public review):

In this manuscript, Aguilera et al. investigate the mechanisms underlying transcriptional repression of constitutively expressed genes during heat stress. While the activation of heat-shock genes has been extensively studied, the mechanisms responsible for widespread transcriptional repression remain poorly understood. The authors propose that the GA-binding transcription factor CLAMP acts as a major regulator of heat-stress-induced transcriptional repression in Drosophila. Using nascent RNA-sequencing approaches, they report that CLAMP contributes to the repression of a large fraction of genes whose transcription decreases upon heat stress. In addition, the authors generate high-resolution Micro-C datasets to analyze changes in chromatin architecture during heat stress and report widespread alterations in chromatin looping associated with transcriptional changes. Based on these results, the study proposes that CLAMP regulates repression through both direct transcriptional mechanisms and modulation of local 3D genome architecture.

The study addresses an important question in gene regulation: how transcription is rapidly repressed during environmental stress. The work is timely because most previous studies have focused on transcriptional activation of heat-shock genes, whereas repression mechanisms remain comparatively less understood. The integration of transcriptional profiling with high-resolution chromatin conformation data is a major strength of the manuscript and provides a valuable resource for the community studying genome organization and stress responses.

The nascent RNA-sequencing experiments appear carefully designed and allow the authors to capture rapid transcriptional responses to heat stress. These data provide convincing evidence that heat stress leads to widespread transcriptional repression of constitutive genes and that CLAMP contributes substantially to this process. The genomic analyses linking CLAMP binding to repressed genes are also informative and support the idea that CLAMP plays a direct regulatory role at many loci.

Another strength of the study is the generation of Micro-C datasets under heat stress conditions. These datasets provide a high-resolution view of chromatin architecture and reveal dynamic changes in local chromatin looping associated with transcriptional responses. The authors' analysis suggests that heat stress induces widespread reorganization of chromatin contacts, and that CLAMP may contribute to these structural changes. This dataset is likely to be useful for future studies exploring how environmental cues influence genome organization.

Despite these strengths, several aspects of the study would benefit from further clarification. First, the mechanism by which CLAMP mediates transcriptional repression remains insufficiently defined. While the data support a role for CLAMP in the repression of a subset of genes during heat stress, the molecular basis of this effect is not fully explored. Second, although the Micro-C dataset represents a valuable resource for studying chromatin architecture during heat stress, the functional interpretation of the observed structural changes could be further developed. In particular, it would be helpful to better establish the relationship between the identified chromatin loops and gene regulation, and to clarify whether these structural changes play a causal role in transcriptional repression or instead reflect broader chromatin reorganization associated with the stress response.

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

sentences that cite other researchers that aren't already highlighted

Venoactive drugs (diosmin, hesperidin, horse chestnut seed extract) may be considered as adjuncts to compression for symptomatic relief in countries where available

Endovenous ablation is contraindicated or relatively unsuitable when venous anatomy precludes catheter-based treatment, specifically: aneurysmal dilation of the GSV close to the saphenofemoral junction, subcutaneous location of truncal veins above the saphenous fascia and close to the skin, and significant tortuosity of the GSV or SSV. [1] In these scenarios, high ligation and stripping is recommended as the preferred alternative (grade 1 strong recommendation

Reviewer #3 (Public review):

I have carefully reviewed the manuscript, the two referee reports, and the authors' detailed responses. I appreciate the substantial effort the authors have invested in addressing the reviewers' comments, and I also recognize the strength and ambition of the work. This is a technically sophisticated study that integrates coarse-grained modeling with live-cell imaging to address an important and timely question regarding HIV-1 capsid inhibition by lenacapavir.

Embedded within Reviewer #2's report are several substantive points that warrant careful consideration, particularly with respect to framing, terminology, and engagement with the broader literature. I view my role here is to distinguish those issues from claims that I do not find to be supported.

First, I do not agree with Reviewer #2's central assertion that the manuscript lacks novelty or fails to present meaningful new findings. While individual elements of the system studied here-capsid docking at the NPC, lenacapavir-induced capsid hyperstabilization, capsid rupture, and competition with FG- nucleoporins-have been observed previously, this work provides a coherent, mechanistic account of how these elements are coupled. In particular, the proposed sequence linking LEN-induced lattice hyperstabilization, preferential pentamer loss at the narrow end, NPC-induced mechanical stress, and failure of nuclear import represents a nontrivial integration that goes beyond prior phenomenological observations. I therefore do not view this work as redundant with existing literature.

That said, Reviewer #2 is correct to note that the manuscript would benefit from broader and more explicit engagement with recent independent studies, including computational and hybrid modeling efforts that address capsid mechanics, nuclear entry, and LEN effects using different frameworks. While the authors' bottom-up coarse-grained approach is clearly distinct and, in many respects, more systematically derived, eLife readers would benefit from a clearer discussion of how the present results relate to, complement, or differ from these other approaches. I strongly encourage the authors to add a short discussion paragraph situating their work within this broader context, without disparaging alternative models.

Second, I find that some mechanistic claims in the manuscript would benefit from more careful language distinguishing model-conditioned interpretation from de novo prediction. This applies in particular to discussions of LEN binding heterogeneity and stoichiometry, as well as to conclusions drawn from biased enhanced-sampling simulations. While I agree with the authors that parameterization does not invalidate mechanistic insight, it is important to be precise about what aspects of the behavior emerge from the simulations versus what is constrained by prior experimental knowledge. Modest tightening/revising of language (e.g., "suggests," "is consistent with," "within the model") would address this concern without weakening the scientific conclusions.

Third, Reviewer #2 raises a legitimate semantic issue regarding the use of the term "elasticity." The manuscript infers changes in capsid mechanical response using heterogeneous elastic network models, which quantify effective stiffness and deformability rather than elasticity in the macroscopic materials sense. I recommend that the authors clarify this definition explicitly in the text to avoid confusion and unnecessary debate.

Finally, I note that several of Reviewer #2's objections-particularly those asserting circular reasoning, misuse of enhanced sampling methods, or invalidity of coarse-grained predictions-reflect a misunderstanding of contemporary bottom-up coarse-grained modeling rather than genuine methodological flaws. I do not believe these points require further rebuttal or revision beyond what the authors have already provided.

In summary, in my view, the manuscript represents a solid contribution to the field, provided that the authors undertake a limited set of targeted revisions aimed at improving framing, clarity, and engagement with the broader literature. Addressing these points will strengthen the manuscript and ensure that its contributions are clearly and fairly communicated to the community.

Reviewer #2 (Public review):

Summary:

The authors are trying to understand why certain mutants of O-GlcNAc transferase (OGT) appear to cause developmental disorders in humans. As an important step towards that goal, the authors generated a mouse model with one of these mutations that disrupts OGT activity. They then go on to test these mice for behavioral differences, finding that the mutant mice exhibit some signs of hyperactivity and differences in learning and memory. They then examine alterations to the structure of the brain and skull, and again find changes in the mutant mice that have been associated with developmental disorders. Finally, they identify proteins that are up or down regulated between the two mice as potential mechanisms to explain the observations.

Strengths:

The major strength of this manuscript is the creation of this mouse model, as a key step in beginning to understand how OGT mutants cause developmental disorders. This line will prove important for not only the authors but other investigators as well, enabling the testing of various hypotheses and potentially treatments. The experiments are also rigorously performed and the conclusions are well supported by the data.

Weaknesses:

The only weakness is a lack of mechanistic insight. However, this certainly may come in the future through more targeted experimentation using this mouse model. I do not recommend that these experiments need to be performed in this manuscript.

Comments on revisions:

The authors have addressed all of my suggestions proactively.

Reviewer #2 (Public review):

Summary:

In this study the authors follow up on a previous suppressor screen of a temperature-sensitive allele of mis4 (mis4-G1487D), the cohesin loading factor in S. pombe, and identify additional suppressor alleles tied to the S. pombe TORC1 complex. Their analysis suggests that these suppressor mutations attenuate TORC1 activity while enhanced TORC1 activity is deleterious in this context. Suppression of TORC1 activity also ameliorates chromosome segregation and spindle defects observed in the mis4-G1487D strain, although some more subtle effects are not reconstituted. The authors provide evidence that this genetic suppression is also tied to the reconstitution of cohesin loading. Moreover, disrupting TORC1 also enhances Mis4/cohesin association with chromatin (likely reflecting enhanced loading) in WT cells while rapamycin treatment can enhance the robustness of chromosome transmission. These effects likely arise directly through TORC1 or its downstream effector kinases as TORC1 co-purifies with Mis4 and Rad21; these factors are also phosphorylated in a TORC1-dependent fashion. Disrupting Sck2, a kinase downstream of TORC1, also suppresses the mis4-G1487D allele while simultaneous disruption of Sck1 and Sck2 enhances cohesin association with chromatin, albeit with differing effects on phosphorylation of Mis4 and Psm1/Scm1. Phosphomutants of Mis4 and Psm1 that mimic observed phosphorylation states identified by mass spectrometry that are TORC1-dependent also suppressed phenotypes observed in the mis4-G1487D background. Lastly, the authors provide evidence that the mis4-G1487D background and TORC1 mutant backgrounds display an overlap in the dysregulation of genes that respond to environmental conditions.

Overall, the authors provide compelling evidence from genetics, biochemistry and cell biology to support a previously unknown mechanism by which nutrient sensing regulates cohesin loading with implications for the stress response. The technical approaches are generally sound, well-controlled, and comprehensive.

The specific points that I raised in the first review have been addressed by changes/additions to the manuscript or have been determined to be beyond the scope of the study by the authors.

One major question that remains open is the relationship between local changes in cohesin loading and gene expression through this TORC1 regulatory signaling pathway and the details of the underlying mechanisms.

Reviewer #2 (Public review):

The work by Spokaite et al describes the discovery of a novel Rab5 binding site present in complex II of class III PI3K using a combination of HDX and Cryo EM. Extensive mutational and sequence analysis define this as the primordial Rab5 interface. The data presented are convincing that this is indeed a biologically relevant interface, and is important in defining mechanistically how vps34 complexes are regulated.

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

They used their increased affinity vps34 mutant to determine the 3.2 ang structure of Rab5 bound to vps34-CII. Clear density was seen for the original Rab5 interface, but an additional site was observed. Based on this structure they mutated out the vps34 interface, allowing for a high resolution structure of the Rab5 bound at the Vps15 interface.

They extensively validated the vps15 interface in the yeast variant of vps34, showing that the Vp215-Rab5 (Vps21) interface identified is critical in controlling complex II vps34 recruitment.

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

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

(1) My main concern has to do a little bit with presentation. My main issue is how the authors use mutant description. They clearly indicate the mutant sequence in the human isoform (for example see Fig 2A, Vps15 described as 579-SHMIT-583>DDMIE), however, when they shift to the yeast version they shift to saying vps15 mutant, but don't define the mutant, Fig 2G). I would recommend they just include the same sequence numbering and WT to mutant replacement every time a new mutant (or species) is described. It is always easier to interpret what is being shown when the authors are jumping between species when the exact mutant is included. This is particularly important in this paper, where we are jumping between both different subunits and different species, so clear description in figure/figure legends makes it much easier to read for non-specialists.

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

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

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

Comments on revisions:

The authors have addressed all of my issues.