Reviewer #1 (Public review):

This study aims to identify the proteins that compose the electrical synapse, which are much less understood than those of the chemical synapse. Identifying these proteins is important to understand how synaptogenesis and conductance are regulated in these synapses.

Using a proteomics approach, the authors identified more than 50 new proteins and used immunoprecipitation and immunostaining to validate their interaction of localization. One new protein, a scaffolding protein (Sipa1l3), shows particularly strong evidence of being an integral component of the electrical synapse. The function of Sipa1l3 remains to be determined.

Another strength is the use of two different model organisms (zebrafish and mice) to determine which components are conserved across species. This approach also expands the utility of this work to benefit researchers working with both species.

The methodology is robust and there is compelling evidence supporting the findings.

Comments on revisions:

I thank the authors for responding to the comments. No further recommendations.

Reviewer #1 (Public review):

Summary:

The authors note that there is a large corpus of research establishing the importance of LC-NE projections to medial prefrontal cortex (mPFC) of rats and mice in attentional set or 'rule' shifting behaviours. However, this is complex behavior and the authors were attempting to gain an understanding of how locus coeruleus modulation of the mPFC contributes to set shifting.

The authors replicated the ED-shift impairment following NE denervation of mPFC by chemogenetic inhibition of the LC. They further showed that LC inhibition changed the way neurons in mPFC responded to the cues, with a greater proportion of individual neurons responsive to 'switching', but the individual neurons also had broader tuning, responding to other aspects of the task (i.e., response choice and response history). The population dynamics was also changed by LC inhibition, with reduced separation of population vectors between early-post-switch trials, when responding was at chance, and later trials when responding was correct. This was what they set out to demonstrate and so one can conclude they achieved their aims.

The authors concluded that LC inhibition disrupted mPFC "encoding capacity for switching" and suggest that this "underlie[s] the behavioral deficits."

Strengths:

The principal strength is combining inactivation of LC with calcium imaging in mPFC. This enabled detailed consideration of the change in behavior (i.e., defining epochs of learning, with an 'early phase' when responding is at chance being compared to a 'later phase' when the behavioral switch has occurred) and how these are reflected in neuronal activity in the mPFC, with and without LC-NE input.

Comments on revised version:

In their response to reviewers, the authors say "We report p values using 2 decimal points and standard language as suggested by this reviewer". However, no changes were made in the manuscript: for example, "P = 4.2e-3" rather than "p = 0.004".

In their response to the reviewers, they wrote: "Upon closer examination of the behavioral data, we exclude several sessions where more trials were taken in IDS than in EDS." If those sessions in which EDSIDS. Most problematic is the fact that the manuscript now reads "Importantly, control mice (pooled from Fig. 1e, 1h, Supp. Fig. 1a, 1b) took more trials to complete EDS than IDS (Trials to criterion: IDS vs. EDS, 10 {plus minus} 1 trials vs. 16 {plus minus} 1 trials, P < 1e-3, Supp. Fig. 1c), further supporting the validity of attentional switching (as in Fig. 1c)" without mentioning that data has been excluded.

Reviewer #1 (Public review):

Summary:

The paper uses rigorous methods to determine phase dynamics from human cortical stereotactic EEGs. It finds that the power of the phase is higher at the lowest spatial phase. The application to data illustrates the solidity of the method and their potential for discovery.

Comments on revised submission:

The authors have provided responses to the previous recommendations.

Reviewer #1 (Public review):

Summary:

The authors scrutinized differences in C-terminal region variant profiles between Rett syndrome patients and healthy individuals and pinpointed that subtle genetic alternation can cause benign or pathogenic output, which harbors important implications in Rett syndrome diagnosis and proposes a therapeutic strategy. This work will be beneficial to clinicians and basic scientists who work on Rett syndrome, and carries the potential to be applied to other Mendelian rare diseases.

Strengths:

Well-designed genetic and molecular experiments translate genetic differences into functional and clinical changes. This is a unique study resolving subtle changes in sequences that give rise to dramatic phenotypic consequences.

Weaknesses:

There are many base-editing and protein-expression changes throughout the manuscript, and they cause confusion. It would be helpful to readers if authors could provide a simple summary diagram at the end of the paper.

Reviewer #1 (Public review):

Summary:

The authors integrate multiple large databases to test whether body sizes were positively associated with which species tolerate urban areas. In general, many plant families showed a positive association between body size and urban tolerance, whereas a smaller, though still non-trivial, percentage of animal families showed the same pattern. Notably, the authors are careful in the interpretation of their findings and provide helpful context for the ways that this analysis can be generative in shaping new hypotheses and theory around how urbanization influences biodiversity at large. They are careful to discuss how body size is an important trait, but the absence of a relationship between body size and urban tolerance in many families suggests a variety of other traits undergird urban success.

Strengths:

The authors aggregated a large dataset, but they also applied robust filters to ensure they had an adequate and representative number of detections for a given species, family, geography, etc. The authors also applied their analysis at multiple taxonomic scales (family and order), which allowed for a better interpretation of the patterns in the data and at what taxonomic scale body size might be important.

Weaknesses:

My main concern is that it is not fully clear how the measure of body size might influence the result. The authors were unable to obtain consistent measures of body size (mean, median, maximum, or sex variation). This, of course, could be very consequential as means and medians can differ quite a bit, and they certainly will differ substantially from a maximum. And of course, sex differences can be marked in multiple directions or absent altogether. The authors do note that they selected the measure that was most common in a family, but it was not clear whether species in that family that did not have that measure were removed or not. This could potentially shape the variability in the dataset and obscure true patterns. This may require additional clarity from the authors and is also a real constraint in compiling large data from disparate sources.

Joint Public Review:

Summary:

This is an excellent, timely study investigating and characterizing the underlying neural activity that generates the neuroendocrine GnRH and LH surges that are responsible for triggering ovulation. Abundant evidence accumulated over the past 20 years implicated the population of kisspeptin neurons in the hypothalamic RP3V region (also referred to as the POA or AVPV/PeN kisspeptin neurons) as being involved in driving the GnRH surge in response to elevated estradiol (E2), also known as the "estrogen positive feedback". However, while former studies used Cfos coexpression as a marker of RP3V kisspeptin neuron activation at specific times and found this correlates with the timing of the LH surge, detailed examination of the live in vivo activity of these neurons before, during, and after the LH surge remained elusive due to technical challenges.

Here, Zhou and colleagues use fiber photometry to measure the long-term synchronous activity of RP3V kisspeptin neurons across different stages of the mouse estrous cycle, including on proestrus when the LH surge occurs, as well as in a well-established OVX+E2 mouse model of the LH surge.

The authors report that RP3V kisspeptin neuron activity is low on estrous and diestrus, but increases on proestrus several hours before the late afternoon LH surge, mirroring prior reports of rising GnRH neuron activity in proestrus female mice. The measured increase in RP3V kisspeptin activation is long, spanning ~13 hours in proestrus females and extending well beyond the end of the LH secretion, and is shown by the authors to be E2 dependent.

For this work, Kiss-Cre female mice received a Cre-dependent AAV injection, containing GCaMP6, to measure the neuronal activation of RP3V Kiss1 cells. Females exhibited periods of increased neuronal activation on the day of proestrus, beginning several hours prior to the LH surge and lasting for about 12 hours. Though oscillations in the pattern of GCaMP fluorescence were occasionally observed throughout the ovarian cycle, the frequency, duration, and amplitude of these oscillations were significantly higher on the day of proestrus. This increase in RP3V Kiss1 neuronal activation that precedes the increase in LH supports the hypothesis that these neurons are critical in regulating the LH surge. The authors compare this data to new data showing a similar increased activation pattern in GnRH neurons just prior to the LH surge, further supporting the hypothesis that RP3V Kiss1 cell activation causes the release of kisspeptin to stimulate GnRH neurons and produce the LH surge.

Strengths:

This study provides compelling data demonstrating that RP3V kisspeptin neuronal activity changes throughout the ovarian cycle, likely in response to changes in estradiol levels, and that neuronal activation increases on the day of the LH surge.

The observed increase in RP3V kisspeptin neuronal activation precedes the LH surge, which lends support to the hypothesis that these neurons play a role in regulating the estradiol-induced LH surge. Continuing to examine the complexities of the LH surge and the neuronal populations involved, as done in this study, is critical for developing therapeutic treatments for women's reproductive disorders.

This innovative study uses a within-subject design to examine neuronal activation in vivo across multiple hormone milieus, providing a thorough examination of the changes in activation of these neurons. The variability in neuronal activity surrounding the LH surge across ovarian cycles in the same animals is interesting and could not be achieved without this within-subjects design. The inclusion and comparison of ovary-intact females and OVX+E2 females is valuable to help test mechanisms under these two valuable LH surge conditions, and allows for further future studies to tease apart minor differences in the LH surge pattern between these 2 conditions.

This study provides an excellent experimental setup able to monitor the daily activity of preoptic kisspeptin neurons in freely moving female mice. It will be a valuable tool to assess the putative role of these kisspeptin neurons in various aspects of altered female fertility (aging, pathologies...). This approach also offers novel and useful insights into the impact of E2 and circadian cues on the electrical activity of RP3V kisspeptin neurons.

An intriguing cyclical oscillation in kisspeptin neural activity every 90 minutes exists, which may offer critical insight into how the RP3V kisspeptin system operates. Interestingly, there was also variability in the onset and duration of RP3V Kisspeptin neuron activity between and within mice in naturally cycling females. Preoptic kisspeptin neurons show an increased activity around the light/dark transition only on the day of proestrus, and this is associated with an increase in LH secretion. An original finding is the observation that the peak of kisspeptin neuron activation continues a few hours past the peak of LH, and the authors hypothesize that this prolonged activity could drive female sexual behaviors, which usually appear after the LH surge.

The authors demonstrated that ovariectomy resulted in very little neuronal activity in RP3V kisspeptin neurons. When these ovarietomized females were treated with estradiol benzoate (EB) and an LH surge was induced, there was an increase in RP3V kisspeptin neuronal activation, as was seen during proestrus. However, the magnitude of the change in activity was greater during proestrus than during the EB-induced LH surge. Interestingly, the authors noted a consistent peak in activity about 90 minutes prior to lights out on each day of the ovarian cycle and during EB treatment, but not in ovariectomized females. The functional purpose of this consistent neuronal activity at this time remains to be determined.

Though not part of this study, the comparison of neuronal activation of GnRH neurons during the LH surge to the current data was convincing, demonstrating a similar pattern of increased activation that precedes the LH surge.

In summary, the study is well-designed, uses proper controls and analyses, has robust data, and the paper is nicely organized and written. The data from these experiments is compelling, and the authors' claims and conclusions are nicely supported and justified by the data. The data support the hypothesis in the field that these RP3V neurons regulate the LH surge. Overall, these findings are important and novel, and lend valuable insight into the underlying neural mechanisms for neuroendocrine control of ovulation.

Weaknesses:

(1) LH levels were not measured in many mice or in robust temporal detail, such as every 30 or 60 min, to allow a more detailed comparison between the fine-scale timing of RP3V neuron activation with onset and timing of LH surge dynamics.

(2) The authors report that the peak LH value occurred 3.5 hours after the first RP3V kisspeptin neuron oscillation. However, it is likely, and indeed evident from the 2 example LH patterns shown in Figures 3A-B, that LH values start to increase several hours before the peak LH. This earlier rise in LH levels ("onset" of the surge) occurs much closer in time to the first RP3V kisspeptin neuron oscillatory activation, and as such, the ensuing LH secretion may not be as delayed as the authors suggest.

(3) The authors nicely show that there is some variation (~2 hours) in the peak of the first oscillation in proestrus females. Was this same variability present in OVX+E2 females, or was the variability smaller or absent in OVX+E2 versus proestrus? It is possible that the variability in proestrus mice is due to variability in the timing and magnitude of rising E2 levels, which would, in theory, be more tightly controlled and similar among mice in the OVX+E2 model. If so, the OVX+E2 mice may have less variability between mice for the onset of RP3V kisspeptin activity.

(4) One concern regarding this study is the lack of data showing the specificity of the AAV and the GCaMP6s signals. There are no data showing that GCaMP6s is limited to the RP3V and is not expressed in other Kiss1 populations in the brain. Given that 2ul of the AAV was injected, which seems like a lot considering it was close to the ventricle, it is important to show that the signal and measured activity are specific to the RP3V region. Though the authors discuss potential reasons for the low co-expression of GCaMP6 and kisspeptin immunoreactivity, it does raise some concern regarding the interpretation of these results. The low co-expression makes it difficult to confirm the Kiss1 cell-specificity of the Cre-dependent AAV injections. In addition, if GFP (GCaMP6s) and kisspeptin protein co-localization is low, it is possible that the activation of these neurons does not coincide with changes in kisspeptin or that these neurons are even expressing Kiss1 or kisspeptin at the time of activation. It is important to remember that the study measures activation of the kisspeptin neuron, and it does not reveal anything specific about the activity of the kisspeptin protein.

(5) One additional minor concern is that LH levels were not measured in the ovariectomized females during the expected time of the LH surge. The authors suggest that the lower magnitude of activation during the LH surge in these females, in comparison to proestrus females, may be the result of lower LH levels. It's hard to interpret the difference in magnitude of neuronal activation between EB-treated and proestrus females without knowing LH levels. In addition, it's possible that an LH surge did not occur in all EB-treated females, and thus, having LH levels would confirm the success of the EB treatment.

(6) This kisspeptin neuron peak activity is abolished in ovariectomized mice, and estradiol replacement restored this activity, but only partially. Circulating levels of estradiol were not measured in these different setups, but the authors hypothesize that the lack of full restoration may be due to the absence of other ovarian signals, possibly progesterone.

(7) Recordings in several mice show inter- and intra-variability in the time of peak onset. It is not shown whether this variability is associated with a similar variability in the timing of the LH surge onset in the recorded mice. The authors hypothesized that this variability indicates a poor involvement of the circadian input. However, no experiments were done to investigate the role of the (vasopressinergic-driven) circadian input on the kisspeptin neuron activation at the light/dark transition. Thus, we suggest that the authors be more tentative about this hypothesis.

Reviewer #1 (Public review):

Summary:

In this paper, Qiu et al. developed a novel spatial navigation task to investigate the formation of multi-scale representations in the human brain. Over multiple sessions and diverse tasks, participants learned the location of 32 objects distributed across 4 different rooms. The key task was a "judgement of relative direction" task delivered in the scanner, which was designed to assess whether object representations reflect local (within-room) or global (across-room) similarity structures. In between the two scanning sessions, participants received extensive further training. The goal of this manipulation was to test how spatial representations change with learning.

Strengths:

The authors designed a very comprehensive set of tasks in virtual reality to teach participants a novel spatial map. The spatial layout is well-designed to address the question of interest in principle. Participants were trained in a multi-day procedure, and representations were assessed twice, allowing the authors to investigate changes in the representation over multiple days.

Weaknesses:

Unfortunately, I see multiple problems with the experimental design that make it difficult to draw conclusions from the results.

(1) In the JRD task (the key task in this paper), participants were instructed to imagine standing in front of the reference object and judge whether the second object was to their left or right. The authors assume that participants solve this task by retrieving the corresponding object locations from memory, rotating their imagined viewpoint and computing the target object's relative orientation. This is a challenging task, so it is not surprising that participants do not perform particularly well after the initial training (performance between 60-70% accuracy). Notably, the authors report that after extensive training, they reached more than 90% accuracy.

However, I wonder whether participants indeed perform the task as intended by the authors, especially after the second training session. A much simpler behavioural strategy is memorising the mapping between a reference object and an associated button press, irrespective of the specific target object. This basic strategy should lead to quite high success rates, since the same direction is always correct for four of the eight objects (the two objects located at the door and the two opposite the door). For the four remaining objects, the correct button press is still the same for four of the six target objects that are not located opposite to the reference object. Simply memorising the button press associated with each reference object should therefore lead to a high overall task accuracy without the necessity to mentally simulate the spatial geometry of the object relations at all.

I also wonder whether the random effect coefficients might reflect interindividual differences in such a strategy shift - someone who learnt this relationship between objects and buttons might show larger increases in RTs compared to someone who did not.

(2) On a related note, the neural effect that appears to reflect the emergence of a global representation might be more parsimoniously explained by the formation of pairwise associations between reference and target objects. Since both objects always came from the same room, an RDM reflecting how many times an object pair acted as a reference-target pair will correlate with the categorical RDM reflecting the rooms corresponding to each object. Since the categorical RDM is highly correlated with the global RDM, this means that what the authors measure here might not reflect the formation of a global spatial map, but simply the formation of pairwise associations between objects presented jointly.

(3) In general, the authors attribute changes in neural effects to new learning. But of course, many things can change between sessions (expectancy, fatigue, change in strategy, but also physiological factors...). Baseline phsiological effects are less likely to influence patterns of activity, so the RSA analyses should be less sensitive to this problem, but especially the basic differences in activation for the contrast of post-learning > pre-learning stages in the judgment of relative direction (JRD) task could in theory just reflect baseline differences in blood oxygenation, possibly due to differences in time of day, caffeine intake, sleep, etc. To really infer that any change in activity or representation is due to learning, an active control would have been great.

(4) RSA typically compares voxel patterns associated with specific stimuli. However, the authors always presented two objects on the screen simultaneously. From what I understand, this is not considered in the analysis ("The β-maps for each reference object were averaged across trials to create an overall β-map for that object."). Furthermore, participants were asked to perform a complex mental operation on each trial ("imagine standing at A, looking at B, then perform the corresponding motor response"). Assuming that participants did this (although see points 1 and 2 above), this means that the resulting neural representation likely reflects a mixture of the two object representations, the mental transformation and the corresponding motor command, and possibly additionally the semantic and perceptual similarity between the two presented words. This means that the βs taken to reflect the reference object representation must be very noisy.

This problem is aggravated by two additional points. Firstly, not all object pairs occurred equally often, because only a fraction of all potential pairs were sampled. If the selection of the object pairs is not carefully balanced, this could easily lead to sampling biases, which RSA is highly sensitive to.

Secondly, the events in the scanner are not jittered. Instead, they are phase-locked to the TR (1.2 sec TR, 1.2 sec fixation, 4.8 sec stimulus presentation). This means that every object onset starts at the same phase of the image acquisition, making HRF sampling inefficient and hurting trial-wise estimation of betas used for the RSA. This likely significantly weakens the strength of the neural inferences that are possible using this dataset.

(5) It is not clear why the authors focus their report of the results in the main manuscript on the preselected ROIs instead of showing whole-brain results. This can be misleading, as it provides the false impression that the neural effects are highly specific to those regions.

(6) I am missing behavioural support for the authors' claims.

Overall, I am not convinced that the main conclusion that global spatial representations emerge during learning is supported by the data. Unfortunately, I think there are some fundamental problems in the experimental design that might make it difficult to address the concerns.

However, if the authors can provide convincing evidence for their claims, I think the paper will have an impact on the field. The question of how multi-scale representations are represented in the human brain is a timely and important one.

Reviewer #1 (Public review):

Summary:

Englert et al. proposed a functional connectivity-based Attractor Neural Network (fcANN) to reveal attractor states and activity flows across various conditions, including resting state, task-evoked, and pathological conditions. The large-scale brain attractors reconstructed by fcANNs are orthogonal organization, which is in line with the free-energy theoretical framework. Additionally, the fcANN demonstrates differences in attractor states between individuals with autism and typically developing individuals.

The study used seven datasets, which ensures robust replication and validation of generalization across various conditions. The study is a representative example that combines experimental evidence based on fcANN and the theoretical framework. The fcANN projection offers an interesting way of visualization, allowing researchers to observe attractor states and activity flow patterns directly. Overall, the study may offer valuable insights into brain dynamics and computational neuroscience.

Comments on revision:

The authors have addressed my previous concerns and substantially improved the manuscript. Fig.4 and Fig.5 still keep fcHNN rather than the updated fcANN.

Reviewer #2 (Public review):

Summary:

Tissue-resident macrophages are more and more thought to exert key homeostatic functions and contribute to physiological responses. In the report of O'Brien and Colleagues, the idea that the macrophage-expressed scavenger receptor MARCO could regulate adrenal corticosteroid output at steady-state was explored. The authors found that male MARCO-deficient mice exhibited higher plasma aldosterone levels and higher lung ACE expression as compared to wild-type mice, while the availability of cholesterol and the machinery required to produce aldosterone in the adrenal gland were not affected by MARCO deficiency. The authors take these data to conclude that MARCO in alveolar macrophages can negatively regulate ACE expression and aldosterone production at steady-state and that MARCO-deficient mice suffer from a secondary hyperaldosteronism.

Strengths:

If properly demonstrated and validated, the fact that tissue-resident macrophages can exert physiological functions and influence endocrine systems would be highly significant and could be amenable to novel therapies.

Major weakness:

The comparison between C57BL/6J wild-type mice and knock-out mice for which a precise information about the genetic background and the history of breedings and crossings is lacking can lead to misinterpretations of the results obtained. Hence, MARCO-deficient mice should be compared with true littermate controls.

Reviewer #1 (Public review):

Summary:

The article presents the details of the high-resolution light-sheet microscopy system developed by the group. In addition to presenting the technical details of the system, its resolution has been characterized and its functionality demonstrated by visualizing subcellular structures in a biological sample.

Strengths:

(1) The article includes extensive supplementary material that complements the information in the main article.

(2) However, in some sections, the information provided is somewhat superficial.

Weaknesses:

(1) Although a comparison is made with other light-sheet microscopy systems, the presented system does not represent a significant advance over existing systems. It uses high numerical aperture objectives and Gaussian beams, achieving resolution close to theoretical after deconvolution. The main advantage of the presented system is its ease of construction, thanks to the design of a perforated base plate.

(2) Using similar objectives (Nikon 25x and Thorlabs 20x), the results obtained are similar to those of the LLSM system (using a Gaussian beam without laser modulation). However, the article does not mention the difficulties of mounting the sample in the implemented configuration.

(3) The authors present a low-cost, open-source system. Although they provide open source code for the software (navigate), the use of proprietary electronics (ASI, NI, etc.) makes the system relatively expensive. Its low cost is not justified.

(4) The fibroblast images provided are of exceptional quality. However, these are fixed samples. The system lacks the necessary elements for monitoring cells in vivo, such as temperature or pH control.

Reviewer #1 (Public review):

Summary:

Zhou and colleagues developed a computational model of replay that heavily builds on cognitive models of memory in context (e.g., the context-maintenance and retrieval model), which have been successfully used to explain memory phenomena in the past. Their model produces results that mirror previous empirical findings in rodents and offers a new computational framework for thinking about replay.

Strengths:

The model is compelling and seems to explain a number of findings from the rodent literature. It is commendable that the authors implement commonly used algorithms from wakefulness to model sleep/rest, thereby linking wake and sleep phenomena in a parsimonious way. Additionally, the manuscript's comprehensive perspective on replay, bridging humans and non-human animals, enhanced its theoretical contribution.

Weaknesses:

This reviewer is not a computational neuroscientist by training, so some comments may stem from misunderstandings. I hope the authors would see those instances as opportunities to clarify their findings for broader audiences.

(1) The model predicts that temporally close items will be co-reactivated, yet evidence from humans suggests that temporal context doesn't guide sleep benefits (instead, semantic connections seem to be of more importance; Liu and Ranganath 2021, Schechtman et al 2023). Could these findings be reconciled with the model or is this a limitation of the current framework?

(2) During replay, the model is set so that the next reactivated item is sampled without replacement (i.e., the model cannot get "stuck" on a single item). I'm not sure what the biological backing behind this is and why the brain can't reactivate the same item consistently. Furthermore, I'm afraid that such a rule may artificially generate sequential reactivation of items regardless of wake training. Could the authors explain this better or show that this isn't the case?

(3) If I understand correctly, there are two ways in which novelty (i.e., less exposure) is accounted for in the model. The first and more talked about is the suppression mechanism (lines 639-646). The second is a change in learning rates (lines 593-595). It's unclear to me why both procedures are needed, how they differ, and whether these are two different mechanisms that the model implements. Also, since the authors controlled the extent to which each item was experienced during wakefulness, it's not entirely clear to me which of the simulations manipulated novelty on an individual item level, as described in lines 593-595 (if any).

As to the first mechanism - experience-based suppression - I find it challenging to think of a biological mechanism that would achieve this and is selectively activated immediately before sleep (somehow anticipating its onset). In fact, the prominent synaptic homeostasis hypothesis suggests that such suppression, at least on a synaptic level, is exactly what sleep itself does (i.e., prune or weaken synapses that were enhanced due to learning during the day). This begs the question of whether certain sleep stages (or ultradian cycles) may be involved in pruning, whereas others leverage its results for reactivation (e.g., a sequential hypothesis; Rasch & Born, 2013). That could be a compelling synthesis of this literature. Regardless of whether the authors agree, I believe that this point is a major caveat to the current model. It is addressed in the discussion, but perhaps it would be beneficial to explicitly state to what extent the results rely on the assumption of a pre-sleep suppression mechanism.

(4) As the manuscript mentions, the only difference between sleep and wake in the model is the initial conditions (a0). This is an obvious simplification, especially given the last author's recent models discussing the very different roles of REM vs NREM. Could the authors suggest how different sleep stages may relate to the model or how it could be developed to interact with other successful models such as the ones the last author has developed (e.g., C-HORSE)? Finally, I wonder how the model would explain findings (including the authors') showing a preference for reactivation of weaker memories. The literature seems to suggest that it isn't just a matter of novelty or exposure, but encoding strength. Can the model explain this? Or would it require additional assumptions or some mechanism for selective endogenous reactivation during sleep and rest?

(5) Lines 186-200 - Perhaps I'm misunderstanding, but wouldn't it be trivial that an external cue at the end-item of Figure 7a would result in backward replay, simply because there is no potential for forward replay for sequences starting at the last item (there simply aren't any subsequent items)? The opposite is true, of course, for the first-item replay, which can't go backward. More generally, my understanding of the literature on forward vs backward replay is that neither is linked to the rodent's location. Both commonly happen at a resting station that is further away from the track. It seems as though the model's result may not hold if replay occurs away from the track (i.e. if a0 would be equal for both pre- and post-run).

(6) The manuscript describes a study by Bendor & Wilson (2012) and tightly mimics their results. However, notably, that study did not find triggered replay immediately following sound presentation, but rather a general bias toward reactivation of the cued sequence over longer stretches of time. In other words, it seems that the model's results don't fully mirror the empirical results. One idea that came to mind is that perhaps it is the R/L context - not the first R/L item - that is cued in this study. This is in line with other TMR studies showing what may be seen as contextual reactivation. If the authors think that such a simulation may better mirror the empirical results, I encourage them to try. If not, however, this limitation should be discussed.

(7) There is some discussion about replay's benefit to memory. One point of interest could be whether this benefit changes between wake and sleep. Relatedly, it would be interesting to see whether the proportion of forward replay, backward replay, or both correlated with memory benefits. I encourage the authors to extend the section on the function of replay and explore these questions.

(8) Replay has been mostly studied in rodents, with few exceptions, whereas CMR and similar models have mostly been used in humans. Although replay is considered a good model of episodic memory, it is still limited due to limited findings of sequential replay in humans and its reliance on very structured and inherently autocorrelated items (i.e., place fields). I'm wondering if the authors could speak to the implications of those limitations on the generalizability of their model. Relatedly, I wonder if the model could or does lead to generalization to some extent in a way that would align with the complementary learning systems framework.

Joint Public Review:

Meiotic recombination begins with DNA double-strand breaks (DSBs) generated by the conserved enzyme Spo11, which relies on several accessory factors that vary widely across eukaryotes. In C. elegans, multiple proteins have been implicated in promoting DSB formation, but their functional relationships and how they collectively recruit the DSB machinery to chromosome axes have remained unclear.

In this study, Raices et al. investigate the biochemical and genetic interactions among known DSB-promoting factors in C. elegans meiosis. Using yeast two-hybrid assays and co-immunoprecipitation, they map pairwise protein interactions and identify a connection between the chromatin-associated protein HIM-17 and the transcription factor XND-1. They also confirm the established interaction between DSB-1 and SPO-11 and show that DSB-1 associates with the nematode-specific factor HIM-5, which is required for X-chromosome DSB formation.

The authors extend these findings with genetic analyses, placing these factors into four epistasis groups based on single- and double-mutant phenotypes. Together, these biochemical and genetic data support a model describing how these proteins engage chromatin loops and localize to chromosome axes. The work provides a clearer view of how C. elegans assembles its DSB-forming machinery and how this process compares to mechanisms in other organisms.

Comment from the Reviewing Editor on the revised version:

The authors have adequately addressed the prior review comments. At this point, after going through multiple rounds of reviews and revisions, the community will be better served by having this paper out in public. This version was assessed by the editors without further input from the reviewers.

Reviewer #1 (Public review):

Summary:

In their article, Guo and coworkers investigate the Ca²⁺ signaling responses induced by Enteropathogenic Escherichia coli (EPEC) in epithelial cells and how these responses regulate NF-κB activation. The authors show that EPEC induces rapid, spatially coordinated Ca²⁺ transients mediated by extracellular ATP released through the type III secretion system (T3SS). Using high-speed Ca²⁺ imaging and stochastic modeling, they propose that low ATP levels trigger "Coordinated Ca²⁺ Responses from IP₃R Clusters" (CCRICs) via fast Ca²⁺ diffusion and Ca²⁺-induced Ca²⁺ release. These responses may dampen TNF-α-induced NF-κB activation through Ca²⁺-dependent modulation of O-GlcNAcylation of p65. The interdisciplinary work suggests a new perspective on calcium-mediated immune response by combining quantitative imaging, bacterial genetics, and computational modeling.

Strengths:

The study provides a new concept for host responses to bacterial infections and introduces the concept of Coordinated Ca²⁺ Responses from IP₃R Clusters (CCRICs) as synchronized, whole-cell-scale Ca²⁺ transients with the fast kinetics typical of local events. This is elegantly done by an interdisciplinary approach using quantitative measurements and mechanistic modelling.

Weaknesses:

(1) The effect of coordination by fast diffusion for small eATP concentrations is explained by the resulting low Ca2+ concentration that is not as strongly affected by calcium buffers compared to higher concentrations. While I agree with this statement on the relative level, CICR is based on the resulting absolute concentration at neighboring IP3Rs (to activate them). Thus, I do not fully agree with the explanation, or at least would expect to use the modelling approach to demonstrate this effect. Simulations for different activation and buffer concentrations could strengthen this point and exclude potential inhibition of channels at higher stimulation levels.

In this respect, I would also include the details of the modelling, such as implementation environment, parameters, and benchmarking. The description in the Supplementary Methods is very similar to the description in the main text. In terms of reproducibility, it would be important to at least provide simulation parameters, and providing the code would align with the emerging standards for reproducible science.

(2) Quantitative characterization of CCRICs:

The paper would benefit from a clearer definition of the term CCRICs and quantitative descriptors like duration, amplitude distribution, frequency, and spatial extent (also in relation to the comment on the EGTA measurements below). Furthermore, it remains unclear to me whether CCRICs represent a population of rapidly propagating micro-waves or truly simultaneous events. Maybe kymographs or wave-front propagation analyses (at least from simulations if experimental resolution is too bad) would strengthen this point.

(3) Specificity of pharmacological tools:

Suramin and U73122 are known to have off-target effects. Control experiments using alternative P2 receptor antagonists like PPADS or inactive U73343 analogs would strengthen the causal link.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The authors have addressed all the weaknesses in this revision.

Reviewer #1 (Public review):

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data clearly demonstrate chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants. To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization.

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays.

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fits-all model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

Reviewer #1 (Public review):

Summary:

The manuscript by Yamamoto et al. presents a model by which the four main axes of the limb are required for limb regeneration to occur in the axolotl. A longstanding question in regeneration biology is how existing positional information is used to regenerate the correct missing elements. The limb provides an accessible experimental system by which to study the involvement of the anteroposterior, dorsoventral, and proximodistal axes in the regenerating limb. Extensive experimentation has been performed in this area using grafting experiments. Yamamoto et al. use the accessory limb model and some molecular tools to address this question. There are some interesting observations in the study. In particular, one strength the potent induction of accessory limbs in the dorsal axis with BMP2+Fgf2+Fgf8 is very interesting. Although interesting, the study makes bold claims about determining the molecular basis of DV positional cues, but the experimental evidence is not definitive and does not take into account the previous work on DV patterning in the amniote limb. Also, testing the hypothesis on blastemas after limb amputation would be needed to support the strong claims in the study.

Strengths:

The manuscript presents some novel new phenotypes generated in axolotl limbs due to Wnt signaling. This is generally the first example in which Wnt signaling has provided a gain of function in the axolotl limb model. They also present a potent way of inducing limb patterning in the dorsal axis by the addition of just beads loaded with Bmp2+Fgf8+Fgf2.

Comments on revised version:

Re-evaluation: The authors have significantly improved the manuscript and their conclusions reflect the current state of knowledge in DV patterning of tetrapod limbs. My only point of consideration is their claim of mesenchymal and epithelial expression of Wnt10b and the finding that Fgf2 and Wnt10b are lowly expressed. It is based upon the failed ISH, but this doesn't mean they aren't expressed. In interpreting the Li et al. scRNAseq dataset, conclusions depend heavily on how one analyzes and interprets it. The 7DPA sample shows a very low representation of epithelial cells compared to other time points, but this is likely a technical issue. Even the epithelial marker, Krt17, and the CT/fibroblast marker show some expression elsewhere. If other time points are included in the analysis, Wnt10b, would be interpreted as relatively highly expressed almost exclusively in the epithelium. By selecting the 7dpa timepoint, which may or may not represent the MB stage as it wasn't shown in the paper, the conclusions may be based upon incomplete data. I don't expect the authors to do more work, but it is worth mentioning this possibility. The authors have considered and made efforts to resolve previous concerns.

Reviewer #1 (Public review):

Summary

The manuscript presents EIDT, a framework that extracts an "individuality index" from a source task to predict a participant's behaviour in a related target task under different conditions. However, the evidence that it truly enables cross-task individuality transfer is not convincing.

Strengths

The EIDT framework is clearly explained, and the experimental design and results are generally well-described. The performance of the proposed method is tested on two distinct paradigms: a Markov Decision Process (MDP) task (comparing 2-step and 3-step versions) and a handwritten digit recognition (MNIST) task under various conditions of difficulty and speed pressure. The results indicate that the EIDT framework generally achieved lower prediction error compared to baseline models and that it was better at predicting a specific individual's behaviour when using their own individuality index compared to using indices from others.

Furthermore, the individuality index appeared to form distinct clusters for different individuals, and the framework was better at predicting a specific individual's behaviour when using their own derived index compared to using indices from other individuals.

Comments on revisions:

I thank the author for the additional analyses. They have fully addressed all of my previous concerns, and I have no further recommendations.

Reviewer #1 (Public review):

Summary:

This study presents compelling new data that combine two FTD-tau mutations P301L/S320F (PL-SF), that reliably induce spontaneous full-length tau aggregation across multiple cellular systems. However, several conclusions would benefit from more validation. Key findings rely on quantification of overexposed immunoblot, and in some experiments, the tau bands shift in molecular weight that are not explained (and in some instances vary between experiments). The effect seems to be driven by the S320F mutation, with the P301L mutation enhancing the effect observed with S320F alone. Although the observation that Hsp70, but not the related Hsc70, enhances aggregation is intriguing, the mechanistic basis for these differences remains unclear despite both Hsp70 and Hsc70 binding to tau. Additional experiments clarifying which PL-SF tau species Hsp70 engages, how this interaction alters tau conformational landscapes, and whether other chaperones or cofactors contribute to this effect would help solidify the conclusions and build a mechanistic picture. Overexpression of Hsp70 in the context of PL tau did not increase tau aggregation, which raises questions about whether the observed effects are specific to the SF mutation. Hsp70 functions in the context of a larger network of chaperones and has been proposed to cooperate with other proteins/machinery to disassemble tau amyloids, perhaps to produce more seeds. This would be consistent with the presented observations. For example, co-IP experiments using Hsp70 as bait combined with proteomics could really help build a more complete picture of what tau species Hsp70 binds and what other factors cooperate to yield the observed increases in aggregation. As it stands, the Hsp70 component of the paper is not fully developed, and additional experiments to address these questions would strengthen this manuscript beyond simply presenting a new tool to study spontaneous tau aggregation.

Strengths:

(1) The PL-SF FL tau mutant aggregates spontaneously in different cellular systems and shows hallmarks of tau pathology linked to disease.

(2) PL-SF 4delta mutant reverses the spontaneous aggregation phenotype, consistent with these residues being critical for tau aggregation.

(3) PL-SF 4delta also loses the ability to recruit Hsp70/Hsc70, consistent with these residues also being critical for chaperone recruitment.

(4) The PL-SF tau mutant establishes a new system to study spontaneous tau assembly and to begin to compare it to seeded tau aggregation processes.

Weaknesses:

(1) Mechanistic insight into how Hsp70 but not Hsc70 increase PL-SF FL tau aggregation/pathology is missing. This is despite both chaperones binding to PL-SF FL tau. What species of tau does Hsp70 bind, and what cofactors are important in this process?

(2) The study relies heavily on densitometry of bands to draw conclusions; in several instances, the blots are overexposed to accurately quantify the signal.

Reviewer #1 (Public review):

Summary:

This study presents convincing findings that oligodendrocytes play a regulatory role in spontaneous neural activity synchronization during early postnatal development, with implications for adult brain function. Utilizing targeted genetic approaches, the authors demonstrate how oligodendrocyte depletion impacts Purkinje cell activity and behaviors dependent on cerebellar function. Delayed myelination during critical developmental windows is linked to persistent alterations in neural circuit function, underscoring the lasting impact of oligodendrocyte activity.

Strengths:

(1) The research leverages the anatomically distinct olivocerebellar circuit, a well-characterized system with known developmental timelines and inputs, strengthening the link between oligodendrocyte function and neural synchronization.

(2) Functional assessments, supported by behavioral tests, validate the findings of in vivo calcium imaging, enhancing the study's credibility.

(3) Extending the study to assess long-term effects of early life myelination disruptions adds depth to the implications for both circuit function and behavior.

Weaknesses:

(1) The study would benefit from a closer analysis of myelination during the periods when synchrony is recorded. Direct correlations between myelination and synchronized activity would substantiate the mechanistic link and clarify if observed behavioral deficits stem from altered myelination timing.

(2) Although the study focuses on Purkinje cells in the cerebellum, neural synchrony typically involves cross-regional interactions. Expanding the discussion on how localized Purkinje synchrony affects broader behaviors-such as anxiety, motor function, and sociality - would enhance the findings' functional significance.

(3) The authors discuss the possibility of oligodendrocyte-mediated synapse elimination as a possible mechanism behind their findings, drawing from relevant recent literature on oligodendrocyte precursor cells. However, there are no data presented supporting these assumptions. The authors should explain why they think the mechanism behind their observation extends beyond the contribution of myelination or remove this point from the discussion entirely.

Comment for resubmission: Although the argument on synaptic elimination has been removed, it has been replaced with similarly unclear speculation about roles for oligodendrocytes outside of conventional myelination or metabolic support, again without clear evidence. The authors measured MBP area but have not performed detailed analysis of oligodendrocyte biology to support the claims made in the discussion. Please consider removing this section or rephrasing it to align with the data presented.

(4) It would be valuable to investigate secondary effects of oligodendrocyte depletion on other glial cells, particularly astrocytes or microglia, which could influence long-term behavioral outcomes. Identifying whether the lasting effects stem from developmental oligodendrocyte function alone or also involve myelination could deepen the study's insights.

(5) The authors should explore the use of different methods to disturb myelin production for a longer time, in order to further determine if the observed effects are transient or if they could have longer-lasting effects.

(6) Throughout the paper, there are concerns about statistical analyses, particularly on the use of the Mann-Whitney test or using fields of view as biological replicates.

Reviewer #1 (Public review):

Summary:

Kashiwagi et al. undertook a population analysis of dendritic spine nanostructure applied to the objective grouping of 8 mouse models of neuropsychiatric disorders. They report that spine morphology in cultured hippocampal neurons shows a higher similarity among schizophrenia mouse models (compared with autism spectrum disorder (ASD) mouse models), and identify an effect of Ecrg4 (encoding small secretory peptides) on spine dynamics and shape in these models.

Strengths:

The study developed a method for objectively comparing spine properties in primary hippocampal neuron cultures from 8 mouse models of psychiatric disorders at the population level using high-resolution structured illumination microscopy (SIM) imaging. This novel technique identified two distinct groups of mouse models according to the population-level spine properties: those with ASD-related gene mutations and those with schizophrenia-related gene mutations. Functional studies, including gene knockdown and overexpression experiments, identified an effect of Ecrg4 on the spine phenotype of the schizophrenia model mice.

Weaknesses:

The main weakness is that the study is wholly in vitro, using cultured hippocampal neurons. The authors present this as an advantage, however, arguing that spine morphology as measured in a reduced culture system can demonstrate direct effects of gene mutations on neuronal phenotypes in the absence of indirect influences from nonneuronal cells or specific environments.

Another weakness is that CaMKIIαK42R/K42R mutant mice are presented as a schizophrenia model, the authors justifying this by saying that "CaMKII-related signaling pathway disruption has been implicated in the working memory deficits found in schizophrenia patients". Since mutations in CAMK2A cause autosomal dominant intellectual developmental disorder-53 (OMIM 617798) and autosomal recessive intellectual developmental disorder-63 (OMIM 618095), and mice carrying the CAMK2A E183V mutation exhibit ASD-related synaptic and behavioral phenotypes (PMID: 28130356), I think it's stretching credibility to refer to the CaMKIIαK42R/K42R mice as a schizophrenia model.

Although the manuscript is largely well written, there are some instances of ambiguous/unspecific language. This extends to the title (Decoding Spine Nanostructure in Mental Disorders Reveals a Schizophrenia-1 Linked Role for Ecrg4), which gives no indication that the work was in vitro on cultured neurons derived from mouse models.

Reviewer #1 (Public review):

Summary of goals:

The authors' stated goal (line 226) was to compare gene expression levels for gut hormones between males and females. As female flies contain more fat than males, they also sought to identify hormones that control this sex difference. Finally, they attempted to place their findings in the broader context of what is already known about established underlying mechanisms.

Strengths:

(1) The core research question of this work is interesting. The authors provide a reasonable hypothesis (neuro/entero-peptides may be involved) and well-designed experiments to address it.

(2) Some of the data are compelling, especially positive results that clearly implicate enteropeptides in sex-biased fat contents (Figures 1 and 3).

Weaknesses:

(1) The greatest weakness of this work is that it falls short of providing a clear mechanism for the regulation of sex-biased fat content by AstC and Tk. By and large, feminization of neurons or enteroendocrine cells with UAS-traF did not increase fat in males (Figure 2). The authors mention that ecdysone, juvenile hormone or Sex-lethal may instead play a role (lines 258-270), but this is speculative, making this study incomplete.

(2) Related to the above point, the cellular mechanisms by which AstC and Tk regulate fat content in males and females are only partially characterized. For example, knockdown of TkR99D in insulin-producing neurons (Figure 4E) but not pan-neuronally (Figure 4B) increases fat in males, but Tk itself only shows a tendency (Figure 3B). In females, the situation is even less clear: again, Tk only shows a tendency (Figure 3B), and pan-neuronal, but not IPC-specific knockdown of TkR99D decreases fat.

(3) The text sometimes misrepresents or contradicts the Results shown in the figures. UAS-traF expression in neurons or enteroendocrine cells did sometimes alter fat contents (Figure 2H, S), but the authors report that sex differences were unaffected (lines 164-166). On the other hand, although knockdown of Tk in enteroendocrine cells caused no significant effect (Figure 3B), the authors report this as a trend towards reduction (lines 182-183). This biased representation raises concerns about the interpretation of the data and the authors' conclusions.

(4) The authors find that in males, neuropeptide expression in the head is higher (Figure 1F-J). This may also play an important role in maintaining lower levels of fat in males, but this finding is not explored in the manuscript.

Appraisal of goal achievement & conclusions:

The authors were successful in identifying hormones that show sex bias in their expression and also control the male vs. female difference in fat content. However, elucidation of the relevant cellular pathways is incomplete. Additionally, some of their conclusions are not supported by the data (see Weaknesses, point 3).

Impact:

It is difficult to evaluate the impact of this study. This is in great part because the authors do not attempt to systematically place their findings about AstC/Tk in the broader context of their previous studies, which investigated the same phenomenon (Wat et al., 2021, eLife and Biswas et al., 2025, Cell Reports). As the underlying mechanisms are complex and likely redundant, it is necessary to generate a visual model to explain the pathways which regulate fat content in males and females.

Reviewer #1 (Public review):

Summary:

This study investigates how the brain processes facial expressions across development by analyzing intracranial EEG (iEEG) data from children (ages 5-10) and post-childhood individuals (ages 13-55). The researchers used a short film containing emotional facial expressions and applied AI-based models to decode brain responses to facial emotions. They found that in children, facial emotion information is represented primarily in the posterior superior temporal cortex (pSTC)-a sensory processing area-but not in the dorsolateral prefrontal cortex (DLPFC), which is involved in higher-level social cognition. In contrast, post-childhood individuals showed emotion encoding in both regions. Importantly, the complexity of emotions encoded in the pSTC increased with age, particularly for socially nuanced emotions like embarrassment, guilt, and pride.The authors claim that these findings suggest that emotion recognition matures through increasing involvement of the prefrontal cortex, supporting a developmental trajectory where top-down modulation enhances understanding of complex emotions as children grow older.

Strengths:

(1) The inclusion of pediatric iEEG makes this study uniquely positioned to offer high-resolution temporal and spatial insights into neural development compared to non-invasive approaches, e.g., fMRI, scalp EEG, etc.

(2) Using a naturalistic film paradigm enhances ecological validity compared to static image tasks often used in emotion studies.

(3) The idea of using state-of-the-art AI models to extract facial emotion features allows for high-dimensional and dynamic emotion labeling in real time.

Weaknesses:

(1) The study has notable limitations that constrain the generalizability and depth of its conclusions. The sample size was very small, with only nine children included and just two having sufficient electrode coverage in the posterior superior temporal cortex (pSTC), which weakens the reliability and statistical power of the findings, especially for analyses involving age. Authors pointed out that a similar sample size has been used in previous iEEG studies, but the cited works focus on adults and do not look at the developmental perspectives. Similar work looking at developmental changes in iEEG signals usually includes many more subjects (e.g., n = 101 children from Cross ZR et al., Nature Human Behavior, 2025) to account for inter-subject variabilities.

(2) Electrode coverage was also uneven across brain regions, with not all participants having electrodes in both the dorsolateral prefrontal cortex (DLPFC) and pSTC, making the conclusion regarding the different developmental changes between DLPFC and pSTC hard to interpret (related to point 3 below). It is understood that it is rare to have such iEEG data collected in this age group, and the electrode location is only determined by clinical needs. However, the scientific rigor should not be compromised by the limited data access. It's the authors' decision whether such an approach is valid and appropriate to address the scientific questions, here the developmental changes in the brain, given all the advantages and constraints of the data modality.

(3) The developmental differences observed were based on cross-sectional comparisons rather than longitudinal data, reducing the ability to draw causal conclusions about developmental trajectories. Also, see comments in point 2.

(4) Moreover, the analysis focused narrowly on DLPFC, neglecting other relevant prefrontal areas such as the orbitofrontal cortex (OFC) and anterior cingulate cortex (ACC), which play key roles in emotion and social processing. Agree that this might be beyond the scope of this paper, but a discussion section might be insightful.

(5) Although the use of a naturalistic film stimulus enhances ecological validity, it comes at the cost of experimental control, with no behavioral confirmation of the emotions perceived by participants and uncertain model validity for complex emotional expressions in children. A non-facial music block that could have served as a control was available but not analyzed. The validation of AI model's emotional output needs to be tested. It is understood that we cannot collect these behavioral data retrospectively within the recorded subjects. Maybe potential post-hoc experiments and analyses could be done, e.g., collect behavioral, emotional perception data from age-matched healthy subjects.

(6) Generalizability is further limited by the fact that all participants were neurosurgical patients, potentially with neurological conditions such as epilepsy that may influence brain responses. At least some behavioral measures between the patient population and the healthy groups should be done to ensure the perception of emotions is similar.

(7) Additionally, the high temporal resolution of intracranial EEG was not fully utilized, as data were downsampled and averaged in 500-ms windows. It seems like the authors are trying to compromise the iEEG data analyses to match up with the AI's output resolution, which is 2Hz. It is not clear then why not directly use fMRI, which is non-invasive and seems to meet the needs here already. The advantages of using iEEG in this study are missing here.

(8) Finally, the absence of behavioral measures or eye-tracking data makes it difficult to directly link neural activity to emotional understanding or determine which facial features participants attended to. Related to point 5 as well.

Comments on revisions:

A behavioral measurement will help address a lot of these questions. If the data continues collecting, additional subjects with iEEG recording and also behavioral measurements would be valuable.

Reviewer #1 (Public review):

Summary:

This is a strong paper that presents a clear advance in multi-animal tracking. The authors introduce an updated version of idtracker.ai that reframes identity assignment as a contrastive representation learning problem rather than a classification task requiring global fragments. This change leads to substantial gains in speed and accuracy and removes a known bottleneck in the original system. The benchmarking across species is comprehensive, the results are convincing, and the work significant.

Strengths:

The main strengths are the conceptual shift from classification to representation learning, the clear performance gains, and the improved robustness of the new version. Removing the need for global fragments makes the software much more flexible in practice, and the accuracy and speed improvements are well demonstrated across a diverse set of datasets. The authors' response also provides further support for the method's robustness.

The comparison to other methods is now better documented. The authors clarify which features are used, how failures are defined, how parameters are sampled, and how accuracy is assessed against human-validated data. This helps ensure that the evaluation is fair and that readers can understand the assumptions behind the benchmarks.

The software appears thoughtfully implemented, with GUI updates, integration with pose estimators, and tools such as idmatcher.ai for linking identities across videos. The overall presentation has been improved so that the limitations of the original idtracker.ai, the engineering optimizations, and the new contrastive formulation are more clearly separated. This makes the central ideas and contributions easier to follow.

Weaknesses:

I do not have major remaining criticisms. The authors have addressed my earlier concerns about the clarity and fairness of the comparison with prior methods, the benchmark design, and the memory usage analysis by adding methodological detail and clearly explaining their choices. At this point I view these aspects as transparent features of the experimental design that readers can take into account, rather than weaknesses of the work.

Overall, this is a high-quality paper. The improvements to idtracker.ai are well justified and practically significant, and the authors' response addresses the main concerns about clarity and evaluation. The conceptual contribution, thorough empirical validation, and thoughtful software implementation make this a valuable and impactful contribution to multi-animal tracking.

Reviewer #1 (Public review):

Olmstead et al. present a single-cell nuclear sequencing dataset that interrogates how hippocampal gene expression changes in response to distinct physiological stimuli and across circadian time. The authors perform single-nucleus RNA sequencing on mouse hippocampal tissue after (1) kainic acid-induced seizure, (2) exposure to an enriched environment, and (3) at multiple circadian phases.

The dataset is rigorously collected, and a major strength is the use of the previously established ABC taxonomy from Yao et al. (2023) to define cell types. The authors further show that this taxonomy is largely independent of activity-driven transcriptional programs. Using these annotations, they examine activity-regulated gene expression across neuronal and glial subclasses. They identify ZT12, corresponding to the transition from the light to the dark period, as transcriptionally distinct from other circadian time points, and show that this pattern is conserved across many cell types. Finally, they test how circadian phase influences activity-dependent gene expression by exposing mice to an enriched environment at different times of day, and report no significant interaction between circadian phase and enriched environment exposure.

A crucial consideration for users of this dataset is the potential confounding effect between circadian phase and locomotor activity. This is particularly relevant because dentate gyrus activity is strongly modulated by locomotion. The authors acknowledge this issue in the Discussion and provide useful guidance for how to interpret their findings, considering this confound.

Taken together, this dataset represents a useful resource for the neuroscience community, particularly for investigators interested in how novel experience and circadian phase shape activity-related and immediate early gene expression in the hippocampus

Reviewer #1 (Public review):

Summary:

The manuscript presents a robust set of experiments that provide new insights into the role of STN neurons during active and passive avoidance tasks. These forms of avoidance have received comparatively less attention in the literature than the more extensively studied escape or freezing responses, despite being extremely relevant to human behaviour and more strongly influenced by cognitive control.

Strengths:

Understanding the neural infrastructure supporting avoidance behaviour would be a fundamental milestone in neuroscience. The authors employ sophisticated methods to delineate the role of STN neurons during avoidance behaviours. The work is thorough and the evidence presented is compelling. Experiments are carefully constructed, well-controlled, and the statistical analyses are appropriate.

Weaknesses:

One possible remaining conceptual concern that might require future work is determining whether STN primarily mediates higher-level cognitive avoidance or if its activation primarily modulates motor tone.

Reviewer #1 (Public review):

Summary:

Artiushin et al. establish a comprehensive 3D atlas of the brain of the orb-web building spider Uloborus diversus. First, they use immunohistochemistry detection of synapsin to mark and reconstruct the neuropils of the brain of six specimen and they generate a standard brain by averaging these brains. Onto this standard 3D brain, they plot immunohistochemical stainings of major transmitters to detect cholinergic, serotonergic, octopaminergic/taryminergic and GABAergic neurons, respectively. Further, they add information on the expression of a number of neuropeptides (Proctolin, AllatostatinA, CCAP and FMRFamide). Based on this data and 3D reconstructions, they extensively describe the morphology of the entire synganglion, the discernable neuropils and their neurotransmitter/neuromodulator content.

Strengths:

While 3D reconstruction of spider brains and the detection of some neuroactive substances have been published before, this seems to be the most comprehensive analysis so far both in terms of number of substances tested and the ambition to analyzing the entire synganglion. Interestingly, besides the previously described neuropils, they detect a novel brain structure, which they call the tonsillar neuropil.

Immunohistochemistry, imaging and 3D reconstruction are convincingly done and the data is extensively visualized in figures, schemes and very useful films, which allow the reader to work with the data. Due to its comprehensiveness, this dataset will be a valuable reference for researchers working on spider brains or on the evolution of arthropod brains.

Weaknesses:

As expected for such a descriptive groundwork, new insights or hypotheses are limited while the first description of the tonsillar neuropil is interesting. The reconstruction of the main tracts of the brain would be a very valuable complementary piece of data.

Reviewer #1 (Public review):

Summary

In their manuscript, Ho and colleagues investigate the importance of thymically-imprinted self-reactivity in determining CD8 T cell pathogenicity in non-obese diabetic (NOD) mice. The authors describe pre-existing functional biases associated with naive CD8 T cell self-reactivity based on CD5 levels, a well characterized proxy for T cell affinity to self-peptide. They find that naive CD5hi CD8 T cells are poised to respond to antigen challenge; these findings are largely consistent with previously published data on the C57Bl/6 background. The authors go on to suggest that naive CD5hi CD8 T cells are more diabetogenic as 1) the CD5hi naive CD8 T cell receptor repertoire has features associated with autoreactivity and contains a larger population of islet-specific T cells, and 2) the autoreactivity of "CD5hi" monoclonal islet-specific TCR transgenic T cells cannot be controlled by phosphatase over-expression. Thus, they implicate CD8 T cells with relatively higher levels of basal self-reactivity in autoimmunity. The data presented offers valuable insights and sets the foundation for future studies, but some conclusions are not yet fully supported.

Specific comments

There is value in presenting phenotypic differences between naive CD5lo and CD5hi CD8 T cells in the NOD background as most previous studies have used T cells harvested from C57Bl/6 mice or peripheral blood from healthy human donors.

The comparison of a marker of self-reactivity, CD5 in this case, on broad thymocyte populations (DN/DP/CD8SP) is cautioned. CD5 is upregulated with signals associated with b-selection and positive selection; CD5 levels will thus vary even among subsets within these broad developmental intermediates. This is a particularly important consideration when comparing CD5 across thymic intermediates in polyclonal versus TCR transgenic thymocytes due to the striking differences in thymic selection efficiency, resulting in different developmental population profiles. The higher levels of CD5 noted in the DN population of NOD8.3 mice, for example, is likely due to the shift towards more mature DN4 post-b-selection cells. Similarly, in the DP population, the larger population of post-positive selection cells in the NOD8.3 transgenic thymus may also skew CD5 levels significantly. Overall, the reported differences between NOD and NOD8.3 thymocyte subsets could be due largely to differences in differentiation/maturation stage rather than affinity for self-antigen during T cell development. The authors have added some additional text to the revised manuscript that acknowledges some of these limitations.

The lack of differences in CD5 levels of post-positive selection DP thymocytes, CD8 SP thymocytes, and CD8 T cells in the pancreas draining lymph nodes from NOD vs NOD8.3 mice also raises questions about the relevance of this model to address the question of basal self-reactivity and diabetogenicity and the authors' conclusion that "that intrinsic high CD5-associated self-reactivity in NOD8.3 T cells overrides the transgenic Pep-mediated protection observed in dLPC/NOD mice"; the phenotype of the polyclonal and NOD8.3 TCR transgenic CD8 T cells that were analyzed in the (spleen and) pancreas draining lymph nodes is not clear (i.e., are these gated on naive T cells?). Furthermore, the rationale for the comparison with NOD-BDC2.5 mice that carry an MHC II-restricted TCR is unclear.

In reference to the conclusion that transgenic Pep phosphatase does not inhibit the diabetogenic potential of "CD5hi" CD8 T cells, there is some concern that comparing diabetes development in mice receiving polyclonal versus TCR transgenic T cells specific for an islet antigen is not appropriate. The increased frequency and number of antigen specific T cells in the NOD8.3 mice may be responsible for some of the observed differences. Further justification for the comparison is suggested.

The manuscript presents an interesting observation that TCR sequences from CD5hi CD8 T cells may share certain characteristics with diabetogenic T cells found in patients (e.g., CDR3 length), and that autoantigen-specific T cells may be enriched within the CD5hi naive CD8 T cell population. However, the percentage of tetramer-positive cells among naive CD8 T cells appears unusually high in the data presented, and caution is warranted when comparing additional T cell receptor features of self-reactivity/auto-reactivity between CD4 and CD8 T cells.

The counts for the KEGG enrichment pathways presented are relatively low, and the robustness of the analysis should be carefully considered, particularly given that several significance values appear borderline. That said, the differentially expressed genes among CD5lo and CD5hi CD8 T cells are generally consistent with previously published datasets.

The manuscript includes some imprecise wording that may be misleading. For example (not exhaustive): The strength of TCR reactivity to foreign antigen is not "contributed by basal TCR signal" per se but rather correlates with sub-threshold TCR signals necessary for T cell development and survival, CD5 is not broadly expressed on all B cells as the text might suggest but is restricted to a specific subset of B cells, some of the proximal signaling molecules downstream of the preTCR are different than for the mature TCR, upregulation of CD127 at early timepoints post T cell activation is not directly suggestive of their "heightened capabilities in memory T cell homeostasis", etc. The statement "Our study exclusively examined female mice because the disease modeled is relevant in females" should be reconsidered. While the use of female NOD mice can be justified by their higher incidence of diabetes than their male counterparts, the current wording could be misleading.

For clarity and transparency, please consider while additional information is provided in the revised manuscript, gating strategies are not always clear (i.e., naive versus total CD8 T cells), and the age/status of the mice from which cells are harvested (i.e., prediabetic?) is not consistently provided as far as this reviewer noted.

Reviewer #1 (Public review):

Summary:

In this study the authors use a Drosophila model to demonstrate that Tachykinin (Tk) expression is regulated by the microbiota. In Drosophila conventionally reared (CR) flies are typically shorter lived than those raised without a microbiota (axenic). Here, knockdown of Tk expression is found to prevent lifespan shortening by the microbiota and the reduction of lipid stores typically seen in CR flies when compared to axenic counterparts. It does so without reducing food intake or fecundity which are often seen as necessary trade-offs for lifespan extension. Further, the strength of the interaction between Tk and the microbiota is found to be bacteria specific and is stronger in Acetobacter pomorum (Ap) mono-associated flies compared to Levilactobacillus brevis (Lb) mono-association. The impact on lipid storage was also only apparent in Ap-flies.

Building on these findings the authors show that gut specific knockdown is largely sufficient to explain these phenotypes. Knockdown of the Tk receptor, TkR99D, in neurons recapitulates the lifespan phenotype of intestinal Tk knockdown supporting a model whereby Tk from the gut signals to TkR99D expressing neurons to regulate lifespan. In addition, the authors show that FOXO may have a role in lifespan regulation by the Tk-microbiota interaction. However, they rule out a role for insulin producing cells or Akh-producing cells suggesting the microbiota-Tk interaction regulates lifespan through other, yet unidentified, mechanisms.

Major comments:

Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. In particular, the impact of TkRNAi on lifespan and lipid levels, the central finding in this study, has been demonstrated multiple times in different experiments and using different genetic tools. As a result, I don't feel that additional experimental work is necessary to support the current conclusions.

However, I find it hard to assess the robustness of the lifespan data from the other manipulations used (TkR99DRNAi, TkRNAi in dFoxo mutants etc.) because information on the population size and whether these experiments have been replicated is lacking. Can the authors state in the figure legends the numbers of flies used for each lifespan and whether replicates have been done? For all other data it is clear how many replicates have been done, and the methods give enough detail for all experiments to be reproduced.

Significance:

Overall, I find the key conclusions of the paper convincing. The authors present an extensive amount of experimental work, and their conclusions are well founded in the data. We have known that the microbiota influence lifespan for some time but the mechanisms by which they do so have remained elusive. This study identifies one such mechanism and as a result opens several avenues for further research. The Tk-microbiota interaction is shown to be important for both lifespan and lipid homeostasis, although it's clear these are independent phenotypes. The fact that the outcome of the Tk-microbiota interaction depends on the bacterial species is of particular interest because it supports the idea that manipulation of the microbiota, or specific aspects of the host-microbiota interaction, may have therapeutic potential.

These findings will be of interest to a broad readership spanning host-microbiota interactions and their influence on host health. They move forward the study of microbial regulation of host longevity and have relevance to our understanding of microbial regulation of host lipid homeostasis. They will also be of significant interest to those studying the mechanisms of action and physiological roles of Tachykinins.

Field of expertise: Drosophila, gut, ageing, microbiota, innate immunity

Reviewer #1 (Public review):

Summary:

The manuscript by Hernandez-Nunez et al. provides a comprehensive characterization of how heart-brain circuits develop in a vertebrate brain, namely the zebrafish. The characterization is performed using a combination of modern and sophisticated imaging and neural manipulation techniques and achieves unprecedented clarity and detail in how the heart-brain communication develops early in life. The paper describes a three-stage program, where first an efferent-circuit from the motor vagus to the heart develops, followed by sympathetic innervation, and lastly sensory neurons innervate the heart.

Strengths:

The paper is very clearly and nicely written. The findings are novel and of high quality and relevance. The presentations are very clear and nicely interpreted. The analyses are well presented and applied.

Weaknesses:

From the heart rate traces, heart rate variability seems to be prominent and changes across days post-fertilization (dpf). That would be a useful dependent variable, considering that the variation captured by the models does not fully explain heart rate, both for sympathetic and parasympathetic efferents. Given the strong autorhythmicity of nodal tissue in neurogenic hearts, modulatory inputs could potentially predict heart rate variability with higher precision.

Reviewer #1 (Public review):

Summary:

This paper demonstrates the first application of voltage imaging using a genetically encoded voltage indicator, ArcLight, for recording the spontaneous activity of the developing spinal cord in zebrafish. This technology enabled better temporal resolution compared to what has been demonstrated with calcium imaging in past studies (Muto et al., 2011; Warp et al., 2012; Wan et al., 2019 ), which led to the discovery of the maturation process of "firing" shapes in spinal neurons. This maturation process occurs simultaneously with axonal elongation and network integration. Thus, voltage imaging revealed new biological details of the development of the spinal circuits.

Strengths:

The use of voltage imaging instead of calcium imaging revealed biological details of the functional maturation of spinal cord neurons in developing zebrafish.

Weaknesses:

This manuscript lacks many basic components and explanations necessary for understanding the methodologies used in this study.

Reviewer #1 (Public review):

Summary:

The manuscript reported a method for deep brain imaging with a GRIN lens that combines "low-NA telecentric scanning (LNTS) of laser excitation with high-NA fluorescence collection" to achieve a larger FOV than conventional approaches.

Strengths:

The manuscript presented in vivo structural images and calcium activity results in side-by-side comparison to wide-field epi fluorescence imaging through a GRIN lens and two-photon scanning imaging.

Weaknesses:

(1) Lack of sufficient technique information on the "high-NA (1.0) fluorescence collection". Is it custom-made or an off-the-shelf component? The only optical schematic, Figure 1, shows two lenses and a Si-PMT as the collection apparatus. There is no information about the lenses and the spacing between each component.

(2) There is no discussion about the speed limitation of the LNTS method, which, as a scanning-based method, is limited by the scanner speed. At a 10 Hz frame rate, the LNTS, although it has a better FOV, is much slower than widefield fluorescence imaging. The 10 Hz speed is not sufficient for some fast calcium activities.

(3) Supplementary Figure 5 is irrelevant to the main claim of the manuscript. This is a preliminary simulation related to the authors' proposed future work.

Reviewer #1 (Public review):

Summary:

This paper proposes a non-decision time (NDT)-informed approach to estimating time-varying decision thresholds in diffusion models of decision making. The manuscript motivates the method well, outlines the identifiability issues it is intended to address, and evaluates it using simulations and two empirical datasets. The aim is clear, the scope is deliberately focused, and the manuscript is well written. The core idea is interesting, technically grounded, and a meaningful contribution to ongoing work on collapsing thresholds.

Strengths:

The manuscript is logically structured and easy to follow. The emphasis on parameter recovery is appropriate and appreciated. The finding that the exponential NDT-informed function produces substantially better recovery than the hyperbolic form is useful, given the importance placed on identifiability earlier in the paper. The threshold visualisations are also helpful for interpreting what the models are doing. Overall, the work offers a well-defined, methodologically oriented contribution that will interest researchers working on time-varying thresholds.

Weaknesses / Areas for Clarification:

A few points would benefit from clarification, additional analysis, or revised presentation:

(1) It would help readers to see a concrete demonstration of the trade-off between NDT and collapsing thresholds, to give a sense of the scale of the identifiability problem motivating the work.

(2) Before moving to the empirical datasets, the manuscript really needs a simulation-based model-recovery comparison, since all major conclusions of the empirical applications rely on model comparison. One approach might be to simulate from (a) an FT model with across-trial drift variability and (b) one of the CT models, then fit both models to each of the simulated data sets. This would address a longstanding issue: sometimes CT models are preferred even when the estimated collapse in the thresholds is close to zero. A recovery study would confirm that model selection behaves sensibly in the new framework.

(3) An additional subtle point is that BIC is defined in terms of the maximised log-likelihood of the model for the data being modelled. In the joint model, the parameter estimates maximise the combined likelihood of behavioural and non-decision-time data. This means the behavioural log-likelihood evaluated at the joint MLEs is not the behavioural MLE. If BIC is being computed for the behavioural data only, this breaks the assumptions underlying BIC. The only valid BIC here would be one defined for the joint model using the joint likelihood.

(4) Table 1 sets up the Study 1 comparisons, but there's no row for the FT model. Similarly, Figures 10 and 13 would be more informative if they included FT predictions. This matters because, in Study 1, the FT model appears to fit aggregate accuracy better than the BIC-preferred collapsing model, currently shown only in Appendix 5. Some discussion of why would strengthen the argument.

(5) In Figure 7, the degree of decay underestimation is obscured by using a density plot rather than a scatterplot, consistent with the other panels of the same figure. Presenting it the same way would make the mis-recovery more transparent. The accompanying text may also need clarification: when data are generated from an FT model with across-trial drift variability, the NDT-informed model seems to infer FT boundaries essentially. If that's correct, the model must be misfitting the simulated data. This is actually a useful result as it suggests across-trial drift variability in FT models is discriminable from collapsing-threshold models. It would be good to make this explicit.

(6) Given the large recovery advantage of the exponential NDT-informed function over the hyperbolic one, the authors may want to consider whether the results favour adopting the former more generally. Given these findings, I would consider recommending the exponential NDT-informed model for future use.

(7) In Study 2 (Figure 13), all models qualitatively miss an interesting empirical pattern: under speed emphasis, errors are faster than corrects, while under accuracy emphasis, errors become slower. The error RT distribution in the speed condition is especially poorly captured. It would be helpful for the authors to comment, as it suggests that something theoretically relevant is missing from all models tested.

(8) The threshold visualisations extend to 3 seconds, yet both datasets show decisions mostly finishing by ~1.5 seconds. Shortening the x-axis would better reflect the empirical RT distributions and avoid unintentionally overstating the timescale of the empirical decision processes.

Reviewer #1 (Public review):

Summary:

Two major breakthroughs in the field of arbuscular mycorrhiza (AM) were the discoveries that first AM fungi obtain lipids (not only carbohydrates) from their plant hosts (Bravo et al 2017; Jiang et al 2017; Keymer et al 2017; Luginbuehl et al 2017) and second that presumably obligate biotrophic AM fungi can produce spores in the absence of host plants when exposed to myristate (Sugiura et al 2020; Tanaka et al 2022).

For this manuscript, Chen et al asked the question of whether myristate in the soil may also play a role in AM symbiosis when AM fungi live in symbiosis with their plant hosts. They show that myristate occurs in natural as well as agricultural soils, probably as a component of root exudates. Further, they treat AM fungi with myristate when grown in symbiosis in a Petri dish system with carrot hairy roots or in pots with alfalfa or rice to describe which effect the exogenous myristate has on symbiosis. Using 13C labelling, they show that myristate is taken up by AM fungi, although they can obtain sugars and lipids from the plant host. They also show that myristate leads to an increase in root colonization as well as expression of fungal genes involved in FA assimilation.

Interestingly, the effect of myristate on colonization depends on the plant species and the level of phosphate fertilization provided to the plant. The reason for this remains unknown.

Strengths:

The findings are interesting and provide an advance in our understanding of lipid use by the extraradical mycelium of AM fungi.

Weaknesses:

However, there are some misconceptions in the writing, and some experimental results remain poorly clear as they are presented in a highly descriptive manner without interpretation or explanation.

Reviewer #1 (Public review):

Summary:

In this work, Okell et al. describe the imaging protocol and analysis pipeline pertaining to the arterial spin labeling (ASL) MRI protocol acquired as part of the UK Biobank imaging study. In addition, they present preliminary analyses of the first 7000+ subjects in whom ASL data were acquired, and this represents the largest such study to date. Careful analyses revealed expected associations between ASL-based measures of cerebral hemodynamics and non-imaging-based markers, including heart and brain health, cognitive function, and lifestyle factors. As it measures physiology and not structure, ASL-based measures may be more sensitive to these factors compared with other imaging-based approaches.

Strengths:

This study represents the largest MRI study to date to include ASL data in a wide age range of adult participants. The ability to derive arterial transit time (ATT) information in addition to cerebral blood flow (CBF) is a considerable strength, as many studies focus only on the latter.

Some of the results (e.g., relationships with cardiac output and hypertension) are known and expected, while others (e.g., lower CBF and longer ATT correlating with hearing difficulty in auditory processing regions) are more novel and intriguing. Overall, the authors present very interesting physiological results, and the analyses are conducted and presented in a methodical manner.

The analyses regarding ATT distributions and the potential implications for selecting post-labeling delays (PLD) for single PLD ASL are highly relevant and well-presented.

Weaknesses:

At a total scan duration of 2 minutes, the ASL sequence utilized in this cohort is much shorter than that of a typical ASL sequence (closer to 5 minutes as mentioned by the authors). However, this implementation also included multiple (n=5) PLDs. As currently described, it is unclear how any repetitions were acquired at each PLD and whether these were acquired efficiently (i.e., with a Look-Locker readout) or whether individual repetitions within this acquisition were dedicated to a single PLD. If the latter, the number of repetitions per PLD (and consequently signal-to-noise-ratio, SNR) is likely to be very low. Have the authors performed any analyses to determine whether the signal in individual subjects generally lies above the noise threshold? This is particularly relevant for white matter, which is the focus of several findings discussed in the study.

Hematocrit is one of the variables regressed out in order to reduce the effect of potential confounding factors on the image-derived phenotypes. The effect of this, however, may be more complex than accounting for other factors (such as age and sex). The authors acknowledge that hematocrit influences ASL signal through its effect on longitudinal blood relaxation rates. However, it is unclear how the authors handled the fact that the longitudinal relaxation of blood (T1Blood) is explicitly needed in the kinetic model for deriving CBF from the ASL data. In addition, while it may reduce false positives related to the relationships between dietary factors and hematocrit, it could also mask the effects of anemia present in the cohort. The concern, therefore, is two-fold: (1) Were individual hematocrit values used to compute T1Blood values? (2) What effect would the deconfounding process have on this?

The authors leverage an observed inverse association between white matter hyperintensity volume and CBF as evidence that white matter perfusion can be sensitively measured using the imaging protocol utilized in this cohort. The relationship between white matter hyperintensities and perfusion, however, is not yet fully understood, and there is disagreement regarding whether this structural imaging marker necessarily represents impaired perfusion. Therefore, it may not be appropriate to use this finding as support for validation of the methodology.

Reviewer #2 (Public review):

Summary:

This is an interesting study exploring methods for reconstructing visual stimuli from neural activity in the mouse visual cortex. Specifically, it uses a competition dataset (published in the Dynamic Sensorium benchmark study) and a recent winning model architecture (DNEM, dynamic neural encoding model) to recover visual information stored in ensembles of mouse visual cortex.

Strengths:

This is a great start for a project addressing visual reconstruction. It is based on physiological data obtained at a single-cell resolution, the stimulus movies were reasonably naturalistic and representative of the real world, the study did not ignore important correlates such as eye position and pupil diameter, and of course, the reconstruction quality exceeded anything achieved by previous studies. There appear to be no major technical flaws in the study, and some potential confounds were addressed upon revision. The study is an enjoyable read.

Weaknesses:

The study is technically competent and benchmark-focused, but without significant conceptual or theoretical advances. The inclusion of neuronal data broadens the study's appeal, but the work does not explore potential principles of neural coding, which limits its relevance for neuroscience and may create some disappointment to some neuroscientists. The authors are transparent that their goal was methodological rather than explanatory, but this raises the question of why neuronal data were necessary at all, as more significant reconstruction improvements might be achievable using noise-less artificial video encoders alone (network-to-network decoding approaches have been done well by teams such as Han, Poggio, and Cheung, 2023, ICML). Yet, even within the methodological domain, the study does not articulate clear principles or heuristics that could guide future progress. The finding that more neurons improve reconstruction aligns with well-established results in the literature that show that higher neuronal numbers improve decoding in general (for example, Hung, Kreiman, Poggio, and DiCarlo, 2005) and thus may not constitute a novel insight.

Specific issues:

(1) The study showed that it could achieve high-quality video reconstructions from mouse visual cortex activity using a neural encoding model (DNEM), recovering 10-second video sequences and approaching a two-fold improvement in pixel-by-pixel correlation over attempts. As a reader, I was left with the question: okay, does this mean that we should all switch to DNEM for our investigations of mouse visual cortex? What makes this encoding model special? It is introduced as "a winning model of the Sensorium 2023 competition which achieved a score of 0.301...single trial correlation between predicted and ground truth neuronal activity," but as someone who does not follow this competition (most eLife readers are not likely to do so, either), I do not know how to gauge my response. Is this impressive? What is the best theoretical score, given noise and other limitations? Is the model inspired by the mouse brain in terms of mechanisms or architecture, or was it optimized to win the competition by overfitting it to the nuances of the data set? Of course, I know that as a reader, I am invited to read the references, but the study would stand better on its own, if it clarified how its findings depended on this model.

The revision helpfully added context to the Methods about the range of scores achieved by other models, but this information remains absent from the Abstract and other important sections. For instance, the Abstract states, "We achieve a pixel-level correlation of 0.57 between the ground truth movie and the reconstructions from single-trial neural responses," yet this point estimate (presented without confidence intervals or comparisons to controls) lacks meaning for readers who are not told how it compares to prior work or what level of performance would be considered strong. Without such context, the manuscript undercuts potentially meaningful achievements.

(2) Along those lines, the authors conclude that "the number of neurons in the dataset and the use of model ensembling are critical for high-quality reconstructions." If true, these principles should generalize across network architectures. I wondered whether the same dependencies would hold for other network types, as this could reveal more general insights. The authors replied that such extensions are expected (since prior work has shown similar effects for static images) but argued that testing this explicitly would require "substantial additional work," be "impractical," and likely not produce "surprising results." While practical difficulty alone is not a sufficient reason to leave an idea untested, I agree that the idea that "more neurons would help" would be unsurprising. The question then becomes: given that this is a conclusion already in the field, what new principle or understanding has been gained in this study?

(3) One major claim was that the quality of the reconstructions depended on the number of neurons in the dataset. There were approximately 8000 neurons recorded per mouse. The correlation difference between the reconstruction achieved by 1000 neurons and 8000 neurons was ~0.2. Is that a lot or a little? One might hypothesize that 7000 additional neurons could contribute more information, but perhaps, those neurons were redundant if their receptive fields are too close together or if they had the same orientation or spatiotemporal tuning. How correlated were these neurons in response to a given movie? Why did so many neurons offer such a limited increase in correlation? Originally, this question was meant to prompt deeper analysis of the neural data, but the authors did not engage with it, suggesting a limited understanding of the neuronal aspects of the dataset.

(4) We appreciated the experiments testing the capacity of the reconstruction process, by using synthetic stimuli created under a Gaussian process in a noise-free way. But this originally further raised questions: what is the theoretical capability for reconstruction of this processing pipeline, as a whole? Is 0.563 the best that one could achieve given the noisiness and/or neuron count of the Sensorium project? What if the team applied the pipeline to reconstruct the activity of a given artificial neural network's layer (e.g., some ResNet convolutional layer), using hidden units as proxies for neuronal calcium activity? In the revision, this concern was addressed nicely in the review in Supplementary Figure 3C. Also, one appreciates that as a follow up, the team produced error maps (New Figure 6) that highlight where in the frames the reconstruction are likely to fail. But the maps went unanalyzed further, and I am not sure if there was a systematic trend in the errors.

(5) I was encouraged by Figure 4, which shows how the reconstructions succeeded or failed across different spatial frequencies. The authors note that "the reconstruction process failed at high spatial frequencies," yet it also appears to struggle with low spatial frequencies, as the reconstructed images did not produce smooth surfaces (e.g., see the top rows of Figures 4A and 4B). In regions where one would expect a single continuous gradient, the reconstructions instead display specular, high-frequency noise. This issue is difficult to overlook and might deserve further discussion.

Reviewer #1 (Public review):

This manuscript introduces a biologically informed RNN (bioRNN) that predicts the effects of optogenetic perturbations in both synthetic and in vivo datasets. By comparing standard sigmoid RNNs (σRNNs) and bioRNNs, the authors make a compelling case that biologically grounded inductive biases improve generalization to perturbed conditions. This work is innovative, technically strong, and grounded in relevant neuroscience, particularly the pressing need for data-constrained models that generalize causally.

Comments on revisions:

The authors have addressed all my concerns.

Reviewer #1 (Public review):

Summary:

The authors analyzed the expression of ATAD2 protein in post-meiotic stages and characterized the localization of various testis-specific proteins in the testis of the Atad2 knockout (KO). By cytological analysis as well as the ATAC sequencing, the study showed that increased levels of HIRA histone chaperone, accumulation of histone H3.3 on post-meiotic nuclei, defective chromatin accessibility and also delayed deposition of protamines. Sperm from the Atad2 KO mice reduces the success of in vitro fertilization. The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin.

Strengths:

The paper describes the role of ATAD2 AAA+ ATPase in the proper localization of sperm-specific chromatin proteins such as protamine, suggesting the importance of the DNA replication-independent histone exchanges with the HIRA-histone H3.3 axis.

Weaknesses:

The work was performed well, and most of the results are convincing. However, this manuscript does not suggest a molecular mechanism for how ATAD2 promotes the formation of testis-specific chromatin.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The study lacks supporting in vivo data, such as gene knockdown experiments, to validate the proposed conclusions at the cellular level.

Reviewer #1 (Public review):

Summary:

In this manuscript, Subhramanian et al. carefully examined how microglia adapt their surveillance strategies during chronic neurodegeneration, specifically in prion-infected mice. The authors used ex vivo time-lapse imaging and in vitro strategies and found that reactive microglia adopt a highly mobile, "kiss-and-ride" behavior, contrasting the more static surveillance typical of homeostatic microglia. The manuscript provides fundamental mechanistic insights into the dynamics of microglia-neuron interactions, implicates P2Y6 signaling in regulating mobility, and suggests that intrinsic reprogramming of microglia might underlie this behavior, the conclusions are therefore compelling.

Strengths:

(1) The novelty of the study is high, particularly the demonstration that microglia lose territorial confinement and dynamically migrate from neuron to neuron under chronic neurodegeneration.

(2) The possible implications of a stimulus-independent high mobility in reactive microglia are particularly striking. Although this is not fully explored.

(3) The use of time-lapse imaging in organotypic slices rather than overexpression models provided a more physiological approach.

(4) Microglia-neuron interactions in neurodegeneration have broad implications for understanding the progression of diseases, such as Alzheimer's and Parkinson's, that are associated with chronic inflammation.

Weaknesses:

Previous weaknesses were addressed.

Reviewer #1 (Public review):

Summary:

Siddiqui et al., investigate the question of how bacterial metabolism contributes to the attraction of C. elegans to specific bacteria. They show that C. elegans prefers three bacterial species when cultured in a leucine-enriched environment. These bacterial species release more isoamyl alcohol, a known C. elegans attractant, when cultured with leucine supplement than without leucine supplement. The study shows correlative evidence that isoamyl alcohol is produced from leucine by the Ehrlich pathway. In addition, they show that SNIF-1 is a receptor for isoamyl alcohol because a null mutant of this receptor exhibits lower chemotaxis to isoamyl alcohol and that chemotaxis to isoamyl alcohol is rescued by expression of snif-1 in AWC.

Strengths:

(1) This study takes a creative approach to examine the question of what specific volatile chemicals released by bacteria may signify to C. elegans by examining both bacterial metabolism and C. elegans preference behavior. Although C. elegans has long been known to be attracted to bacterial metabolites, this study may be one of the first to examine the possible role of a specific bacterial metabolic pathway in mediating attraction.

(2) A strength of the paper is the identification of SNIF-1 as a receptor for isoamyl alcohol. The ligands for very few olfactory receptors have been identified in C. elegans and so this is a significant addition to the field. The SNIF-1 null mutant strain will likely be a useful reagent for many labs examining olfactory and foraging behaviors.

Weaknesses:

(1) The authors write that the leucine metabolism via the Ehrlich pathway is required for production of isoamyl alcohol by three bacteria (CEent1, JUb66, BIGb0170), but their evidence for this is correlation and not causation. They show that the gene, ilvE (which is part of the Ehrlich pathway) is upregulated in CEent1 bacteria upon exposure to leucine. Although this indicates that the ilvE gene may be involved in leucine metabolism, it does not show causation. To show causation, they need to knockout ilvE from one of these strains, show that the bacteria does not have increased isoamyl alcohol production when cultured on leucine, and that the bacteria is no longer attractive to C. elegans.

(2) Although the authors do show that the three bacterial strains they focus on (CEent1, JUb66, and BIGb0170) are more attractive to C. elegans when supplemented with leucine. Some other strains such as BIGb0393 are also more attractive with leucine supplementation and produce isoamyl alcohol (Fig 1B and Supp Table 2). It is unclear why these other strains are not included with the selected three strains.

(3) The behavioral evidence that snif-1 gene encodes a receptor for isoamyl alcohol is compelling because of the mutant phenotype and rescue experiments. The evidence would be stronger with calcium imaging of AWC neurons in response to isoamyl alcohol in the receptor mutant with the expectation that the response would be reduced or abolished in the mutant compared to wildtype.

Reviewer #1 (Public review):

Summary:

This work by Al-Jezani et al. focused on characterizing clonally derived MSC populations from the synovium of normal and osteoarthritis (OA) patients. This included characterizing the cell surface marker expression in situ (at time of isolation), as well as after in vitro expansion. The group also tried to correlate marker expression with trilineage differential potential. They also tested the ability of the different sub-populations for their efficacy in repairing cartilage in a rat model of OA. The main finding of the study is that CD47hi MSCs may have a greater capacity to repair cartilage than CD47lo MSCs, suggesting that CD47 may be a novel marker of human MSCs that have enhanced chondrogenic potential.

Strengths:

Studies on cell characterization of the different clonal populations isolated indicate that the MSC are heterogenous and traditional cell surface markers for MSCs do not accurately predict the differentiation potential of MSCs. While this has been previously established in the field of MSC therapy, the authors did attempt to characterize clones derived from single cells, as well as evaluate the marker profile at the time of isolation. While the outcome of heterogeneity is not surprising, the methods used to isolate and characterze the cells were well developed. The interesting finding of the study is the identification of CD47 as a potential MSC marker that could be related to chondrogenic potential. The authors suggest that MSCs with high CD47 repaired cartilage more effectively than MSC with low CD47 in a rat OA model.

Comments on revisions:

Thank you for addressing the comments from the first review. No additional revisions.

Reviewer #1 (Public review):

This is a highly original and impactful study that significantly advances our understanding of transcriptional regulation, in particular RNAPII pausing, during early heart development. The Chen lab has a long history of producing influential studies in cardiac morphogenesis, and this manuscript represents another thorough and mechanistically insightful contribution. The authors have thoroughly addressed this Reviewer's concerns and incorporated all of my suggestions in the revised manuscript. In addition, their responses to the other reviewer's comments are also very clear. As it is, this work is of great interest to the readership of Elife, as well as to the general scientific community.

The authors reveal a fundamentally new role for Rtf1-a component of the PAF1 complex-in governing promoter-proximal RNAPII pausing in the context of myocardial lineage specification. While transcriptional pausing has been implicated in stress responses and inducible gene programs, its developmental relevance has remained poorly defined. This study fills that gap with rigorous in vivo evidence demonstrating that Rtf1-dependent pausing is indispensable for activating the cardiac gene program from the lateral plate mesoderm.

Importantly, the study also provides compelling therapeutic implications. Showing that CDK9 inhibition-using either flavopiridol or targeted knockdown-can restore promoter-proximal pausing and rescue cardiomyocyte formation in Rtf1-deficient embryos suggests that modulation of pause-release kinetics may represent a new avenue for correcting transcriptionally driven congenital heart defects. Given that many CDK inhibitors are clinically approved or in active development, this connection significantly elevates the translational impact of the findings.

In sum, this study is rigorous, innovative, and transformative in its implications for developmental biology and cardiac medicine. I strongly support its publication.

Reviewer #1 (Public review):

The authors used fluorescence microscopy, image analysis, and mathematical modeling to study the effects of membrane affinity and diffusion rates of MinD monomer and dimer states on MinD gradient formation in B. subtilis. To test these effects, the authors experimentally examined MinD mutants that lock the protein in specific states, including Apo monomer (K16A), ATP-bound monomer (G12V) and ATP-bound dimer (D40A, hydrolysis defective), and compared to wild-type MinD. Overall, the experimental results support the conclusions that reversible membrane binding of MinD is critical for the formation of the MinD gradient, but the binding affinities between monomers and dimers are similar.

The modeling part is a new attempt to use the Monte Carlo method to test the conditions for the formation of the MinD gradient in B. subtilis. The modeling results provide good support for the observations and find that the MinD gradient is sensitive to different diffusion rates between monomers and dimers. This simulation is based on several assumptions and predictions, which raises new questions that need to be addressed experimentally in the future.

Reviewer #1 (Public review):

Summary:

This manuscript investigates the biological mechanism underlying the assembly and transport of the AcrAB-TolC efflux pump complex. By combining endogenous protein purification with cryo-EM analysis, the authors show that the AcrB trimer adopts three distinct conformations simultaneously and identify a previously uncharacterized lipoprotein, YbjP, as a potential additional component of the complex. The work aims to advance our understanding of the AcrAB-TolC efflux system in near-native conditions and may have broader implications for elucidating its physiological mechanism.

Strengths:

Overall, the manuscript is clearly presented, and several of the datasets are of high quality. The use of natively isolated complexes is a major strength, as it minimizes artifacts associated with reconstituted systems and enables the discovery of a novel subunit. The authors also distinguish two major assemblies-the TolC-YbjP sub-complex and the complete pump-which appear to correspond to the closed and open channel states, respectively. The conceptual advance is potentially meaningful, and the findings could be of broad interest to the field.

Weaknesses:

(1) As the identification of YbjP is a key contribution of this work, a deeper comparison with functional "anchor" proteins in other efflux pumps is needed. Including an additional supplementary figure illustrating these structural comparisons would be valuable.

(2) The observation of the LTO states in the presence of TolC represents an important extension of previous findings. A more detailed discussion comparing these LTO states to those reported in earlier structural and biochemical studies would improve the clarity and significance of this point.

Reviewer #1 (Public review):

Summary:

The manuscript by Lu and colleagues demonstrates convincingly that PRRT2 interacts with brain voltage-gated sodium channels to enhance slow inactivation in vitro and in vivo. The work is interesting and rigorously conducted. The relevance to normal physiology and disease pathophysiology (e.g., PRRT2-related genetic neurodevelopmental disorders) seems high. Some simple additional experiments could elevate the impact and make the study more complete.

Strengths:

Experiments are conducted rigorously, including experimenter blinding and appropriate controls. Data presentation is excellent and logical. The paper is well written for a general scientific audience.

Weaknesses:

There are a few missing experiments and one place where data are over-interpreted.

(1) An in vitro study of Nav1.6 is conspicuously absent. In addition to being a major brain Na channel, Nav1.6 is predominant in cerebellar Purkinje neurons, which the authors note lack PRRT2 expression. They speculate that the absence of PRRT2 in these neurons facilitates the high firing rate. This hypothesis would be strengthened if PRRT2 also enhanced slow inactivation of Nav1.6. If a stable Nav1.6 cell were not available, then simple transient co-transfection experiments would suffice.

(2) To further demonstrate the physiological impact of enhanced slow inactivation, the authors should consider a simple experiment in the stable cell line experiments (Figure 1) to test pulse frequency dependence of peak Na current. One would predict that PRRT2 expression will potentiate 'run down' of the channels, and this finding would be complementary to the biophysical data.

(3) The study of one K channel is limited, and the conclusion from these experiments represents an over-interpretation. I suggest removing these data unless many more K channels (ideally with measurable proxies for slow inactivation) were tested. These data do not contribute much to the story.

(4) In Figure 2, the authors should confirm that protein is indeed expressed in cells expressing each truncated PRRT2 construct. Absent expression should be ruled out as an explanation for absent enhancement of slow inactivation.

Reviewer #1 (Public review):

Summary:

This work provides valuable new insights into the Paleocene Asian mammal recovery and diversification dynamics during the first ten million years post-dinosaur extinction. Studies that have examined the mammalian recovery and diversification post-dinosaur extinction have primarily focused on the North American mammal fossil record, and it's unclear if patterns documented in North America are characteristic of global patterns. This study examines dietary metrics of Paleocene Asian mammals and found that there is a body size disparity increase before dietary niche expansion and that dietary metrics track climatic and paleobotanical trends of Asia during the first 10 million years after the dinosaur extinction.

Strengths:

The Asian Paleocene mammal fossil record is greatly understudied, and this work begins to fill important gaps. In particular, the use of interdisciplinary data (i.e., climatic and paleobotanical) is really interesting in conjunction with observed dietary metric trends.

Weaknesses:

While this work has the potential to be exciting and contribute greatly to our understanding of mammalian evolution during the first 10 million years post-dinosaur extinction, the major weakness is in the dental topographic analysis (DTA) dataset.

There are several specimens in Figure 1 that have broken cusps, deep wear facets, and general abrasion. Thus, any values generated from DTA are not accurate and cannot be used to support their claims. Furthermore, the authors analyze all tooth positions at once, which makes this study seem comprehensive (200 individual teeth), but it's unclear what sort of noise this introduces to the study. Typically, DTA studies will analyze a singular tooth position (e.g., Pampush et al. 2018 Biol. J. Linn. Soc.), allowing for more meaningful comparisons and an understanding of what value differences mean. Even so, the dataset consists of only 48 specimens. This means that even if all the specimens were pristinely preserved and generated DTA values could be trusted, it's still only 48 specimens (representing 4 different clades) to capture patterns across 10 million years. For example, the authors note that their results show an increase in OPCR and DNE values from the middle to the late Paleocene in pantodonts. However, if a singular tooth position is analyzed, such as the lower second molar, the middle and late Paleocene partitions are only represented by a singular specimen each. With a sample size this small, it's unlikely that the authors are capturing real trends, which makes the claims of this study highly questionable.

Reviewer #1 (Public review):

Summary:

This is an interesting and useful review highlighting the complex pathways through which pulmonary colonisation or infection with Mycobacterium tuberculosis (Mtb) may progress to develop symptomatic disease and transmit the pathogen. I found the section on immune correlates associated with individuals who have clearly been exposed to and reacted to Mtb but did not develop latent infections particularly valuable. However, several aspects would benefit from clarification.

Strengths:

The main strengths lie in the arguments presented for a multiplicity of immune pathways to TB disease.

Weaknesses:

The main weaknesses lie in clarity, particularly in the precise meanings of the three figures.

I accept that there is a 'goldilocks zone' that underpins the majority of TB cases we see and predominantly reflects different patterns of immune response, but the analogies used need to be more clearly thought through.

Reviewer #1 (Public review):

In this important study, the authors characterized the transformation of neural representations of olfactory stimuli from primary sensory cortex to multisensory regions in the medial temporal lobe and investigated how they were affected by non-associative learning. The authors used high-density silicon probe recordings from five different cortical regions while familiar vs. novel odors were presented to a head-restrained mouse. This is a timely study because unlike other sensory systems (e.g., vision), the progressive transformation of olfactory information is still poorly understood. The authors report that both odor identity and experience are encoded by all of these five cortical areas but nonetheless, some themes emerge. Single neuron tuning of odor identity is broad in the sensory cortices but becomes narrowly tuned in hippocampal regions. Furthermore, while experience affects neuronal response magnitudes in early sensory cortices, it changes the proportion of active neurons in hippocampal regions. Thus, this study is an important step forward in the ongoing quest to understand how olfactory information is progressively transformed along the olfactory pathway.

The study is well-executed. The direct comparison of neuronal representations from five different brain regions is impressive. Conclusions are based on single neuronal level as well as population level decoding analyses. Among all the reported results, one stands out for being remarkably robust. The authors show that the anterior olfactory nucleus (AON), which receives direct input from the olfactory bulb output neurons, was far superior at decoding odor identity as well as novelty compared to all the other brain regions. This is perhaps surprising because the other primary sensory region - the piriform cortex - has been thought to be the canonical site for representing odor identity. A vast majority of studies have focused on aPCx, but direct comparisons between odor coding in the AON and aPCx are rare. The experimental design of this current study allowed the authors to do so and the AON was found to convincingly outperform aPCx. Although this result goes against the canonical model, it is consistent with a few recent studies including one that predicted this outcome based on anatomical and functional comparisons between the AON-projecting tufted cells vs. the aPCx-projecting mitral cells in the olfactory bulb.

Future experiments are needed to probe the circuit mechanisms underlying the differential importance of the two primary olfactory cortices, as well as their potential causal roles in odor identification. Moreover, future work should test whether the decoding accuracy of odor identity and experience from neural data (as reported here) can predict the causal contributions of these regions, as revealed through perturbations during behavioral tasks that explicitly probe odor identification and/or experience.

Reviewer #1 (Public review):

Summary:

This manuscript describes critical intermediate reaction steps of a HA synthase at the molecular level; specifically, it examines the 2nd step, polymerization, adding GlcA to GlcNAc to form the initial disaccharide of the repeating HA structure. Unlike the vast majority of known glycosyltransferases, the viral HAS (a convenient proxy extrapolated to resemble the vertebrate forms) uses a single pocket to catalyze both monosaccharide transfer steps. The authors' work illustrates the interactions needed to bind & proof-read the UDP-GlcA using direct and '2nd layer' amino acid residues. This step also allows the HAS to distinguish the two UDP-sugars; this is very important as the enzymes are not known or observed to make homopolymers of only GlcA or GlcNAc, but only make the HA disaccharide repeats GlcNAc-GlcA.

Strengths:

Overall, the strengths of this paper lie in its techniques & analysis.

The authors make significant leaps forward towards understanding this process using a variety of tools and comparisons of wild-type & mutant enzymes. The work is well presented overall with respect to the text and illustrations (especially the 3D representations), and the robustness of the analyses & statistics is also noteworthy.

Furthermore, the authors make some strides towards creating novel sugar polymers using alternative primers & work with detergent binding to the HAS. The authors tested a wide variety of monosaccharides and several disaccharides for primer activity and observed that GlcA could be added to cellobiose and chitobiose, which are moderately close structural analogs to HA disaccharides. Did the authors also test the readily available HA tetramer (HA4, [GlcA-GlcNAc]2) as a primer in their system? This is a highly recommended experiment; if it works, then this molecule may also be useful for cryo-EM studies of CvHAS as well.

Weaknesses:

In the past, another report describing the failed attempt of elongating short primers (HA4 & chitin oligosaccharides larger than the cello- or chitobiose that have activity in this report) with a vertebrate HAS, XlHAS1, an enzyme that seems to behave like the CvHAS ( https://pubmed.ncbi.nlm.nih.gov/10473619/); this work should probably be cited and briefly discussed. It may be that the longer primers in the 1999 paper and/or the different construct or isolation specifics (detergent extract vs crude) were not conducive to the extension reaction, as the authors extracted recombinant enzyme.

There are a few areas that should be addressed for clarity and correctness, especially defining the class of HAS studied here (Class I-NR) as the results may (Class I-R) or may not (Class II) align (see comment (a) below), but overall, a very nicely done body of work that will significantly enhance understanding in the field.

Reviewer #2 (Public review):

Summary:

This study investigated whether the identity of a peripheral saccade target object is fed back to the foveal retinotopic cortex during saccade preparation, a critical prediction of the foveal prediction hypothesis proposed by Kroell & Rolfs (2022). To achieve this, the authors leveraged a gaze-contingent fMRI paradigm, where the peripheral saccade target was removed before the eyes landed near it, and used multivariate decoding analysis to quantify identity information in the foveal cortex. The results showed that the identity of the saccade target object can be decoded based on foveal cortex activity, despite the fovea never directly viewing the object, and that the foveal feedback representation was similar to passive viewing and not explained by spillover effects. Additionally, exploratory analysis suggested IPS as a candidate region mediating such foveal decodability. Overall, these findings provide neural evidence for the foveal cortex processing the features of the saccade target object, potentially supporting the maintenance of perceptual stability across saccadic eye movements.

Strengths:

This study is well-motivated by previous theoretical findings (Kroell & Rolfs, 2022), aiming to provide neural evidence for a potential neural mechanism of trans-saccadic perceptual stability. The question is important, and the gaze-contingent fMRI paradigm is a solid methodological choice for the research goal. The use of stimuli allowing orthogonal decoding of stimulus category vs stimulus shape is a nice strength, and the resulting distinctions in decoded information by brain region are clean. The results will be of interest to readers in the field, and they fill in some untested questions regarding pre-saccadic remapping and foveal feedback.

Weaknesses:

The authors have done a nice job addressing the previous weaknesses. The remaining weaknesses / limitations are appropriately discussed in the manuscript. E.g., the use of only 4 unique stimuli in the experiment. The findings are intriguing and relevant to saccadic remapping and foveal feedback, but somewhat limited in terms of the ability to draw theoretical distinctions between these related phenomena.

Specifics:

The revised manuscript is much improved in terms of framing and discussion of the prior literature, and the theoretical claims are now stated with appropriate nuance.

I have two remaining minor suggestions/comments, which the authors may optionally respond to:

(1) In the parametric modulation analysis, the authors' additional analyses nicely addresses my concern and strengthens the claim. However, the description in the revised manuscript (Pg 7 Ln 190-191) is minimal and may be difficult to grasp what the control analysis is about and how it rules out alternative explanations to the IPS findings. The authors may wish to elaborate on the description in the text.

(2) Out of curiosity (not badgering): The authors argued that the findings of Harrison et al. (2013) and Szinte et al. (2015) can be explained by feature integration between the currently attended location and its future, post-saccadic location. Couldn't the same argument apply in the current paradigm, where attention at the saccade target gets remapped to the pre-saccadic fovea (see also Rolfs et al., 2011 Fig 5), thus leading to the observed feature remapping?

Reviewer #2 (Public review):

Summary:

The author proposes a novel method for mapping single-cell data to specific locations with higher resolution than several existing tools.

Strengths:

The spatial mapping tests were conducted on various tissues, including the mouse cortex, human PDAC, and intestinal villus.

Comments on revised version:

I have no additional comments regarding the current version of the manuscript.

Reviewer #1 (Public review):

Summary:

This study examines whether changes in pupil size index prediction-error-related updating during associative learning, formalised as information gain via Kullback-Leibler (KL) divergence. Across two independent tasks, pupil responses scaled with KL divergence shortly after feedback, with the timing and direction of the response varying by task. Overall, the work supports the view that pupil size reflects information-theoretic processes in a context-dependent manner.

Strengths:

This study provides a novel and convincing contribution by linking pupil dilation to information-theoretic measures, such as KL divergence, supporting Zénon's hypothesis that pupil responses reflect information gain during learning. The robust methodology, including two independent datasets with distinct task structures, enhances the reliability and generalisability of the findings. By carefully analysing early and late time windows, the authors capture the timing and direction of prediction-error-related responses, offering new insights into the temporal dynamics of model updating. The use of an ideal-learner framework to quantify prediction errors, surprise, and uncertainty provides a principled account of the computational processes underlying pupil responses. The work also highlights the critical role of task context in shaping the direction and magnitude of these effects, revealing the adaptability of predictive processing mechanisms. Importantly, the conclusions are supported by rigorous control analyses and preprocessing sanity checks, as well as convergent results from frequentist and Bayesian linear mixed-effects modelling approaches.

Weaknesses:

Some aspects of directionality remain context-dependent, and on current evidence cannot be attributed specifically to whether average uncertainty increases or decreases across trials. Differences between the two tasks (e.g., sensory modality and learning regime) limit direct comparisons of effect direction and make mechanistic attribution cautious. In addition, subjective factors such as confidence were not measured and could influence both prediction-error signals and pupil responses. Importantly, the authors explicitly acknowledge these limitations, and the manuscript clearly frames them as areas for future work rather than settled conclusions.

Reviewer #2 (Public review):

Summary

This study investigates the role of GMCL1 in regulating the mitotic surveillance pathway (MSP), a protective mechanism that activates p53 following prolonged mitosis. The authors identify a physical interaction between 53BP1 and GMCL1, but not with GMCL2. They propose that the ubiquitin ligase complex CRL3-GMCL1 targets 53BP1 for degradation during mitosis, thereby preventing the formation of the "mitotic stopwatch" complex (53BP1-USP28-p53) and subsequent p53 activation. The authors show that high GMCL1 expression correlates with resistance to paclitaxel in cancer cell lines that express wild-type p53. Importantly, loss of GMCL1 restores paclitaxel sensitivity in these cells, but not in p53-deficient lines. They propose that GMCL1 overexpression enables cancer cells to bypass MSP-mediated p53 activation, promoting survival despite mitotic stress. Targeting GMCL1 may thus represent a therapeutic strategy to re-sensitize resistant tumors to taxane-based chemotherapy.

Strengths

This manuscript presents potentially interesting observations. The major strength of this article is the identification of GMCL1 as 53BP1 interaction partner. The authors identified relevant domains and show that GMCL1 controls 53BP1 stability. The authors further show a potentially interesting link between GMCL1 status and sensitivity to Taxol.

Weaknesses

A major limitation of the original manuscript was that the functional relevance of GMCL1 in regulating 53BP1 within an appropriate model system was not clearly demonstrated. In the revised version, the authors attempt to address this point. However, the new experiment is insufficiently controlled, making it difficult to interpret the results. State-of-the-art approaches would typically rely on single-cell tracking to monitor cell fate following release from a moderately prolonged mitosis.

In contrast, the authors use a population-based assay, but the reported rescue from arrest is minimal. If the assay were functioning robustly, one would expect that nearly all cells depleted of USP28 or 53BP1 should have entered S-phase at a defined time after release. Thus, the very small rescue effect of siTP53BP1 suggests that the current assay is not suitable. It is also likely that release from a 16-hour mitotic arrest induces defects independent of the 53BP1-dependent p53 response.

Furthermore, the cell-cycle duration of RPE1 cells is less than 20 hours. It is therefore unclear why cells are released for 30 hours before analysis. At this time point, many cells are likely to have progressed into the next cell cycle, making it impossible to draw conclusions regarding the immediate consequences of prolonged mitosis. As a result, the experiment cannot be evaluated due to inadequate controls.

To strengthen this part of the study, I recommend that the authors first establish an assay that reliably rescues the mitotic-arrest-induced G1 block upon depletion of p53, 53BP1, or USP28. Once this baseline is validated, GMCL1 knockout can then be introduced to quantify its contribution to the response.

A broader conceptual issue is that the evidence presented does not form a continuous line of reasoning. For example, it is not demonstrated that GMCL1 interacts with or regulates 53BP1 in RPE1 cells-the system in which the limited functional experiments are conducted.

There are also a number of inconsistencies and issues with data presentation that need to be addressed:

(1) Figure 2C: p21 levels appear identical between GMCL1 KO and WT rescue. If GMCL1 regulates p53 through 53BP1, p21 should be upregulated in the KO.

(2) Figure 2A vs. 2C: GMCL1 KO affects chromatin-bound 53BP1 in Figure 2A, yet in Figure 2C it affects 53BP1 levels specifically in G1-phase cells. This discrepancy requires clarification.

(3) Figure 2C quantification: The three biological repeats show an unusual pattern, with one repeat's data points lying exactly between the other two. It is unclear what the line represents; please clarify.

(4) Figure nomenclature: Some abbreviations (e.g., FLAG-KI in Fig. 1F, WKE in Fig. 1C-D, ΔMFF in Fig. 1E) are not defined in the figure legends. All abbreviations must be explained.

(5) Figure 2D: Please indicate how many times the experiment was reproduced. Quantification with statistical testing would strengthen the result. Pull-downs of 53BP1 with calculation of the ubiquitinated/total ratio could also support the conclusion.

(6) Figures 3A and 3C: The G1 bars share the same color as the error bars, making the graphs difficult to interpret. Please adjust the color scheme.

Reviewer #1 (Public review):

Summary:

This paper introduces a dual-pathway model for reconstructing naturalistic speech from intracranial ECoG data. It integrates an acoustic pathway (LSTM + HiFi-GAN for spectral detail) and a linguistic pathway (Transformer + Parler-TTS for linguistic content). Output from the two components is later merged via CosyVoice2.0 voice cloning. Using only 20 minutes of ECoG data per participant, the model achieves high acoustic fidelity and linguistic intelligibility.

Strengths:

(1) The proposed dual-pathway framework effectively integrates the strengths of neural-to-acoustic and neural-to-text decoding and aligns well with established neurobiological models of dual-stream processing in speech and language.

(2) The integrated approach achieves robust speech reconstruction using only 20 minutes of ECoG data per subject, demonstrating the efficiency of the proposed method.

(3) The use of multiple evaluation metrics (MOS, mel-spectrogram R², WER, PER) spanning acoustic, linguistic (phoneme and word), and perceptual dimensions, together with comparisons against noise-degraded baselines, adds strong quantitative rigor to the study.

Weaknesses:

(1) It is unclear how much the acoustic pathway contributes to the final reconstruction results, based on Figures 3B-E and 4E. Including results from Baseline 2 + CosyVoice and Baseline 3 + CosyVoice could help clarify this contribution.

(2) As noted in the limitations, the reconstruction results heavily rely on pre-trained generative models. However, no comparison is provided with state-of-the-art multimodal LLMs such as Qwen3-Omni, which can process auditory and textual information simultaneously. The rationale for using separate models (Wav2Vec for speech and TTS for text) instead of a single unified generative framework should be clearly justified. In addition, the adaptor employs an LSTM architecture for speech but a Transformer for text, which may introduce confounds in the performance comparison. Is there any theoretical or empirical motivation for adopting recurrent networks for auditory processing and Transformer-based models for textual processing?

(3) The model is trained on approximately 20 minutes of data per participant, which raises concerns about potential overfitting. It would be helpful if the authors could analyze whether test sentences with higher or lower reconstruction performance include words that were also present in the training set.

(4) The phoneme confusion matrix in Figure 4A does not appear to align with human phoneme confusion patterns. For instance, /s/ and /z/ differ only in voicing, yet the model does not seem to confuse these phonemes. Does this imply that the model and the human brain operate differently at the mechanistic level?

(5) In general, is the motivation for adopting the dual-pathway model to better align with the organization of the human brain, or to achieve improved engineering performance? If the goal is primarily engineering-oriented, the authors should compare their approach with a pretrained multimodal LLM rather than relying on the dual-pathway architecture. Conversely, if the design aims to mirror human brain function, additional analysis, such as detailed comparisons of phoneme confusion matrices, should be included to demonstrate that the model exhibits brain-like performance patterns.

Reviewer #1 (Public review):

Summary:

Here, the authors address the organization of reach-related activity in layer 2/3 across a broad swath of anterodorsal neocortex that included large subregions of M1, M2, and S1. In mice performing a novel variant water-reaching task, the authors measured activity using two-photon fluorescence imaging of a GECI expressed in excitatory projection neurons. The authors found a substantial diversity of response patterns using a number of metrics they developed for characterizing the PETHs of neurons across reach conditions (target locations). By mapping single-neuron properties across the cortex, the authors found substantial spatial variation, only some of which aligned with traditional boundaries between cortical regions. Using Gaussian mixture models, the authors found evidence of distinct response types in each region, with several types prominent in multiple cortical regions. Aggregating across regions, four primary subpopulations were apparent, each distinct in its average response properties. Strikingly, each subpopulation was observed in multiple regions, but subpopulation members from different regions exhibited largely similar response properties.

Strengths:

The work addresses a fundamental question in the field that has not previously been addressed at cellular resolution across such a broad cortical extent. I see this as truly foundational work that will support future investigation of how the rodent brain drives and controls reaching.

The quantification is thoughtful and rigorous. It is great that the authors provide an explanation for and intuition behind their response metrics, rather than burying everything in the Methods.

The Discussion and general contextualization of the results are thorough, thoughtful, and strong. It is great that the authors avoid the common over-interpretation of classical observations regarding cortical organization that are endemic in the field.

All things considered, this is the best paper regarding spatial structure in the motor system I have ever read. The breadth of cellular resolution activity measurement, the rigor of the quantification, and the clear and open-minded interrogation of the data collectively have produced a very special piece of work.

Weaknesses:

The behavioral task is very impressive and an important contribution to the field in its own right. However, given that it appears substantially different from the one used in the previous paper, the characterization of the behavior provided in the Results is too brief. More illustration of the behavior would be helpful. For example, it is rather deep into the paper when the authors reveal that the mice can whisk to help localize the target location. That should be expressed at the outset when the behavior is first described. Other suggestions for elaborating the behavior description are included below.

Statistical support for key claims is lacking. For example, "The five areas of interest varied in the fraction of neurons that were modulated: M2 had 14%, M1 had 23%, S1-fl had 30%, S1-hl had 25%, and S1-tr had 27%" - I cannot locate the statistical tests showing that these values are actually different. Another example is Figure 7, where a key observation is that distributions of PETH features are distinct across regions. It is clear that at least some distributions are not overlapping, but a clearer statistical basis for this key claim should be provided.

I understand that the authors are planning a follow-up study that addresses the relation between activity patterns and kinematics. One question about interpreting the results here though, is how much the activity variation across target locations may relate to the kinematic differences across these different conditions, as opposed to true higher-order movement features like reach direction.

Reviewer #1 (Public review):

WIPI1 is a PROPPIN family protein that has been implicated in Retromer-mediated membrane fission events. Although the cargos that it has been tested to be important for are diverse, one of the cargos that is unaffected is Beta1-Integrin. This leads the authors to assess another PROPPIN family protein - WIPI2, which is a homolog of WIPI1. KD using siRNA is effective and had no consequences on LAMP1, EGFR trafficking or GLUT1 trafficking. Integrin-B1, however, had a large and significant defect in its recycling from the endosome, with a clear endosomal colocalisation. Complementation experiments with WT WIPI2 recovered the phenotype, but various mutant WIPI2 complements resulted in elongated tubules, and there was also a dominant negative effect of the mutant. Integrin is a classic retreiver cargo, so the authors rationalise that WIPI2 may be playing a role with retreiver that WIPI1 plays with retromer. To assess this, they perform a set of immunoprecipitations. SNX17, the retreiver-associated sorting nexin, co-IPs with WIPI2 in a VPS26C-dependent manner. VPS26C but not VPS26 co-IPs with WIPI2, and the reciprocal with WIPI1. These interactions were not present for the FSSS mutation of WIPI2. WIPI2 localises to Rab11 endosomes mainly, as does retriever. Mutations of WIPI2 not only affected WIPI2 localisation, but also VPS35L mutations, indicating that there is a functional relationship between the two.

On the whole, I find the manuscript compelling. The manuscript is very clearly written, the results are convincing and well performed. The flow of experiments is logical, and although not comprehensive in the subsequent mechanistic understanding, the fundamental findings are important and convincing. My comments below are, on the whole, minor and are intended to support the communication of the findings to the field.

(1) The IP interaction data were convincing; however, for me and some others, an interaction is only convincing when performed in vitro, and understood at a structural level. I do not suggest the authors do that in this case; however, I think, at a minimum, some sensible moderation of claims would be useful here.

(2) I found the final localisation data and its interpretation confusing. My interpretation of that data would not be that the retreiver is relocalised, but rather that there is less of both recruited to the membrane and the remaining localisation distribution is shifted. In addition, I am not quite sure of the model here - is the idea that WIPI2 recruits retreiver, if that is the case, I find it hard to resolve with its role as a mediator of fission. Clarity would be appreciated here.

(3) I am concerned that the repeats being compared for statistical analysis are not biological repeats but technical repeats (cells in the same experiment). I should think the idea of the statistical comparison is to show experimental reproducibility and variability across biological repeats. Therefore, I would expect an appropriate number of biological repeats (3 or more minimum), to be the data compared in the statistical analysis and graphs. I think it is appropriate to average the technical repeats from each biological repeat. I find these to be useful resources https://doi.org/10.1083/jcb.202401074, https://doi.org/10.1083/jcb.200611141

Reviewer #1 (Public review):

A summary of what the authors were trying to achieve:

(1) Identify probiotic candidates based on the phylogenetic proximity and their presence in the lower respiratory tract based on phylogenetic analysis and on meta-analysis of 16S rRNA sequencing of mouse lung samples.

(2) Predefine probiotic candidates with overlapping and competing metabolic profiles based on a simple and easy-to-applicable score, taking carbon source use into consideration.

(3) Confirm the functionality of these candidate probiotics in vitro and define their mechanism of action (niche exclusion by either metabolic competition or active antibacterial strategies).

(4) Confirm the probiotic action in vivo.

Strengths:

The authors attempt to go the whole 9 yards from rational choice of phylogenetic close lower respiratory tract probiotics, over in silico modelling of niche index based on use of similar carbon sources with in vitro confirmation, to in vivo competition experiments in mice.

Weaknesses:

(1) The use of a carbon source is defined as growth to OD600 two SD above the blank level. While allowing a clear cutoff, this procedure does not take into account larger differences in the preferences of carbon sources between the pathogen and the probiotic candidate. If the pathogen is much better at taking up and processing a carbon source, the competition by the probiotic might be biologically irrelevant.

(2) The authors do not take into account the growth of candidate probiotics in the presence of Bt. In monoculture, three of the four most potent candidate probiotics grow to comparable levels as Bt in LSM.

(3) Niche exclusion in vivo is not shown. Mortality of hosts after infection with Bt is not a measure for competition of CP with the pathogen. Only Bt titers would prove a competitive effect. For CP17, less than half of the mice were actually colonized, but still, there is 100% protection. Activation of the host immune system would explain this and has to be excluded as an alternative reason for improved host survival.

Appraisal:

(1) Based on phylogenetic comparison and published resources on lower respiratory tract colonizing bacteria, the authors find a reasonably good number of candidate probiotics that grow in LSM and successfully compete with the pathogenic target bacterium Bt in vitro.

(2) In vivo, only host survival was tested, and a direct competition of CP with Bt by testing for Bt titers was not shown.

Impact:

Niche exclusion based on competition for environmentally provided metabolites is not a new concept and was experimentally tested, e.g. in the intestine. The authors show here that this concept could be translated into the resource-poor environment of the respiratory tract. It remains to be tested if the LSM growth-based competition data in vitro can be translated into niche exclusion in vivo.

Reviewer #1 (Public review):

Summary:

This research group has consistently performed cutting-edge research aiming to understand the role of hormones in the control of social behaviors, specifically by utilizing the genetically-tractable teleost fish, medaka, and the current work is no exception. The overall claim they make, that estrogens modulate social behaviors in males is supported, with important caveats. For one, there is no evidence these estrogens are generated by "neurons" as would be assumed by their main claim that it is NEUROestrogens that drive this effect. While indeed the aromatase they have investigated is expressed solely in the brain, in most teleosts, brain aromatase is only present in glial cells (astrocytes, radial glia). The authors should change this description so as not to mislead the reader. Below I detail more specific strengths and weaknesses of this manuscript.

Strengths:

Excellent use of the medaka model to disentangle the control of social behavior by sex steroid hormones

The findings are strong for the most part because deficits in the mutants are restored by the molecule (estrogens) that was no longer present due to the mutation

Presentation of the approach and findings are clear, allowing the reader to make their own inferences and compare them with the authors'

Includes multiple follow-up experiments, which leads to tests of internal replication and an impactful mechanistic proposal

Findings are provocative not just for teleost researchers, but for other species since, as the authors point out, the data suggest mechanisms of estrogenic control of social behaviors may be evolutionary ancient

Weaknesses:

The experimental design for studying aggression in males has flaws, but it appears a typical resident-intruder type assay is not appropriate for medaka. seems other species may be better for studying aggression in teleosts.

Reviewer #1 (Public review):

Summary:

This paper provides a novel method to improve the accuracy of predictions of the impact of ITN strategies, by using sub-national estimates of the duration of ITN access and use over time from cross-sectional survey data and annual country ITNs received.

Strengths:

The approach is novel, makes use of available data, and has considered all of the relevant components of ITN distributions.

Weaknesses:

(1) The main message of the paper was not very clear, and did not seem to fit the title. The title focuses on sub-national tailoring of ITN, but the abstract did not feature results directly about SNT. It was not very clear what the main result of the paper was - there are several ITN observations in the results and discussion. Most did not seem to be directly about SNT, but rather sub-national differences in use and access were accounted for in the analyses. It was not clear if the same conclusions would be reached without accounting for sub-national differences, but the estimates and predictions could be expected to be more accurate.

(2) Some of the results seemed to me to be apparent even without a modelling exercise (eg high coverage could not be maintained between campaigns, use would be higher with 2-yearly distributions rather than 3-yearly) or were not in themselves new insights (eg estimates of the duration of use). It would be helpful to clearly state what the novel results are in the abstract, the first paragraph of the discussion and the conclusions, and to make sure that the title is consistent.

(3) On L236, the link to SNT is stated: "the models indicate trends that can support sub-national tailoring of ITNs". They could indeed, but SNT itself is not done in this paper. It seems to be about improving sub-national predictions of the impact of single ITN strategies, by taking into account sub-national variation in access and use duration. This is useful, and the model developed has novel aspects.

(4) Individual countries may have records on when nets were distributed to the regions rather than needing to use the annual country number of nets together with the DHS data. It could be helpful to say what the analysis steps would be in that case.

(5) There were several assumptions that needed to be made in building the model. There is some validation of the timing of the distributions (L633 "verified where possible through discussion with interested parties nationally and internationally") and the fit of estimated access and use to survey data, and agreement between predictions of prevalence and MAP estimates. It would be helpful to say which assumptions are important for the results (and would be key knowledge gaps) and which would not make a difference. It might be possible to validate the net timing model using a country where net distributions are known reasonably well.

(6) What was assumed about what happens to old nets after a mass campaign was not clear. This assumption is likely to affect the predictions of access for the biennial distributions.

(7) L312 and elsewhere: That use given access declines with net age is plausible. However, I wondered if this could be partly a consequence of the assumptions in the model (eg the two exponential decays for access and use, the possible assumption that new nets displace the current ones when there is a mass campaign).

(8) The Methods section on Estimating historical use and access seemed to be aimed at readers familiar with formulae, but I think it could lose other interested readers. It could be useful to explain a little more about what is happening at each step and also why.

(9) The model was fitted to MAP estimates of PfPR2-10, which themselves come from a model. It may be that there is different uncertainty in the MAP estimates for different regions. I couldn't see this on the graph, but maybe the uncertainty is small. Was this taken into account in the fitting?

(10) Was uncertainty from each estimated component integrated into the other components?

(11) Eyeballing Figure 2 (Burkina Faso), there is a general pattern of decline in all the regions, some differences between the regions and some differences in how well the model fits between the regions. If possible, it could be helpful to say how much better the fit was when using region-specific compared to countrywide parameter values for access and use, and how different the results would be.

(12) The question of moving from a campaign every three to every two years may not be the most pertinent question in the current funding landscape. I realise that a paper is in development for a long time, but it would be helpful to comment on what else the model could be used for when fewer rather than more nets are likely to be available.

Reviewer #1 (Public review):

Summary:

Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from simple steatosis, steatohepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma. In the current study, the authors aimed to determine the early molecular signatures differentiating patients with MASLD associated fibrosis from those patients with early MASLD but no symptoms. The authors recruited 109 obese individuals before bariatric surgery. They separated the cohorts as no MASLD (without histological abnormalities) and MASLD. The liver samples were then subjected to transcriptomic and metabolomic analysis. The serum samples were subjected to metabolomic analysis. The authors identified dysregulated lipid metabolism, including glyceride lipids, in the liver samples of MASLD patients compared to the no MASLD ones. Circulating metabolomic changes in lipid profiles slightly correlated with MASLD, possibly due to the no MASLD samples derived from obese patients. Several genes involved in lipid droplet formation were also found elevated in MASLD patients. Besides, elevated levels of amino acids, which are possibly related to collagen synthesis, were observed in MASLD patients. Several antioxidant metabolites were increased in MASLD patients. Furthermore, dysregulated genes involved in mitochondrial function and autophagy were identified in MASLD patients, likely linking oxidative stress to MASLD progression. The authors then determined the representative gene signatures in the development of fibrosis by comparing this cohort with the other two published cohorts. Top enriched pathways in fibrotic patients included GTPas signaling and innate immune responses, suggesting the involvement of GTPas in MASLD progression to fibrosis. The authors then challenged human patient derived 3D spheroid system with a dual PPARa/d agonist and found that this treatment restored the expression levels of GTPase-related genes in MASLD 3D spheroids. In conclusion, the authors suggested the involvement of upregulated GTPase-related genes during fibrosis initiation.

Concerns from first round of review:

(1) A recent study, via proteomic and transcriptomic analysis, revealed that four proteins (ADAMTSL2, AKR1B10, CFHR4 and TREM2) could be used to identify MASLD patients at risk of steatohepatitis (PMID: 37037945). It is not clear why the authors did not include this study in their comparison.

(2) The authors recruited 109 patients but only performed transcriptomic and metabolomic analysis in 94 liver samples. Why did the authors exclude other samples?

(3) The authors mentioned clinical data in Table 1 but did not present the table in this manuscript.

(4) The generated metabolomic data could be a very useful resource to the MASLD community. However, it is very confusing how the data was generated in those supplemental tables. There is no clear labeling of human clinical information in those tables. Also, what do those values mean in columns 47-154? This reviewer assumed that they are the raw data of metabolomic analysis in plasma samples. However, without clear clinical information in these patients, it is impossible that any scientist can use the data to reproduce the authors' findings.

(5) In Fig. 5B, the authors excluded the steatosis and fibrosis overlapped genes. Steatosis and fibrosis specific genes could simply reflect the outcomes rather than causes. In this case, the obtained results might not identify the gene signatures related to fibrosis initiation.

(6 In Fig. 6D, the authors used 3D liver spheroid to validate their findings. However, there is no images showing the 3D liver spheroid formation before and after PPARa/d agonist treatment. It is not clear whether the 3D liver spheroid was successfully established.

(7) The authors suggested that targeting LX-2 cells with Rac1 and Cdc42 inhibitors could reduce collagen production. Did the authors observe these two genes upregulated in mRNA and protein expression levels in their cohort when compared MASLD patients with and without fibrosis?

(8) Did the authors observe that the expression levels of Rac1 and Cdc42 are correlated with fibrosis progression in MASLD patients?

(9) Other studies have revealed several metabolite changes related to MASLD progression (PMID: 35434590, PMID: 22364559). However, the authors did not discuss the discrepancies between their findings with the previous studies.

Significance:

Overall, the current study might provide some new resources regarding transcriptomic and metabolomic data derived from obese patients with and without MASLD. The MASLD research community will be interested in the resource data.

Comments on revised version:

Thank you for the authors' responses to my concerns. I do not have any further comments.

for - Medium article - cogress - Part 1 - progress trap - James Gien Wong - definition - cogress - to - Medium article cogress - Part 2 - progress trap - James Gien Wong - https://hyp.is/t8FhpDGAEfC4J7f0NEFujg/medium.com/@gien_SRG/human-cogress-part-2-d6fd075a55c7 - to - Stop Reset Go hypothesis annotations - progress trap - Ronald Wright - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=ronald+wright - General - https://jonudell.info/h/facet/?max=100&expanded=true&user=stopresetgo&exactTagSearch=true&any=progress+trap - from - youtube - Planet Critical interview - Samuel Miller MacDonald - The Myth of Progress - https://hyp.is/r-hmFtjKEfCd8odATbINbA/www.youtube.com/watch?v=BEhmWEDkZUQ

Reviewer #1 (Public Review):

Summary:

Argunşah et al. describe and investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains in the whisker-related primary somatosensory cortex (S1). Upon repeated stimulation, the authors report that the response ratio between multi- and single-whisker stimulation increases in layer (L) 4 neurons of the septal domain, while remaining constant in barrel L4 neurons. The authors attribute this divergence to differences in short-term synaptic plasticity, particularly within somatostatin-expressing (SST⁺) interneurons. This interpretation is supported by 1) the increased density of SST+ neurons in L4 of the septa compared to barrel domain, 2) the stronger response of (L2/3) SST+ neurons to repeated multi- vs single-whisker stimulation and 3) the reduced functional difference in single- versus multi-whisker response ratios across barrel and septal domains in Elfn1 KO mice, which lack a synaptic protein that confers characteristic short-term plasticity, notably in SST+ neurons. Consistently, a decoder trained on WT data fails to generalize to Elfn1 KO responses. Finally, the authors report a relative enrichment of S2- and M1-projecting cell densities in L4 of the septal domain compared to the barrel domain, suggesting that septal and barrel circuits may differentially route information about single vs multi-whisker stimulation downstream of S1.

Strengths:

This paper describes and aims to study a circuit underlying differential response between barrel columns and septal domains of the primary somatosensory cortex. This work supports the view these two domains contribute distinctly to the processing single versus multi-whisker inputs and highlight the role of SST+ neuron and their short-term plasticity. Together, this study suggests that the barrel cortex multiplexes whisker-derived sensory information across its domains, enabling parallel processing within S1.

Weaknesses:

Although the divergence in responses to repeated single- versus multi-whisker stimulation between barrel and septal domains is consistent with a role for SST⁺ neuron short-term plasticity, the evidence presented does not conclusively demonstrate that this mechanism is the critical driver of the difference. The lack of targeted recordings and manipulations limits the strength of this conclusion: SST⁺ neuron activity is not measured in L4, nor is it assessed in a domain-specific manner. The Elfn1 knockout manipulation does not appear to selectively affect either stimulus condition, domain or interneuron subtype. Finally, all experiments were performed under anesthesia, which raises concerns about how well the reported dynamics generalize to awake cortical processing.

Reviewer #2 (Public review):

Summary:

In this manuscript, Pereira de Castro and coworkers are studying potential competition between a more standard splicing factor SF1 and an alternative splicing factor called QK1. This is interesting because they bind to overlapping sequence motifs and could potentially have opposing effects on promoting the splicing reaction. To test this idea, the authors KD either SF1 or QK1 in mammalian cells and uncover several exons whose splicing regulation follows the predicted pattern of being promoted for splicing by SF1 and repressed by QK1. Importantly, these have introns enriched in SF1 and QK1 motifs. The authors then focus on one exon in particular with two tandem motifs to study the mechanism of this in greater detail and their results confirm the competition model. Mass spec analysis largely agrees with their proposal; however, it is complicated by apparently quick transition of SF1 bound complexes to later splicing intermediates. An inspired experiment in yeast shows how QK1 competition could potentially have a determinental impact on splicing in an orthogonal system. Overall these results show how splicing regulation can be achieved by competition between a "core" and alternative splicing factor and provide additional insight into the complex process of branch site recognition. The manuscript is exceptionally clear and the figures and data very logically presented. The work will be valuable to those in the splicing field who are interested in both mechanism and bioinformatics approaches to deconvolve any apparent "splicing code" being used by cells to regulate gene expression.

Strengths:

(1) The main discovery of the manuscript involving evidence for SF1/QK1 competition is quite interesting and important for this field. This evidence has been missing and may change how people think about branch site recognition.

(2) The experiments and the rationale behind them are clearly and logically presented.

(3) The experiments are carried out to a high standard and well-designed controls are included.

(4) The extrapolation of the result to yeast in order to show the potentially devastating consequences of QK1 competition was creative and informative.

Weaknesses:

Overall the weaknesses are relatively minor and involve cases where conclusions could potentially have been strengthened with additional experimentation. For example, pull-down of the U2 snRNP could be strengthened by detection of the snRNA whereas the proteins may themselves interact with these factors in the absence of the snRNA. In addition the discussion is a bit speculative given the data, but compelling nonetheless.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate the nanoscopic distribution of glycine receptor subunits in the hippocampus, dorsal striatum, and ventral striatum of the mouse brain using single-molecule localization microscopy (SMLM). They demonstrate that only a small number of glycine receptors are localized at hippocampal inhibitory synapses. Using dual-color SMLM, they further show that clusters of glycine receptors are predominantly localized within gephyrin-positive synapses. A comparison between the dorsal and ventral striatum reveals that the ventral striatum contains approximately eight times more glycine receptors and this finding is consistent with electrophysiological data on postsynaptic inhibitory currents. Finally, using cultured hippocampal neurons, they examine the differential synaptic localization of glycine receptor subunits (α1, α2, and β). This study is significant as it provides insights into the nanoscopic localization patterns of glycine receptors in brain regions where this protein is expressed at low levels. Additionally, the study demonstrates the different localization patterns of GlyR in distinct striatal regions and its physiological relevance using SMLM and electrophysiological experiments. However, several concerns should be addressed.

Specific comments on the original version:

(1) Colocalization analysis in Figure 1A. The colocalization between Sylite and mEos-GlyRβ appears to be quite low. It is essential to assess whether the observed colocalization is not due to random overlap. The authors should consider quantifying colocalization using statistical methods, such as a pixel shift analysis, to determine whether colocalization frequencies remain similar after artificially displacing one of the channels.

(2) Inconsistency between Figure 3A and 3B. While Figure 3B indicates an ~8-fold difference in the number of mEos4b-GlyRβ detections per synapse between the dorsal and ventral striatum, Figure 3A does not appear to show a pronounced difference in the localization of mEos4b-GlyRβ on Sylite puncta between these two regions. If the images presented in Figure 3A are not representative, the authors should consider replacing them with more representative examples or providing an expanded images with multiple representative examples. Alternatively, if this inconsistency can be explained by differences in spot density within clusters, the authors should explain that.

(3) Quantification in Figure 5. It is recommended that the authors provide quantitative data on cluster formation and colocalization with Sylite puncta in Figure 5 to support their qualitative observations.

(4) Potential for pseudo replication. It's not clear whether they're performing stats tests across biological replica, images, or even synapses. They often quote mean +/- SEM with n = 1000s, and so does that mean they're doing tests on those 1000s? Need to clarify.

(5) Does mEoS effect expression levels or function of the protein? Can't see any experiments done to confirm this. Could suggest WB on homogenate, or mass spec?

(6) Quantification of protein numbers is challenging with SMLM. Issues include i) some of FP not correctly folded/mature, and ii) dependence of localisation rate on instrument, excitation/illumination intensities, and also the thresholds used in analysis. Can the authors compare with another protein that has known expression levels- e.g. PSD95? This is quite an ask, but if they could show copy number of something known to compare with, it would be useful.

(7) Rationale for doing nanobody dSTORM not clear at all. They don't explain the reason for doing the dSTORM experiments. Why not just rely on PALM for coincidence measurements, rather than tagging mEoS with a nanobody, and then doing dSTORM with that? Can they explain? Is it to get extra localisations- i.e. multiple per nanobody? If so, localising same FP multiple times wouldn't improve resolution. Also, no controls for nanobody dSTORM experiments- what about non-spec nb, or use on WT sections?

(8) What resolutions/precisions were obtained in SMLM experiments? Should perform Fourier Ring Correlation (FRC) on SR images to state resolutions obtained (particularly useful for when they're presenting distance histograms, as this will be dependent on resolution). Likewise for precision, what was mean precision? Can they show histograms of localisation precision.

(9) Why were DBSCAN parameters selected? How can they rule out multiple localisations per fluor? If low copy numbers (<10), then why bother with DBSCAN? Could just measure distance to each one.

(10) For microscopy experiment methods, state power densities, not % or "nominal power".

(11) In general, not much data presented. Any SI file with extra images etc.?

(12) Clarification of the discussion on GlyR expression and synaptic localization: The discussion on GlyR expression, complex formation, and synaptic localization is sometimes unclear, and needs terminological distinctions between "expression level", "complex formation" and "synaptic localization". For example, the authors state: "What then is the reason for the low protein expression of GlyRβ? One possibility is that the assembly of mature heteropentameric GlyR complexes depends critically on the expression of endogenous GlyR α subunits." Does this mean that GlyRβ proteins that fail to form complexes with GlyRα subunits are unstable and subject to rapid degradation? If so, the authors should clarify this point. The statement "This raises the interesting possibility that synaptic GlyRs may depend specifically on the concomitant expression of both α1 and β transcripts." suggests a dependency on α1 and β transcripts. However, is the authors' focus on synaptic localization or overall protein expression levels? If this means synaptic localization, it would be beneficial to state this explicitly to avoid confusion. To improve clarity, the authors should carefully distinguish between these different aspects of GlyR biology throughout the discussion. Additionally, a schematic diagram illustrating these processes would be highly beneficial for readers.

(13) Interpretation of GlyR localization in the context of nanodomains. The distribution of GlyR molecules on inhibitory synapses appears to be non-homogeneous, instead forming nanoclusters or nanodomains, similar to many other synaptic proteins. It is important to interpret GlyR localization in the context of nanodomain organization.

Significance:

The paper presents biological and technical advances. The biological insights revolve mostly on the documentation of Glycine receptors in particular synapses in forebrain, where they are typically expressed at very low levels. The authors provide compelling data indicating that the expression is of physiological significance. The authors have done a nice job of combining genetically tagged mice with advanced microscopy methods to tackle the question of distributions of synaptic proteins. Overall, these advances are more incremental than groundbreaking.

Comments on revised version:

The authors have addressed the majority of the significant issues raised in the review and revised the manuscript appropriately. One issue that can be further addressed relates to the issue of pseudo-replication. The authors state in their response that "All experiments were repeated at least twice to ensure reproducibility (N independent experiments). Statistical tests were performed on pooled data across the biological replicates; n denotes the number of data points used for testing (e.g., number of synaptic clusters, detections, cells, as specified in each case).". This suggests that they're not doing their stats on biological replicates, and instead are pseudo replicating. It's not clear how they have ensured reproducibility, when the stats seem to have been done on pooled data across repeats.

Reviewer #1 (Public review):

Summary:

This very thorough anatomical study addresses the innervation of the Drosophila male reproductive tract. Two distinct glutamatergic neuron types were classified: serotonergic (SGNs) and octopaminergic (OGNs). By expansion microscopy, it was established that glutamate and serotonin /octopamine are co-released. The expression of different receptors for 5-HT and OA in muscles and epithelial cells of the innervation target organs was characterized. The pattern of neurotransmitter receptor expression in the target organs suggests that seminal fluid and sperm transport and emission are subjected to complex regulation. While silencing of abdominal SGNs leads to male infertility and prevents sperm from entering the ejaculatory duct, silencing of OGNs does not render males infertile.

Strengths:

The studied neurons were analysed with different transgenes and methods, as well as antibodies against neurotransmitter synthesis enzymes, building a consistent picture of their neurotransmitter identity. The careful anatomical description of innervation patterns together with receptor expression patterns if the target organs provides a solid basis for advancing the understanding how seminal fluid and sperm transport and emission are subjected to complex regulation. The functional data showing that SGNs are required for male fertility and for the release of sperm from the seminal vesicle into the ejaculatory duct is convincing.

Weaknesses:

The functional analysis of the characterized neurons is not as comprehensive as the anatomical description and phenotypic characterization was limited to simple fertility assays. It is understandable that a full functional dissection is beyond the scope of the present work. The paper contains experiments showing neuron-independent peristaltic waves in the reproductive tract muscles, which are thematically not very well integrated into the paper. Although very interesting, one wonders if these experiments would not fit better into a future work that also explores these peristaltic waves and their interrelation with neuromodulation mechanistically.

Comments on revisions:

The manuscript has improved after fixing many small issues/errors. The new sections in the discussion are likewise adding to the quality of the manuscript.

Reviewer #1 (Public review):

Summary:

In this paper, the authors developed a chemical labeling reagent for P2X7 receptors, called X7-uP. This labeling reagent selectively labels endogenous P2X7 receptors with biotin based on ligand-directed NASA chemistry. After labeling the endogenous P2X7 receptor with biotin, the receptor can be fluorescently labeled with streptavidin-AlexaFluor647. The authors carefully examined the binding properties and labeling selectivity of X7-uP to P2X7, characterized the labeling site of P2X7 receptors, and demonstrated fluorescence imaging of P2X7 receptors. The data obtained by SDS-PAGE, Western blot, and fluorescence microscopy clearly shows that X7-uP labels the P2X7 receptor. Finally, the authors fluorescently labeled the endogenous P2X7 in BV2 cells, which are a murine microglia model, and used dSTORM to reveal a nanoscale P2X7 redistribution mechanism under inflammatory conditions at high resolution.

Strengths:

X7-uP selectively labels endogenous P2X7 receptors with biotin. Streptavidin-AlexaFluor647 binds to the biotin labeled to the P2X7 receptor, allowing visualization of endogenous P2X7 receptors.

Reviewer #1 (Public review):

Summary:

The co-localization of large conductance calcium- and voltage activated potassium (BK) channels with voltage-gated calcium channels (CaV) at the plasma membrane is important for the functional role of these channels in controlling cell excitability and physiology in a variety of systems. An important question in the field is where and how do BK and CaV channels assemble as 'ensembles' to allow this coordinated regulation - is this through preassembly early in the biosynthetic pathway, during trafficking to the cell surface or once channels are integrated into the plasma membrane. These questions also have broader implications for assembly of other ion channel complexes. Using an imaging based approach, this paper addresses the spatial distribution of BK-CaV ensembles using both overexpression strategies in tsa201 and INS-1 cells and analysis of endogenous channels in INS-1 cells using proximity ligation and superesolution approaches. In addition, the authors analyse the spatial distribution of mRNAs encoding BK and Cav1.3. The key conclusion of the paper that BK and CaV1.3 are co-localised as ensembles intracellularly in the ER and Golgi is well supported by the evidence. The experiments and analysis are carefully performed and the findings are very well presented.

Reviewer #1 (Public review):

Summary:

Adult (4mo) rats were tasked to either press one lever for an immediate reward or another for a delayed reward. The task had an adjusting amount structure in which (1) the number of pellets provided on the immediate reward lever changed as a function of the decisions made, (2) rats were prevented from pressing the same lever three times in a row.

While the authors have been very responsive to the reviews, and I appreciate that, unfortunately, the new analyses reported in this revision actually lead me to deeper concerns about the adequacy of the data to support the conclusions. In this revision, it has become clear that the conclusions are forced and not supported by the data. Alternative theories are not considered or presented. This revision has revealed deep problems with the task, the analyses, and the modeling.

Data Weaknesses

Most importantly, the inclusion of the task behavior data has revealed a deep problem with the entire structure of the data. As is obvious in Figure 1D, there is a slow learning effect that is changing over the sessions as the animals learn to stop taking the delayed outcome. Unfortunately, the 8s delays came *after* the 4s. The first 20 sessions contain 19 4s delays and 1 8s delay, while the last 20 sessions contain 14 8s delays and 6 4s delays. Given the changes across sessions, it is likely that a large part of the difference is due to across-session learning (which is never addressed or considered).

These data are not shown by subject and I suspect that individual subjects did all 4s then all 8s and some subjects switched tasks at different times. If my suspicion is true, then any comparisons between the 4s and 8s conditions (which are a major part of the author's claims) may have nothing to do with the delays, but rather with increased experience on the task.

Furthermore, the four "groups", which are still poorly defined, seem to have been assessed at a session-by-session level. So when did each animal fall into a given group? Why is Figure 1D not showing which session fell into which group and why are we not seeing each animal's progression? They also admit that animals used a mixture of strategies, which implies that the "group" assignment is an invalid analysis, as the groups do not accommodate strategy mixing.

Figure 2 shows that none of the differences of the group behavior against random choice with a basic p(delay) are significant. The use a KS test to measure these differences. KS tests are notoriously sensitive as KS tests simply measure whether there are any statistical differences between two distributions. They do not report the full statistics for Figure 2, but only say that the 4HI group was not significant (KS p-value = 0.72) and the 8LO showed a p-value of 0.1 (which they interpret as significant). p=0.1 is not significant. They don't report the value of the 4LO or 8HI groups (why not?), but say they are in-between these two extremes. That means *none* of the differences are significant.

They then test a model with additional parameters, and say that the model includes more than the minimal p_D parameter, but never report BIC or AIC model comparisons. In order to claim that the model is better than the bare p_D assumption, they should be reporting model-comparison statistics. But given that the p_D parameters are enough (q.v. Figure 2), this entire model seems unnecessary

It took me a while to determine what was being shown in Figure 3, but I was eventually able to determine that 0 was the time after the animal made the choice to wait out the delay side, so the 4s in Figure 3A1 with high power in the low-frequency (<5 Hz) range is the waiting time. They don't show the full 8s time. Nor do they show the spectrograms separated by group (assuming that group is the analytical tool they are using). In B they show only show theta power, but it is unclear how to interpret these changes over time.

In Figure 4, panel A is mostly useless because it is just five sample sessions showing firing rate plotted on the same panels as the immediate reward amount. If they want to claim correlation, they should show and test it. But moreover, this is not how neural data should be presented - we need to know what the cells are doing, population-wise. We need to have an understanding of the neural ensemble. These data are clearly being picked and chosen, which is not OK.

Figure 4, panels B and C show that the activity trivially reflects the reward that has been delivered to the animal, if I am understanding the graphs correctly. (The authors do not interpret it this way, but the data is, to my eyes, clear.) The "immediate" signal shows up immediately at choice and reflects the size of the immediate reward (which is varying). The "delay" signal shows up after the delay and does not, which makes sense as the animals get 6 pellets on the delayed side no matter what. In fact, the max delayed side activity = the max immediate side activity, which is 6 pellets. This is just reward-related firing.

Figure 5 is poorly laid out, switching the order in 5C to be 2 1 3 in E and F. (Why?!) The statistics for Figure 5 on page 17 should be asking whether there are differences between neuron types, not whether there is a choice x time interaction in a given neuron type. When I look at Figure 5F1-3, all three types look effectively similar with different levels of noise. It is unclear why they are doing this complicated PC analysis or what we should be drawing from it.

Figure 6 mis-states pie charts as "total number" rather than proportions.

Interpretation Weaknesses

The separation of cognitive effort into "resource-based" and "resistance-based" seems artificial to me. I still do not understand why the ability to resist a choice does not also depend on resource or why using resources are not a form of resistance. Doesn't every action in the end depend on the resources one has available? And doesn't every use of a resource resist one option by taking another? Even if one buys these two separate cognitive control processes (which at this point in reading the revision, I do not), the paper starts from the assumption that a baseline probability of waiting out the delays is a "resistance-based cognitive control" (why?) and a probability of choice that takes into account the size of the immediate value (confusingly abbreviated as ival) is a "resource-based cognitive control" (again, why?)

Reviewer #1 (Public review):

The paper from Hudait and Voth details a number of coarse-grained simulations as well as some experiments focused on the stability of HIV capsids in the presence of the drug lenacapavir. The authors find that LEN hyperstabilizes the capsid, making it fragile and prone to breaking inside the nuclear pore complex.

I found the paper interesting. I have a few suggestions for clarification and/or improvement.

(1) How directly comparable are the NPC-capsid and capsid-only simulations? A major result rests on the conclusion that the kinetics of rupture are faster inside the NPC, but are the numbers of LENs bound identical? Is the time really comparable, given that the simulations have different starting points? I'm not really doubting the result, but I think it could be made more rigorous/quantitative.

(2) Related to the above, it is stated on page 12 that, based on the estimated free-energy barrier, pentamer dissociation should occur in ~10 us of CG time. But certainly, the simulations cover at least this length of time?

(3) At first, I was surprised that even in a CG simulation, LEN would spontaneously bind to the correct site. But if I read the SI correctly, LEN was parameterized specifically to bind to hexamers and not pentamers. This is fine, but I think it's worth describing in the main text.

Reviewer #1 (Public review):

Summary:

Spinal projection neurons in the anterolateral tract transmit diverse somatosensory signals to the brain, including touch, temperature, itch, and pain. This group of spinal projection neurons is heterogeneous in their molecular identities, projection targets in the brain, and response properties. While most anterolateral tract projection neurons are multimodal (responding to more than one somatosensory modality), it has been shown that cold-selective projection neurons exist in lamina I of the spinal cord dorsal horn. Using a combination of anatomical and physiological approaches, the authors discovered that the cold-selective lamina I projection neurons are heavily innervated by Trpm8+ sensory neuron axons, with calb1+ spinal projection neurons primarily capturing these cold-selective lamina I projection neurons. These neurons project to specific brain targets, including the PBNrel and cPAG. This study adds to the ongoing effort in the field to identify and characterize spinal projection neuron subtypes, their physiology, and functions.

Strengths:

(1) The combination of anatomical and physiological analyses is powerful and offers a comprehensive understanding of the cold-selective lamina I projection neurons in the spinal cord dorsal horn. For example, the authors used detailed anatomical methods, including EM imaging of Trpm8+ axon terminals contacting the Phox2a+ lamina I projection neurons. Additionally, they recorded stimulus-evoked activity in Trpm8-recipient neurons, carefully selected by visual confirmation of tdTomato and GFP juxtaposition, which is technically challenging.

(2) This study identifies, for the first time, a molecular marker (calb1) that labels cold-selective lamina I projection neurons. Although calb1+ projection neurons are not entirely specific to cold-selective neurons, using an intersectional strategy combined with other genes enriched in this ALS group or cold-induced FosTRAP may further enhance specificity in the future.

(3) This study shows that cold-selective lamina I projection neurons specifically innervate certain brain targets of the anterolateral tract, including the NTS, PBNrel, and cPAG. This connectivity provides insights into the role of these neurons in cold sensation, which will be an exciting area for future research.

Weaknesses:

(1) The sample size for the ex vivo electrophysiology is small. Given the difficulty and complexity of the preparation, this is understandable. However, a larger sample size would have strengthened the authors' conclusions.

(2) The authors used tdTomato expression to identify brain targets innervated by these cold-selective lamina I projection neurons. Since tdTomato is a soluble fluorescent protein that fills the entire cell, using synaptophysin reporters (e.g., synaptophysin-GFP) would have been more convincing in revealing the synaptic targets of these projection neurons.

(3) The summary cartoon shown in Figure 7 can be misleading because this study did not determine whether these cold-selective lamina I projection neurons have collateral branches to multiple brain targets or if there are anatomical subtypes that may project exclusively to specific targets. For example, a recent study (Ding et al., Neuron, 2025) demonstrated that there are PBN-projecting spinal neurons that do not project to other rostral brain areas. Furthermore, based on the authors' bulk labeling experiments, the three main brain targets are NTS, PBNrel, and cPAG. The VPL projection is very sparse and almost negligible.

Reviewer #1 (Public review):

This manuscript provides several important findings that advance our current knowledge about the function of the gustatory cortex (GC). The authors used high-density electrophysiology to record neural activity during a sucrose/NaCl mixture discrimination task. They observed population-based activity capable of representing different mixtures in a linear fashion during the initial stimulus sampling period, as well as representing the behavioral decision (i.e., lick left or right) at a later time point. Analyzing this data at the single neuron level, they observed functional subpopulations capable of encoding the specific mixture (e.g., 45/55), tastant (e.g., sucrose), and behavioral choice (e.g., lick left). To test the functional consequences of these subpopulations, they built a recurrent neural network model in order to "silence" specific functional subpopulations of GC neurons. The virtual ablation of these functional subpopulations altered virtual behavioral performance in a manner predicted by the subpopulation's presumed contribution.

Strengths:

Building a recurrent neural network model of the gustatory cortex allows the impact of the temporal sequence of functionally identifiable populations of neurons to be tested in a manner not otherwise possible. Specifically, the author's model links neural activity at the single neuron and population level with perceptual ability. The electrophysiology methods and analyses used to shape the network model are appropriate. Overall, the conclusions of the manuscript are well supported.

Weaknesses:

One potential concern is the apparent mismatch between the neural and behavioral data. Neural analyses indicate a clear separation of the activity associated with each mixture that is independent of the animal's ultimate choice. This would seemingly indicate that the animals are making errors despite correctly encoding the stimulus. Based solely on the neural data, one would expect the psychometric curve to be more "step-like" with a significantly steeper slope. One potential explanation for this observation is the concentration of the stimuli utilized in the mixture discrimination task. The authors utilize equivalent concentrations, rather than intensity-matched concentrations. In this case, a single stimulus can (theoretically) dominate the perception of a mixture, resulting in a biased behavioral response despite accurate concentration coding at the single neuron level. Given the difficulty of isointensity matching concentrations, this concern is not paramount. However, the apparent mismatch between the neural and behavioral data should be acknowledged/addressed in the text.

Reviewer #1 (Public review):

Summary:

In this manuscript, Green et al. attempt to use large-scale protein structure analysis to find signals of selection and clustering related to antibiotic resistance. This was applied to the whole proteome of Mycobacterium tuberculosis, with a specific focus on the smaller set of known antibiotic-resistance-related proteins.

Strengths:

The use of geospatial analysis to detect signals of selection and clustering on the structural level is really intriguing. This could have a wider use beyond the AMR-focussed work here and could be applied to a more general evolutionary analysis context. Much of the strength of this work lies in breaking ground into this structural evolution space, something rarely seen in such pathogen data. Additional further research can be done to build on this foundation, and the work presented here will be important for the field.

The size of the dataset and use of protein structure prediction via AlphaFold, giving such a consistent signal within the dataset, is also of great interest and shows the power of these approaches to allow us to integrate protein structure more confidently into evolution and selection analyses.

Weaknesses:

There are several issues with the evolutionary analysis and assumptions made in the paper, which perhaps overstate the findings, or require refining to take into account other factors that may be at play.

(1) The focus on antimicrobial resistance (AMR) throughout the paper contains the findings within that lens. This results in a few different weaknesses:

(a) While the large size of the analysis is highlighted in the abstract and elsewhere, in reality, only a few proteins are studied in depth. These are proteins already associated with AMR by many other studies, somewhat retreading old ground and reducing the novelty.

(b) Beyond the AMR-associated proteins, the proteome work is of great interest, but only casually interrogated and only in the context of AMR. There appears to be an assumption that all signals of positive selection detected are related to AMR, whereas something like cas10 is part of the CRISPR machinery, a set of proteins often under positive selection, and thus unlikely to be AMR-related.

(2) The strength of the signal from the structural information and the novelty of the structural incorporation into prediction are perhaps overstated.

(a) A drop of 13% in F1 for a gain of 2% in PPV is quite the trade-off. This is not as indicative of a strong predictor that could be used as the abstract claims. While the approach is novel and this is a good finding for a first attempt at such complex analysis, this is perhaps not as significant as the authors claim

(b) In relation to this, there is a lack of situating these findings within the wider research landscape. For instance, the use of structure for predicting resistance has been done, for example, in PncA (https://academic.oup.com/jacamr/article/6/2/dlae037/7630603, https://www.sciencedirect.com/science/article/pii/S1476927125003664, https://www.nature.com/articles/s41598-020-58635-x) and in RpoB (https://www.nature.com/articles/s41598-020-74648-y). These, and other such works, should be acknowledged as the novelty of this work is perhaps not as stark as the authors present it to be.

(3) The authors postulate that neutral AA substitutions would be randomly distributed in the protein structure and thus use random mutations as a negative control to simulate this neutral evolution. However, I am unsure if this is a true negative control for neutral evolution. The vast majority of residues would be under purifying selection, not neutral selection, especially in core proteins like rpoB and gyrA. Therefore, most of these residues would never be mutated in a real-world dataset. Therefore, you are not testing positive selection against neutral selection; you are testing positive against purifying, which will have a much stronger signal. This is likely to, in turn, overestimate the signal of positive selection. This would be better accounted for using a model of neutral evolution, although this is complex and perhaps outside the scope. Still, it needs to be made clear that these negative controls are not representative of neutral evolution.

(4) In a similar vein, the use of 15 Å as a cut-off for stating co-localisation feels quite arbitrary. The average radius of a globular protein is about 20 Å, so this could be quite a large patch of a protein. I think it may be good to situate the cut-off for a 'single location' within a size estimator of the entire protein, as 15 Å could be a neighbourhood in a large protein, but be the whole protein for smaller ones.

Reviewer #1 (Public review):

Summary:

This manuscript by Xie and colleagues presents an intriguing behavioral finding for the field of perceptual learning (PL): combining the reactivation-based training paradigm with anodal tDCS induces complete generalization of the learning effect. Notably, this generalization is achieved without compromising the magnitude of learning effects and with an 80% reduction in total training time. The experimental design is well-structured, and the observed complete generalization is robustly replicated across two stimulus dimensions (orientation and motion direction).

However, while the empirical results are methodologically valid and scientifically surprising, the theoretical framework proposed to explain them appears underdeveloped and, in some cases, difficult to reconcile with the existing literature. Several arguments are insufficiently justified. In addition, the introduction of a non-standard metric (NGI: normalized learning gain index) raises concerns about the interpretability and comparability with existing PL literature.

Strengths:

(1) Rigorous experimental design

In this study, Xie and colleagues employed a 2×2 factorial design (Training paradigm: Reactivation vs. Full-Practice × tDCS protocols: Anodal vs. Sham), which allowed clear dissociation of the main and interaction effects.

(2) High statistical credibility

Sample sizes were predetermined using G*Power, non-significant effects were evaluated using the Bayes factor, and the core behavioral findings were replicated in a second stimulus dimension. These strengthen the credibility of the findings.

(3) Strong translational potential

The observed complete generalization could have useful implications for sensory rehabilitation. The large reduction (80%) in total training time is particularly compelling.

Weaknesses:

(1) NGI (Normalized learning gain index) is a non-standard behavioral metric and may distort interpretability.

NGI (pre - post / ((pre + post) / 2)) is rarely used in PL studies to measure learning effects. Almost all PL studies rely on raw thresholds and percent improvements (pre - post / pre), making it difficult to contextualize the current NGI-based results within the broader field. The current manuscript provides no justification for adopting NGI.

A more critical issue is the NGI's nonlinearity: by normalizing to the mean of pre- and post-test thresholds, it disproportionately inflates learning effects for participants with lower post-test thresholds. Notably, the "complete generalization" claims are illustrated mainly with NGI plots. Although the authors also analyze thresholds directly and the results also support the core claim, the interpretation in the text relies heavily on NGI.

The authors may consider rerunning key analyses using the standard percent improvement metric. If retaining NGI, the authors should provide explicit justification for why NGI is superior to standard measures.

(2) The proposed theoretical framework is sometimes unclear and insufficiently supported.

The authors propose the following mechanistic chain:

(a) reactivation-based learning depends on offline consolidation mediated by GABA (page 4 line 73);

(b) online a-tDCS reduces GABA (page 4, line 76), thereby disrupting offline consolidation (page 11, line 225);

(c) disrupted offline consolidation reduces perceptual overfitting (page 4, line 77; page 11, line 225), thereby enabling generalization;

(d) under full-practice training, a-tDCS increases specificity via a different mechanism (page 11 line 235).

While this framework is plausible in broad terms, several components are speculative at best in the absence of neurochemical or neural measurements.

(3) Several reasoning steps require further clarification.

(a) Mechanisms of Reactivation-based Learning.

The manuscript focuses on the neurochemical basis of reactivation-based learning. However, reactivation-induced neurochemical changes differ across brain regions. In the motor cortex, Eisenstein et al. (2023) reported that after reactivation, increased GABA and decreased E/I ratio were associated with offline gains. In contrast, Bang et al. (2018) demonstrated that, in the visual cortex, reactivation decreased GABA and increased E/I ratio. While both studies are consistent with GABA involvement, the direction of GABA modulation differs. The authors should clarify this discrepancy.<br /> More importantly, Bang et al. (2018) demonstrated that reactivation-based (3 blocks) and full-practice (16 blocks) training produced similar time courses of E/I ratio changes in V1: an initial increase followed by a decrease. Given this similarity, the manuscript would benefit from a more thorough discussion of how the two paradigms diverge mechanistically. For example, behaviorally, Song et al. (2021) reported greater generalization with reactivation-based training than with full-practice training, aligning with Kondat et al. (2025). Neurally, Kondat et al. (2024) showed that reactivation-based training increased activity in higher-order brain regions (e.g., IPS), whereas full practice training reduced connectivity between temporal and parietal regions.

(b) tDCS Mechanisms and Protocols.

The effect of a-tDCS on GABA is not consistent across brain regions. While a-tDCS reliably reduces GABA in the motor cortex, recently, a more related work (Abuleli et al., 2025) reports no significant modulation of GABA or Glx in V1, challenging the authors' assumption of tDCS-induced GABA reduction in the visual cortex.

The manuscript proposes that online a-tDCS disrupts offline consolidation is somewhat difficult to interpret conceptually. Online tDCS typically modulates processes occurring during stimulation (e.g., encoding process, attentional state), whereas consolidation occurs afterward. Thus, stating that online tDCS protocols only disrupt offline consolidation without considering the possibility that they first modulate the encoding process is difficult to interpret. Even if tDCS has prolonged effects, the link between online stimulation and disruption of offline consolidation remains unelucidated.

(c) Missing links between GABA modulation and perceptual overfitting.

The proposed chain ("tDCS disrupts consolidation → reduced overfitting → improved generalization") skips a critical step: how GABA modulation translates to changes in neural representational properties (e.g., tuning width, representational overlap between trained/untrained stimuli) that define "perceptual overfitting." The PL literature has not established a link between GABA levels and these representational changes, leaving a key component of the mechanistic explanation underspecified.

(d) Insufficient explanation of the opposite effects.

The manuscript does not fully explain why the same a-tDCS promotes generalization in reactivation-based training but increases specificity in full-practice training. Both paradigms engage offline consolidations, and, as mentioned above, the time courses of E/I ratio changes are similar for 3-block reactivation-based or 16-block training. Thus, if offline consolidation mechanisms (and their associated E/I changes) are comparable across paradigms, it is unclear why identical a-tDCS would produce opposite outcomes in the two paradigms.

Reviewer #1 (Public review):

Summary:

The authors generated mouse and zebrafish models for DeSanto-Shinawi Syndrome, caused by loss-of-function variants in the WAC gene. Using these vertebrate systems, they demonstrate conserved craniofacial and social-behavioral phenotypes that parallel human clinical features, along with deficits in GABAergic markers. They observe increased seizure susceptibility and male-biased brain volumetric changes in Wac mutant mice. Together, these findings begin to define the biological consequences of Wac haploinsufficiency and provide valuable resources for future mechanistic studies.

Strengths:

WAC is a high-confidence neurodevelopmental disorder gene and one of the genes identified by large-scale exome sequencing efforts, including the Satterstrom et al. (2020) autism spectrum disorder cohort. This study establishes the first vertebrate Wac models, addressing a major gap in the understanding of DeSanto-Shinawi Syndrome, and provides a framework for studying other syndromic forms of autism. The models generated will be impactful and useful to the community to study and understand DeSanto-Shinawi Syndrome.

The cross-species analysis is important and well executed, and reveals both conserved and divergent phenotypes. The behavioral and anatomical assays are rigorously executed and well-controlled, and the inclusion of RNA-sequencing analyses adds valuable insights into the mechanisms underlying brain function in Wac mutants. Notably, the RNA-seq data reveal upregulation of several clustered protocadherins, genes central to neuronal identity and cell-cell interactions, which are known to be regulated by dynamic developmental regulation of chromatin architecture. This observation provides an intriguing hint that could link Wac function to higher-order chromatin organization and neuronal connectivity.

Weaknesses:

The evidence is solid, but the study remains incomplete in its mechanistic depth and molecular interpretation. The authors compellingly describe behavioral, anatomical, and transcriptomic phenotypes associated with WAC loss, yet do not explore how WAC mechanistically regulates chromatin or transcription. Given prior evidence that WAC interacts with the RNF20/40 ubiquitin ligase complex and promotes histone H2B ubiquitination and transcriptional elongation, the paper would benefit from a discussion of these functions as a potential link between Wac haploinsufficiency and the observed changes in neuronal gene expression. Similarly, the authors mention WAC's WW and coiled-coil domains but do not consider how these domains could mediate nuclear interactions or recruitment of transcriptional cofactors that shape gene regulation and chromatin organization in neurons.

The transcriptomic analysis is rich but largely descriptive. Although the upregulation of clustered protocadherins is particularly intriguing, these findings are not validated or localized to specific neuronal populations. The study would be strengthened by independently validating the most significant RNA-seq changes, such as protocadherin gamma genes, using in situ hybridization methods to confirm the spatial and cellular specificity of expression changes.

Finally, while the behavioral and MRI results add valuable breadth, their interpretation would be improved by clearer reporting of sample sizes, statistical corrections, and effect sizes to support claims of sex-specific and regional brain volume differences.

Reviewer #1 (Public review):

Summary:

This manuscript investigates whether newborns can use speaker identity to separate verbal memories, aiming to shed light on the earliest mechanisms of language learning and memory formation. The authors employ a well-designed experimental paradigm using functional near-infrared spectroscopy (fNIRS) to measure neural responses in newborns exposed to familiar and novel words, with careful counterbalancing and acoustic controls. Their main finding is that newborns show differential neural activation to novel versus familiar words, particularly when speaker identity changes, suggesting that even at birth, infants can use indexical cues to support memory.

Strengths:

Major strengths of the work include its innovative approach to a longstanding question in developmental science, the use of appropriate and state-of-the-art neuroimaging methods for this age group, and a thoughtful experimental design that attempts to control for order and acoustic confounds. The study addresses a significant gap in our understanding of how infants process and remember speech, and the data are presented transparently, with clear reporting of both significant and non-significant results.

Weaknesses:

However, there are notable weaknesses that limit the strength of the conclusions. The main recognition effect is restricted to a specific subgroup of participants and emerges only during a particular testing window, raising questions about the robustness and generalizability of the findings. The sample size, while typical for infant neuroimaging, is modest, and the statistical power is further reduced by missing data and group-dependent effects. Additionally, the claims regarding episodic memory and evolutionary implications are somewhat overstated, as the paradigm primarily demonstrates memory retention over a few minutes without evidence of the rich, contextually bound recall characteristic of fully developed episodic memory.

Overall, the authors have achieved their primary aim of demonstrating that speaker identity can facilitate memory separation in newborns, providing valuable preliminary evidence for early indexical processing in language learning. The results are intriguing and likely to stimulate further research, but the limitations in effect robustness and theoretical interpretation mean that the findings should be viewed as an important step forward rather than a definitive answer. The methods and data will be of interest to researchers studying infant cognition, memory, and language, and the study highlights both the promise and the challenges of probing complex cognitive processes in the earliest stages of life.

Reviewer #1 (Public review):

Working memory affects sensory processing. Observers make faster and more accurate perceptual decisions at remembered locations, and corresponding regions of retinotopic visual cortex display enhanced response gain and modulations in oscillatory activity and spike-phase coupling.

Roshanaei et al investigate the relationship between working memory, oscillatory activity, and response gain by reanalyzing extracellular laminar probe recordings from area MT of rhesus monkeys performing a spatial working memory task. During the memory period, visual probes were flashed in the receptive field of the recorded neurons, allowing a comparison of visual responses when memory overlapped with this receptive field (IN) or a location in the opposite hemifield (OUT). They first replicate a range of findings, including increased power in lower frequency bands (theta and alpha/beta) and increased visually-evoked responses in the IN condition. The authors next deployed a spectral technique (MODWT) to decompose the local field potential on single trials into 6 non-arbitrary component frequency bands. This approach allows the authors to observe shifts in peak spectral frequencies across IN and OUT trials. Finally, these single-trial spectral decompositions allowed the authors to relate frequency band power and response gain. This analysis revealed that response gain tended to increase with power in lower (alpha, beta, and theta) frequency bands, and this effect minimally interacted with the remembered location.

Together, these interesting results provide correlational evidence that the effect of working memory on response gain may be mediated by oscillatory power. As the authors note, these results are also consistent with theories positing that lower frequency oscillatory activity primarily reflects working-memory related feedback signals from prefrontal and parietal cortex.

These findings also suggest opportunities for further exploration. From a methodological perspective, it's not clear if the particular spectral decomposition highlighted here is necessary for obtaining these results, or if applying more standard approaches to single trials (as in Lundqvist et al., 2016) would have provided similar sensitivity. Additionally, although the relationship among working memory, oscillatory power, and response gain explored here is necessarily correlational, it could be of interest to subject these factors to a mediation analysis in this or future studies. Finally, the careful analysis of oscillatory phenomena reported here can ideally be used to inform large-scale circuit models and constrain the underlying mechanism.

Reviewer #1 (Public review):

Summary:

In this paper, the authors develop a biologically plausible recurrent neural network model to explain how the hippocampus generates and uses barcode-like activity to support episodic memory. They address key questions raised by recent experimental findings: how barcodes are generated, how they interact with memory content (such as place and seed-related activity), and how the hippocampus balances memory specificity with flexible recall. The authors demonstrate that chaotic dynamics in a recurrent neural network can produce barcodes that reduce memory interference, complement place tuning, and enable context-dependent memory retrieval, while aligning their model with observed hippocampal activity during caching and retrieval in chickadees.

Strengths:

(1) The manuscript is well-written and structured.

(2) The paper provides a detailed and biologically plausible mechanism for generating and utilizing barcode activity through chaotic dynamics in a recurrent neural network. This mechanism effectively explains how barcodes reduce memory interference, complement place tuning, and enable flexible, context-dependent recall.

(3) The authors successfully reproduce key experimental findings on hippocampal barcode activity from chickadee studies, including the distinct correlations observed during caching, retrieval, and visits.

(4) Overall, the study addresses a somewhat puzzling question about how memory indices and content signals coexist and interact in the same hippocampal population. By proposing a unified model, it provides significant conceptual clarity.

Weaknesses:

The recurrent neural network model incorporates assumptions and mechanisms, such as the modulation of recurrent input strength, whose biological underpinnings remain unclear. The authors acknowledge some of these limitations thoughtfully, offering plausible mechanisms and discussing their implications in depth. It may be worth exploring the robustness of the results to certain modeling assumptions. For instance, the choice to run the network for a fixed amount of time and then use the activity at the end for plasticity could be relaxed.

Reviewer #1 (Public review):

The authors did a good job addressing my concerns. This is an important paper on the basis of appetitive associative learning.

Reviewer #1 (Public review):

Summary:

The authors presented a simplified E. coli cell-free protein synthesis (eCFPS) system that reduces core reaction components from 35 to 7, improving protein expression levels. They also presented a "fast lysate" protocol that simplifies extract preparation, enhancing accessibility and robustness for diverse applications.

Strengths:

The authors present a valuable new protocol for eCFPS, which simplifies its application.

Weaknesses:

The authors only provided the data for optimization, leaving the underlying mechanism that explains the phenomena unexplained.

Reviewer #1 (Public review):

Summary:

The authors have created a new model of KCNC1-related DEE in which a pathogenic patient variant (A421V) is knocked into mouse in order to better understand the mechanisms through which KCNC1 variants lead to DEE.

Strengths:

(1) The creation of a new DEE model of KCNC1 dysfunction.

(2) InVivo phenotyping demonstrates key features of the model such as early lethality and several types of electrographic seizures.

(3) The ex vivo cellular electrophysiology is very strong and comprehensive including isolated patches to accurately measure K+ currents, paired recording to measure evoked synaptic transmission, and the measurement of membrane excitability at different timepoint and in two cell types.

(4) 2P imaging relates the cellular dysfunction in PV neurons to epilepsy.

Reviewer #1 (Public review):

Sandkuhler et al. re-evaluated the biological functions of TANGO2 homologs in C. elegans, yeast, and zebrafish. Compared to the previously reported role of TANGO2 homologs in transporting heme, Sandkuhler et al. expressed a different opinion on the biological functions of TANGO2 homologs. With the support of some results from their tests, they conclude that 'there is insufficient evidence to support heme transport as the primary function of TANGO2', in addition to the evidence that C. elegans TANGO2 helps counteract oxidative stress.. While the differences are reported in this study, more work is needed to elucidate the intuitive biological function of TANGO2.

Strengths:

(1) This work revisits a set of key experiments, including the toxic heme analog GaPP survival assay, the fluorescent ZnMP accumulation assay, and the multi-organismal investigations documented by Sun et al. in Nature (2022), which are critical for comparing the two works. Meanwhile, the authors also highlight the differences in reagents and methods between the two studies, demonstrating significant academic merit.

(2) This work reported additional phenotypes for the C. elegans mutant of the TANGO2 homologs, including lawn avoidance, reduced pharyngeal pumping, smaller brood size, faster exhaustion under swimming test, and a shorter lifespan. These phenotypes are important for understanding the biological function of TANGO2 homologs, while they were missing from the report by Sun et al.

(3) Investigating the 'reduced GaPP consumption' as a cause of increased resistance against the toxic GaPP for the TANGO2 homologs, hrg-9 hrg-10 double null mutant provides a valuable perspective for studying the biological function of TANGO2 homologs.

(4) The induction of hrg-9 gene expression by paraquat indicates a strong link between TANGO2 and mitochondrial function.

(5) This work thoroughly evaluated the role of TANGO2 homologs in supporting yeast growth using multiple yeast strains and also pointed out the mitochondrial genome instability feature of the yeast strain used by Sun et al.

Weakness:

It is always a challenge to replicate someone else's work, but it is worthwhile to take on the challenge, provide evidence, and raise concerns about it. These authors attempted to replicate the experiment using the same biological material as that used by Sun et al. in Nature (2022), despite some experimental differences between the two studies. This study does not have many technical weaknesses, but it can become a much better project by focusing on the new phenotypes discovered here.

Reviewer #1 (Public review):

Summary:

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeat-containing intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Strengths and Weaknesses:

The manuscript was previously reviewed through Review Commons. As noted there, the experiments are well controlled, the claims are well supported, and the methods are clearly described. The conclusions are convincing. All concerns I raised have been addressed except one (minor point #8):

"The way the authors mapped the ChIP-seq data is potentially problematic when analyzing the same repeat type in different genomic regions. Reads with multiple equally good mapping positions were assigned randomly. This is fine when analyzing repeats by type, independent of genomic position, which is what the authors do to reach their main conclusions. However, several figures (Fig. 3B, Fig. 4B, Fig. 5B, Fig. 7) show the same repeat type at specific genomic locations." Due to the random assignment, all of these regions merely show the average signal for the given repeat. I find it misleading that this average is plotted out at "specific" genomic regions.<br /> Initially, I suggested a workaround, but the authors clarified why the workaround was not feasible, and their explanation is reasonable to me. That said, the figures still show a signal at positions where they can't be sure it actually exists. If this cannot be corrected analytically, it should at least be noted in the figure legends, Results, or Discussion.

Importantly, the authors' conclusions do not hinge on this point; they are appropriately cautious, and their interpretations remain valid regardless.

Significance:

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with mini-chromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Comments on revised version:

All my recommendations have been addressed.

Reviewer #1 (Public review):

Summary:

The authors set out on the ambitious task of establishing the reproducibility of claims from the Drosophila immunity literature. Starting out from a corpus of 400 articles from 1959 and 2011, the authors sought to determine whether their claims were confirmed or contradicted by previous or subsequent publications. Additionally, they actively sought to replicate a subset of the claims for which no previous replications were available (although this set was not representative of the whole sample, as the authors focused on suspicious and/or easily testable claims). The focus of the article is on inferential reproducibility; thus, methods don't necessarily map exactly to the original ones.

The authors present a large-scale analysis of the individual replication findings, which are presented in a companion article (Westlake et al., 2025. DOI 10.1101/2025.07.07.663442). In their retrospective analysis of reproducibility, the authors find that 61% of the original claims were verified by the literature, 7.5% were partialy verified, and only 6.8% were challenged, with 23.8% having no replication available. This is in stark contrast with the result of their prospective replications, in which only 16% of claims were successfully reproduced.

The authors proceed to investigate correlates of replicability, with the most consistent finding being that findings stemming from higher-ranked universities (and possibly from very high impact journals) were more likely to be challenged.

Strengths:

(1) The work presents a large-scale, in-depth analysis of a particular field of science that includes authors with deep domain expertise of the field. This is a rare endeavour to establish the reproducibility of a particular subfield of science, and I'd argue that we need many more of these in different areas.

(2) The project was built on a collaborative basis (https://ReproSci.epfl.ch/), using an online database (https://ReproSci.epfl.ch/), which was used to organize the annotations and comments of the community about the claims. The website remains online and can be a valuable resource to the Drosophila immunity community.

(3) Data and code are shared in the authors' GitHub repository, with a Jupyter notebook available to reproduce the results.

Main concerns:

(1) Although the authors claim that "Drosophila immunity claims are mostly replicable", this conclusion is strictly based on the retrospective analysis - in which around 84% of the claims for which a published verification attempt was found. This is in very stark contrast with the findings that the authors replicate prospectively, of which only 16% are verified.

Although this large discrepancy may be explained by the fact that the authors focused on unchallenged and suspicious claims (which seems to be their preferred explanation), an alternative hypothesis is that there is a large amount of confirmation bias in the Drosophila immunity literature, either because attempts to replicate previous findings tend to reach similar results due to researcher bias, or because results that validate previous findings are more likely to be published.

Both explanations are plausible (and, not being an expert in the field, I'd have a hard time estimating their relative probability), and in the absence of prospective replication of a systematic sample of claims - which could determine whether the replication rate for a random sample of claims is as high as that observed in the literature -, both should be considered in the manuscript.

(2) The fact that the analysis of factors correlating with reproducibility includes both prospective and retrospective replications also leads to the possibility of confusion bias in this analysis. If most of the challenged claims come from the authors' prospective replications, while most of the verified ones come from those that were replicated by the literature, it becomes unclear whether the identified factors are correlated with actual reproducibility of the claims or with the likelihood that a given claim will be tested by other authors and that this replication will be published.

(3) The methods are very brief for a project of this size, and many of the aspects in determining whether claims were conceptually replicated and how replications were set up are missing.

Some of these - such as the PubMed search string for the publications and a better description of the annotation process - are described in the companion article, but this could be more explicitly stated. Others, however, remain obscure. Statements such as "Claims were cross-checked with evidence from previous, contemporary and subsequent publications and assigned a verification category" summarize a very complex process for which more detail should be given - in particular because what constitutes inferential reproducibility is not a self-evident concept. And although I appreciate that what constitutes a replication is ultimately a case-by-case decision, a general description of the guidelines used by the authors to determine this should be provided. As these processes were done by one author and reviewed by another, it would also be useful to know the agreement rates between them to have a general sense of how reproducible the annotation process might be.

The same gap in methods descriptions holds for the prospective replications. How were labs selected, how were experimental protocols developed, and how was the validity of the experiments as a conceptual replication assessed? I understand that providing the methods for each individual replication is beyond the scope of the article, but a general description of how they were developed would be important.

(4) As far as I could tell, the large-scale analysis of the replication results was not preregistered, and many decisions seem somewhat ad hoc. In particular, the categorization of journals (e.g. low impact, high impact, "trophy") and universities (e.g. top 50, 51-100, 101+) relies on arbitrary thresholds, and it is unclear how much the results are dependent on these decisions, as no sensitivity analyses are provided.

Particularly, for analyses that correlate reproducibility with continuous variable (such as year of publication, impact factor or university ranking, I'd strongly favor using these variables as continuous variables in the analysis (e.g. using logistic regression) rather than performing pairwise comparisons between categories determined by arbitrary cutoffs. This would not only reduce the impact of arbitrary thresholds in the analysis, but would also increase statistical power in the univariate analyses (as the whole sample can be used in at once) and reduce the number of parameters in the multivariate model (as they will be included as a single variable rather than multiple dummy variables when there are more than two categories).

(5) The multivariate model used to investigate predictors of replicability includes unchallenged claims along with verified ones in the outcome, which seems like an odd decision. If the intention is to analyze which factors are correlated with reproducibility, it would make more sense to remove the unchallenged findings, as these are likely uninformative in this sense. In fact, based on the authors' own replications of unchallenged findings, they may be more likely to belong the "challenged" category than to the "unchallenged" one if they were to be verified.

Reviewer #1 (Public review):

One of the roadblocks in PfEMP1 research has been the challenges in manipulating var genes to incorporate markers to allow the transport of this protein to be tracked and to investigate the interactions taking place within the infected erythrocyte. In addition, the ability of Plasmodium falciparum to switch to different PfEMP1 variants during in vitro culture has complicated studies due to parasite populations drifting from the original (manipulated) var gene expression. Cronshagen et al have provided a useful system with which they demonstrate the ability to integrate a selectable drug marker into several different var genes that allows the PfEMP1 variant expression to be 'fixed'. This on its own represents a useful addition to the molecular toolbox and the range of var genes that have been modified suggests that the system will have broad application. As well as incorporating a selectable marker, the authors have also used selective linked integration (SLI) to introduce markers to track the transport of PfEMP1, investigate the route of transport and probe interactions with PfEMP1 proteins in the infected host cell.

One of the major strengths of this paper is that the authors have not only put together a robust system for further functional studies, but they have used it to produce a range of interesting findings including:

Co-activation of rif and var genes when in a head-to-head orientation.

The reduced control of expression of var genes in the 3D7-MEED parasite line.

More support for the PTEX transport route for PfEMP1.<br /> Identification of new proteins involved in PfEMP1 interactions in the infected erythrocyte, including some required for cytoadherence.

In most cases the experimental evidence is straightforward, and the data support the conclusions strongly. The authors have been very careful in the depth of their investigation, and where unexpected results have been obtained, they have looked carefully at why these have occurred.

A weakness of the paper is, as mentioned above, that the results are sometimes not as clear as might have been expected, for example, in the requirement for panning modified parasites to produce binding to EPCR. Where this has happened, the authors take a robust and thoughtful approach, and acknowledge that (as in most research) there are more questions to address. Being able to select specific var gene switches using drug markers will provide some useful starting points to understand how switching happens in P. falciparum. However, our trypanosome colleagues might remind us that forcing switches may show us some mechanisms, but perhaps not all.

Despite these sometimes complicated findings, the authors have achieved their aim as stated in the title of the paper, and in doing so have provided an excellent resource to themselves and other researchers in the field to answer some important questions.

Overall, the authors have produced a useful and robust system to support functional studies on PfEMP1, which provides a platform for future studies manipulating the domain content in var genes. They have used this system to produce a range of interesting findings and to support its use by the research community.

Comments on revisions:

I have no further recommendations for changes by the authors. They have addressed my concerns, and the paper reads very well.

Reviewer #1 (Public review):

Summary:

This study resolves a cryo-EM structure of the GPCR, GPR30, in the presence of bicarbonate, which the author's lab recently identified as the physiological ligand. Understanding the ligand and the mechanism of activation is of fundamental importance to the field of receptor signaling. This solid study provides important insight into the overall structure and suggests a possible bicarbonate binding site.

Strengths:

The overall structure, and proposed mechanism of G-protein coupling are solid. Based on the structure, the authors identify a binding pocket that might accommodate bicarbonate. Although assignment of the binding pocket is speculative, extensive mutagenesis of residues in this pocket identifies several that are important to G-protein signaling. The structure shows some conformational differences with a previous structure of this protein determined in the absence of bicarbonate (PMC11217264). To my knowledge, bicarbonate is the only physiological ligand that has been identified for GPR30, making this study an important contribution to the field. However, the current study provides novel and important circumstantial evidence for the bicarbonate binding site based on mutagenesis and functional assays.

Weaknesses:

Bicarbonate is a challenging ligand for structural and biochemical studies, and because of experimental limitations, this study does not elucidate the exact binding site. Higher resolution structures would be required for structural identification of bicarbonate. The functional assay monitors activation of GPR30, and thus reports on not only bicarbonate binding, but also the integrity of the allosteric network that transduces the binding signal across the membrane. However, biochemical binding assays are challenging because the binding constant is weak, in the mM range.

The authors appropriately acknowledge the limitations of these experimental approaches, and they build a solid circumstantial case for the bicarbonate binding pocket based on extensive mutagenesis and functional analysis. However, the study does fall short of establishing the bicarbonate binding site.

Reviewer #1 (Public review):

Summary:

This study presents a new Bayesian approach to estimate importation probabilities of malaria combining epidemiological data, travel history, and genetic data through pairwise IBD estimates. Importation is an important factor challenging malaria elimination, especially in low transmission settings. This paper focus on Magude and Matutuine, two districts in south Mozambique with very low malaria transmission. The results show isolation-by-distance in Mozambique, with genetic relatedness decreasing with distances larger than 100 km, and no spatial correlation for distances between 10 and 100 km. But again strong spatial correlation in distances smaller than 10 km. They report high genetic relatedness between Matutuine and Inhambane, higher than between Matutuine and Magude. Inhambane is the main source of importation in Matutuine, accounting for 63.5% of imported cases. Magude, on the other hand, shows smaller importation and travel rates than Matutuine, as it is a rural area with less mobility. Additionally, they report higher levels of importation and travel in the dry season, when transmission is lower. Also, no association with importation was found for occupation, sex and other factors. These data have practical implications for public health strategies aiming malaria elimination, for example, testing and treating travelers from Matutuine in the dry season.

Strengths:

The strength of this study relies in the combination of different sources of data - epidemiological, travel and genetic data - to estimate importation probabilities, the statistical analyses.

Weaknesses:

The authors recognize the limitations related to sample size and the biases of travel reports.

Reviewer #1 (Public review):

Summary:

Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. Colonoscopy and fecal immunohistochemical testing are among the early diagnostic tools that have significantly enhanced patient survival rates in CRC. Methylation dysregulation has been identified in the earliest stages of CRC, offering a promising avenue for screening, prediction, and diagnosis. The manuscript entitled "Early Diagnosis and Prognostic Prediction of Colorectal Cancer through Plasma Methylation Regions" by Zhu et al. presents that a panel of genes with methylation pattern derived from cfDNA (27 DMRs), serving as a noninvasive detection method for CRC early diagnosis and prognosis.

Strengths:

The authors provided evidence that the 27 DMRs pattern worked well in predicting CRC distant metastasis, and the methylation score remarkably increased in stages III-IV. Additionally, compared with the traditional tumor marker CEA, 27 DMRs prediction showed a superior sensitivity, highlighting the potential clinical application.

Weaknesses:

The major concerns are the design of DMRs screening, the relatively low sensitivity of this DMRs' pattern in detecting early-stage of CRC, the limited size of the cohorts, and the lack of comparison with the traditional diagnosis test.

Comments on revisions:

All my concerns have been cleared, and I have no further questions.

Reviewer #1 (Public review):

Summary:

Taylar Hammond and colleagues identified new regulators of the G1/S transition of the cell cycle. They did so by screening publicly available data from the Cancer Dependency Map and identified FAM53C as a positive regulator of the G1/S transition. Using biochemical assays they then show that FAM53 interacts with the DYRK1A kinase to inhibit its function. They show in RPE1 cells that loss of FAMC53 leads to a DYRK1A + P53-dependent cell cycle arrest. Combined inactivation of FAM53C and DYRK1A in a TP53-null background caused S-phase entry with subsequent apoptosis. Finally the authors assess the effect of FAM53C deletion in a cortical organoid model, and in Fam53c knockout mice. Whereas proliferation of the organoids is indeed inhibited, mice show virtually no phenotype.

The authors have revised the manuscript, and I respond here point-by-point to indicate which parts of the revision I found compelling, and which parts were less convincing. So the numbering is consistent with the numbering in my first review report.

(1) The p21 knockdowns are a valuable addition, and the claim that other p53 targets than p21 are involved in the FAMC53 RNAi-mediated arrest is now much more solid. Minor detail: if S4D is a quantification of S4C, it is hard to believe that the quantification was done properly (at least the DYRK1Ai conditions). Perhaps S4C is not the best representative example, or some error was made?

(2a) I appreciate the decision to remove the cyclin D1 phosphorylation data. A more nuanced model now emerges. It is not clear to me however why the Protein Simple immunoassay was used for experiments with RPE cells, and not the cortical organoids. Even though no direct claims are made based on the phospho-cyclin D data in Figure 5E+G, showing these data suggests that FAM53C deletion increases DYRK1A-mediated cyclin D1 phosphorylation. I find it tricky to show these data, while knowing now that this effect could not be shown in the RPE1 cells.<br /> (2b) The quantifications of the immunoassays are not convincing. In multiple experiments, the HSP90 levels vary wildly, which indicates big differences in protein loading if HSP90 is a proper loading control. This is for example problematic for the interpretation of figure 3F and S3I. The cyclin D1 "bands" look extremely similar between siCtrl and siFAM53C (Fig S3I), in fact the two series of 6 samples with different dosages of DYRK1Ai look seem an identical repetition of each other. I did not have to option to overlay them, but it would be important to check if a mistake was made here. The cyclin D1 signals aside, the change in cycD1/HSP90 ratios seems to be entirely caused by differences in HSP90 levels. Careful re-analysis of the raw data and more equal loading seem necessary. The same goes (to a lesser extent) for S3J+K.<br /> (2c) the new model in Fig S4L: what do the arrows at the right FAM53C and p53 that merge a point straight towards S-phase mean? They suggest that p53 (and FAM53C) directly promote S-phase progression, but most likely this is not what the authors intended with it.

(3) Clear; nicely addressed.

(4) Thank you for correcting.

(5) I appreciate that the authors are now more careful to call the IMPC analysis data preliminary. This is acceptable to me, but nevertheless, I suggest the authors to seriously consider taking this part entirely out. The risk of chance finding and the extremely skewed group sizes (as reviewer #2 had pointed out) hamper the credibility of this statistical analysis.

Reviewer #1 (Public review):

Summary:

Cotton et al. investigated the role of tusB in antibiotic tolerance in Yersinia pseudotuberculosis. They used the IP2226 strain and introduced appropriate mutations and complementation constructs. Assays were performed to measure growth rates, antibiotic tolerance, tRNA modification, gene expression and proteomic profiles. In addition, experiments to measure ribosome pausing and bioinformatic analysis of codon usage in ribosomal proteins provided in-depth mechanistic support for the conclusions.

Strengths:

The findings are consistent with the authors having uncovered new mechanistic insights into bacterial antibiotic tolerance mediated by reducing ribosomal protein abundance.

Weaknesses:

Since the WT strain grows faster than the tusB mutant, there is a question of how growth rate, per se, impacts some of the analysis done. The authors should address this issue. In addition, it may not be essential, but would analysis of another slow-growing mutant (in some other antibiotic tolerance pathway if available) serve as a good control in this context?

Reviewer #1 (Public review):

Summary:

This unique study reports original and extensive behavioral data collected by the authors on 21 living mammal taxa in zoo conditions (primates, tree shrew, rodents, carnivorans, and marsupials) on how descent along a vertical substrate can be done effectively and securely using gait variables. Ten morphological variables reflecting head size and limb proportions are examined in relationship to vertical descent strategies and then applied to reconstruct modes of vertical descent in fossil mammals.

Strengths:

This is a broad and data-rich comparative study, which requires a good understanding of the mammal groups being compared and how they are interrelated, the kinematic variables that underlie the locomotion used by the animals during vertical descent, and the morphological variables that are associated with vertical descent styles. Thankfully, the study presents data in a cogent way with clear hypotheses at the beginning, followed by results and a discussion that addresses each of those hypotheses using the relevant behavioral and morphological variables, always keeping in mind the relationships of the mammal groups under investigation. As pointed out in the study, there is a clear phylogenetic signal associated with vertical descent style. Strepsirrhine primates much prefer descending tail first, platyrrhine primates descend sideways when given a choice, whereas all other mammals (with the exception of the raccoon) descend head first. Not surprisingly, all mammals descending a vertical substrate do so in a more deliberate way, by reducing speed, and by keeping the limbs in contact for a longer period (i.e., higher duty factors).

Reviewer #2 (Public review):

The Revision title and abstract are not updated enough to distinguish the special niche piRNA clusters from the more prominent major dual strand piRNA clusters that are widely known in the field for Drosophila, like 42AB and 38C. This revision mainly adds the term "piRNA source loci (piSL)" that is too vague and not a well-accepted name that would distinguish just these particularly niche piRNA clusters from major dual strand piRNA clusters like 42AB and 38C. This piSL term is problematic because it seems to imply these piSL's are connected to or would eventually become major dual strand piRNA clusters, but there is zero evidence in this study for any genetic or evolutionary connection between these two distinct types of piRNA sources. This revision still lacks the necessary changes needed to point out like in the abstract that major dual strand piRNA clusters like 42AB, 38C, 80F, and 102F in Drosophila that make up the bulk of piRNAs cannot be shown to be impacted by changes aimed at depleting ADMA-histones from these loci, and the authors' current evidence is still only limited to showing in these few 'niche' piRNA clusters that ADMA-histones may exhibit a direct interaction with Rhino as supported only by the knockdown of Drosophila Art4.

The author's rebuttal letter argues that 42AB and 38C are just conserved piRNA clusters that may no longer be regulated by ADMA. This is still a weak claim for dismissing the potential genetic redundancy problem when this study can only report strong knockdown of Art4. First, the dual strand 42AB piRNA cluster's conservation as a Drosophilid piRNA cluster is actually still a relatively recent evolutionary innovation in just D.simulans and D.melanogaster that are less than 3MYA diverged. This 42AB cluster is no longer conserved in D.sechelia and is also younger than the uni-strand Flamenco piRNA cluster that is conserve to 7MYA. The evolutionary arguments by the authors are not well-grounded. Second, the 42AB and 38C are the largest major dual strand piRNA clusters with very significant localization of Rhino and impact from Rhino loss of function, and if this paper's central thesis is that ADMA-histones directed by Art1 or Art4 is critical for the expression of dual-strand piRNA cluster loci by impacting Rhino, the current data still remain weak with no new experiments to help bolster their claims.

The author's rebuttal letter argues that the challenges they faced in trying to knock down Art1 in the fly was thwarted by reagent issues, and the explanations are unsatisfactory. They claim they only tested two RNAi cross lines to try to knock down Art1: the strain BDSC #36891, y[1] sc[*] v[1] sev[21]; P{y[+t7.7], v[+t1.8]=TRiP.GL01072}attP2/TM3, Sb[1] that they said they could not obtain this strain to be alive from the stock center? And then testing an alternative line VDRC #v110391P{KK101196}VIE-260B that displayed mediocre knockdown, the authors seemed to suggest they have given up trying to make this very important experiment work? They should have tried to figure out with the BDSC, a venerable stock center for Drosophila genetic tools, why they could not receive that fly strain alive (shipping flies at the economy rate internationally may be cheaper but often is too strenuous for flies to survive), and the authors have not acknowledged testing two other available knockdown lines for Art1: BDSC #31348, y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF01306}attP2 dsRNA and VDRC #w1118 P{GD11959}v40388. Trying to get good knockdown of Art1 would be a critical must-have experiment to address whether this arginine methyltransferase has an in vivo impact on ADMA-histones in the Drosophila ovary and showing an impact on 42AB and 38C. The revision does not address this major deficiency in impact on these two major dual strand piRNA clusters, only the very few niche piRNA clusters that are responsive to Art4 knockdown.

The rebuttal letter argues that "Therefore, conserved clusters such as 42AB and 38C may no longer be regulated by ADMA." but then the revision discussion is still speculating much too wildly that the piRNA source loci are then precursors for the eventual large piRNA clusters of 42AB and 38C. This renaming of the term piRNA source loci and the model in Fig. 7C is still misleading because 42AB and 38C are the main largest dual-strand piRNA clusters, and the pictures depict the ADMA-histones as recruiting Rhino and then Kipferl at a piRNA cluster. The term "piRNA source loci" does not sound distinct enough to separate it from the main piRNA clusters of 42AB and 38C, and I had suggested calling them 'niche piRNA clusters' to denote they are very special and distinct to only be responsive to Drosophila Art4 knockdown.

In regards to the revision's changing of gene names, the convention for gene names is to use the previous name designation. Rather than calling the gene DART1, the conventional name of this gene in Flybase is Art1 (CG6554). There is the same problem with using the new name DART4 when in Flybase the gene is called Art4 (CG5358). Alternatively, the authors should clarify the re-naming up front and make it consistent with Drosophila genetics nomenclature, perhaps dArt1 or dArt4 would be more appropriate.

Reviewer #1 (Public review):

Summary:

Even though mutations in LRRK2 and GBA1 (which encodes the protein GCase) increase the risk of developing Parkinson's disease (PD), the specific mechanisms driving neurodegeneration remain unclear. Given their known roles in lysosomal function, the authors investigate how LRRK2 and GCase activity influence the exocytosis of the lysosomal lipid BMP via extracellular vesicles (EVs). They use fibroblasts carrying the PD-associated LRRK2-R1441G mutation and pharmacologically modulate LRRK2 and GCase activity.

Strengths:

The authors examine both proteins at endogenous levels, using MEFs instead of cancer cells. The study's scope is potentially interesting and could yield relevant insights into PD disease mechanisms.

Weaknesses:

Many of the authors' conclusions are overstated and not sufficiently supported by the data. Several statistical errors undermine their claims. Pharmacological treatment is very long, leading to potential off target effects. Additionally, the authors should be more rigorous when using EV markers.

Comments on revisions:

The authors have not addressed most of my concerns. For example, instead of trying with a 1-2 hour MLi2 treatment, they cited all the papers that use extremely long time points for LRRK2 inhibition; the fact that other groups do it does not mean it is biologically correct. They also refused to quantify their western blots in a proper manner, without the "hyper-normalization" claiming that it is an accepted way to quantify western blots. Again, it is statistically incorrect and biologically impossible. They also do not have a satisfactory explanation as to why the R1441G cells (which increase LRRK2 kinase activity) have no effect on EV release, but they still claim it is LRRK2 kinase activity dependent.

Overall, I am very confused by the model proposed by the authors. They only see increased EV release in the G2019S expressing cells, but not the R1441G cells, yet they claim that the increase of EV release is LRRK2 kinase activity dependent. Then, they claim that the presence of BMP (unchanged in R1441G vs CTL) in EVs is also LRRK2 kinase activity dependent. Finally, they perform TIRF with pHluorin-CD63 construct and observed an increase in G2019S cells vs CTL "further confirming that BMP release is associated with EV secretion". First, I could not see the increase in BMP release in G2019S cells (if I missed it, I apologize). And second, why didn't they do this experiment in R1441G cells? As, the R1441G cells have not displayed an increase in EV release compared to CTL cells, it could also be possible that the BMP release might be more abundant through lysosomal exocytosis (which could explain the pHluorin results) than EVs. Overall, the authors nicely demonstrate that the R1441G cells have more BMP species, likely due to increase CLN5 expression, but the release of the BMP is still not clear to this reviewer.

Reviewer #1 (Public review):

Summary:

Marchand et al. seek to understand how basement membrane (BM) is initially assembled around developing vasculature (and by extension basement membrane assembly generally progresses). To do this, they use an established cell culture system that is amenable to advanced microscopy techniques, including high-resolution fluorescence imaging and atomic force microscopy. This allows them to produce very high-quality imaging data that includes both protein localization and matrix topography in 3D. They show that fibronectin (FN) is remodeled as collagen IV (Col IV) assembles. Lysyl oxidase-like-2 (LOXL2) is needed for this process, and without it, BM does not form correctly, cells cannot adhere to BM, and cells also don't correctly form junctions with other cells.

Detailed Review:

The authors provide quantitative measures of BM fibril assembly at the earliest timepoints. They show two phases of growth - initial deposition, elongation, and interconnection of short fibers; the second is a significant thickening. As the BM forms, FN is immediately associated with filaments, but laminin and Col IV are not associated with fibers as detected by AFM. LOXL2 is associated with fibers, similar to FN. At a later time point, Col IV becomes associated with fibers, but laminin never does. Likely FN templates LOXL2, which crosslink Col IV into fibrils over time. Could the authors comment on how this data fits with in vivo data from model organisms? Also, I would like to know if they can uncouple LOXL2 from the FN matrix? Could you express a mutated form of LOXL2 that cannot interact with FN but still is able to crosslink Col IV?)

Depletion of LOXL2 supports this mechanism. Without it, Col IV and FN are uncoupled and accumulate as large aggregates rather than a complex fibrous network. Further, long-term thickening/growth of the fibronectin network is inhibited, indicating LOXL2 and/or the Col IV network positively reinforces fibronectin assembly. (Does LOXL2 directly act on FN, or is this effect dependent on Col IV? The nature of the molecular interactions between COL IV, LOXL2, and FN will be an important future research area.)

Next, Marchand et al. ask if failure to produce mature BM (induced by LOXL2 depletion) has consequences for underlying cells. They demonstrate a clear shift in the orientation of actin towards a linear alignment, and similarly, cells change shape from round to very elongated. Cell junctions also shifted to a linear arrangement in LOXL2 depletion. This fits with the known balance between cell-ECM and cell-cell adhesion. The changes in actin network and cell shape/adhesion correlate with a change in B1 integrin localization upon LOXL2 depletion. B1 integrin colocalized with sparse early FN fibers, but was absent from large FN aggregates that occur if LOXL2 is depleted. Similar reorganization of integrin adhesion components (FAK, Vinc, Pax). Clearly, there is feedback between BM assembly and cell junction organization. But I think the authors might emphasize to the reader that this normally reinforces the epithelial fate of these cells. It's less a balance and more like a tipping point. (Related to this section, I could not read Figure 4C graphs unless I enlarged them to 300%.)

Finally, they culture cells on micro groove plates, with or without LOXL2. The grooved substrate can orient the cells, and they show this is superseded by BM once it assembles. Without LOXL2 cells on micro-grooved substrates become disorganized, similar to their observation on flat surfaces (elongated cells, linear actin, etc.). This demonstrates a switch from external topographical cues to self-generated BM. This is consistent with the idea of reorganizing junctions to produce a stable epithelial tube. I was very interested in their 3D culture. The effect of BM assembly on tube diameter makes sense. But how does BM assembly support complex capillary functions like branching? (Can they force branching with targeted mutations that decouple integrin from the BM?) Is this a question of change to cell fate? (Are other remodeling enzymes activated after initial BM assembly that could support growth and/or branching?)

Reviewer #1 (Public review):

Summary:

The authors utilize genetic code expansion to tag TDP-43 and G3BP1, and evaluate this protein tagging system (ANAP) compared to antibodies, and evaluate protein trafficking and stress granule formation in response to stress with sodium arsenite treatment. They find similar staining to antibodies in HeLa cells, mouse embryonic stem cells, and primary mouse cortical neurons. This is a useful study that demonstrates the utility of ANAP tagging to evaluate ALS proteins.

Strengths:

Rescue of cell survival by ANAP-tagged TDP-43 is compelling

Weaknesses:

While the ANAP-tagged proteins had similar distributions to antibody staining, there were some discrepancies that may be more explained by the technique than by novel findings, as the authors suggested. The inclusion of additional controls to evaluate this would be helpful.

Reviewer #2 (Public review):

This paper proposes two changes to classic RSA, a popular method to probe neural representation in neuroimaging experiments: computing RSA at row/column level of RDM, and using linear mixed modeling to compute second level statistics, using the individual row/columns to estimate a random effect of stimulus. The benefit of the new method is demonstrated using simulations and a re-analysis of a prior fMRI dataset on object perception and memory encoding.

The author's claim that tRSA is a promising approach to perform more complete modeling of cogneuro data, and to conceptualize representation at the single trial/event level (cf Discussion section on P42), is appealing.

In their revised manuscript, the authors have addressed some previous concerns, now referencing more literature aiming to improve RSA and its associated statistical inferences, and providing more guidance on methodological considerations in the Discussion. However, I wish the authors had more extensively edited the Introduction to better contextualize the work and clarify the specific settings in which they see the method as being beneficial over classic RSA. For example, some of the limitations of cRSA mentioned on page 6, e.g. related to presenting the same stimuli to multiple subjects, seem to be quite specific to settings where the researcher expects differential responses across subjects to fundamentally alter the interpretation, rather than something that will just average out by repeatedly offering the same stimulus, or combining data across subjects. It's not clear to me how the switch from 'matrix-level' to 'row-level' analysis in tRSA necessarily addresses this problem. I would be very helpful if the authors would more explicitly outline what problem the row-level aspect of tRSA is solving; what problem statistical inference via LMM is solving; and walk the reader through a very specific use case (perhaps a toy version of the real-data experiment which is now at the end of the paper). Explaining the utility of tRSA for experimental settings in which assessing representational strength for a single-events is crucial would clarify the contribution of this new method better.

A few weaknesses mentioned in my previous review were not adequately addressed. To demonstrate the utility of the method on real neural recordings, only a single dataset is used with a quite complicated experimental design; it's not clear if there is any benefit of using tRSA on a simpler real dataset. Moreover, the cells of an RDM/RSM reflect pairwise comparisons between response patterns. Because the response patterns are repeatedly compared, the cells of this matrix are not independent of one another. While the authors show examples that failure to meet independence assumptions do not affect results in their specific dataset, it does not get acknowledged as a problem at a more fundamental level. Finally, while the paper now states that 'simulations and example tRSA code' are publicly available, the link points to the lab's general github page containing many lab repositories, in which I could not identify a specific repository related to this paper. This is disappointing given that the main goal of this manuscript is to provide a new method that they encourage others to use; a clear pointer to available code is only a minimal requirement to achieve that goal. A dedicated repository, including documentation, READMEs and tutorials/demo's to run simulations, compare methods, etc. would greatly enhance the paper's contribution.

Reviewer #2 (Public review):

Summary:

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

Significance:

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

Reviewer #1 (Public review):

Summary:

The authors attempted to clarify the impact of N protein mutations on ribonucleoprotein (RNP) assembly and stability using analytical ultracentrifugation (AUC) and mass photometry (MP). These complementary approaches provide a more comprehensive understanding of the underlying processes. Both SV-AUC and MP results consistently showed enhanced RNP assembly and stability due to N protein mutations.<br /> The overall research design appears well planned, and the experiments were carefully executed.

Strengths:

SV-AUC, performed at higher concentrations (3 µM), captured the hydrodynamic properties of bulk assembled complexes, while MP provided crucial information on dissociation rates and complex lifetimes at nanomolar concentrations. Together, the methods offered detailed insights into association states and dissociation kinetics across a broad concentration range. This represents a thorough application of solution physicochemistry.

Weaknesses:

Unlike AUC, MP observes only a part of solution. In MP, bound molecules are accumulated on the glass surface (not dissociated) thus concentration in solution should change as time develops. How does such concentration change impact the result shown here?

Comments on revisions:

The response from the authors is appropriate and reasonable.

Reviewer #1 (Public review):

Summary:

Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

Strengths:

The CATailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue.

Reviewer #1 (Public review):

This study uses a new 'hidden multivariate pattern method' to parse in time and space the neural events intervening between stimulus and response in an immediately-reported perceptual decision, and use the resultant neural event timing information to show quite convincingly that Pieron's and Fechner's laws can apply in concert at distinct processing levels.

They designed a clever contrast comparison paradigm in which the contrast difference is kept constant while widely manipulating mean contrast, so that sensory encoding of the overall stimulus would be boosted with increasing mean contrast, whereas decision difficulty and hence duration would increase. With this, they found that the time intervening between early sensory-evoked components, up to an 'N200'-type component associated with launching the decision process, varies inversely with contrast according to Pieron's law. Meanwhile, the time intervals running up to neural events peaking near the time of response, consistent with decision termination, increases with contrast, fitting Fechner's law. Further, a diffusion model whose drift rates are scaled by Fechner's law, fit to RT, predicts the observed proportion of correct responses very well.

In the process of review and revision it was highlighted that presumably the full sequence of neural events intervening between stimulus and response is massively task dependent, but;

(1) The method is intended to capture all key components that specifically covary with RT, as opposed to each and every component in general, and

(2) The main conclusions of the study mentioned above do not change whether the method is set up to track three neural events, or five, as was done in the final analysis.

The propensity for topographic parsing algorithms to potentially lump-together distinct processes that partially co-evolve was acknowledged, but a key clarification in review was that even though the method entails a specification of neural event duration - which was changed from 50 to 25 ms - the success of the method is not strongly contingent on the actual underlying neural events in question having that very duration - indeed, the components extracted using that short template duration can be observed to evolve over a longer time frame associated with the Fechner diffusion process.

Notably, standard average event-related potential analysis was able to show expected amplitude effects - where sensory signals increased with contrast but decision signals decreased - but assessment of the by-trial distribution of their timings was grealy aided by the HMP method.

One of the stages of processing implicated in the parsing analysis was linked to attention orientation, and the authors speculate on whether this might reflect a spatially-selective deployment of attention or a resource allocation, but sensibly refrain from speculating too far since the focus here was on the sensory and decision process durations and their respective adherence to Pieron and Fechner's laws.

Reviewer #1 (Public review):

Summary:

The authors report the results of a tDCS brain stimulation study (verum vs sham stimulation of left DLPFC; between-subjects) in 46 participants, using an intense stimulation protocol over 2 weeks, combined with an experience-sampling approach, plus follow-up measures after 6 months.

Strengths:

The authors are studying a relevant and interesting research question using an intriguing design, following participants quite intensely over time and even at a follow-up time point. The use of an experience-sampling approach is another strength of the work.

Weaknesses:

There are quite a few weaknesses, some related to the actual study and some more strongly related to the reporting about the study in the manuscript. The concerns are listed roughly in the order in which they appear in the manuscript.

(1) In the introduction, the authors present procrastination nearly as if it were the most relevant and problematic issue there is in psychology. Surely, procrastination is a relevant and study-worthy topic, but that is also true if it is presented in more modest (and appropriate) terms. The manuscript mentions that procrastination is a main cause of psychopathology and bodily disease. These claims could possibly be described as 'sensationalized'. Also, the studies to support these claims seem to report associations, not causal mechanisms, as is implied in the manuscript.

(2) It is laudable that the study was pre-registered; however, the cited OSF repository cannot be accessed and therefore, the OSF materials cannot be used to (a) check the preregistration or to (b) fill in the gaps and uncertainties about the exact analyses the authors conducted (this is important because the description of the analyses is insufficiently detailed and it is often unclear how they analyzed the data).

(3) Related to the previous point: I find it impossible to check the analyses with respect to their appropriateness because too little detail and/or explanation is given. Therefore, I find it impossible to evaluate whether the conclusions are valid and warranted.

(4) Why is a medium effect size chosen for the a priori power analysis? Is it reasonable to assume a medium effect size? This should be discussed/motivated. Related: 18 participants for a medium effect size in a between-subjects design strikes me as implausibly low; even for a within-subjects design, it would appear low (but perhaps I am just not fully understanding the details of the power analysis).

(5) It remains somewhat ambiguous whether the sham group had the same number of stimulation sessions as the verum stimulation group; please clarify: Did both groups come in the same number of times into the lab? I.e., were all procedures identical except whether the stimulation was verum or sham?

(6) The TDM analysis and hyperbolic discounting approach were unclear to me; this needs to be described in more detail, otherwise it cannot be evaluated.

(7) Coming back to the point about the statistical analyses not being described in enough detail: One important example of this is the inclusion of random slopes in their mixed-effects model which is unclear. This is highly relevant as omission of random slopes has been repeatedly shown that it can lead to extremely inflated Type 1 errors (e.g., inflating Type 1 errors by a factor of then, e.g., a significant p value of .05 might be obtained when the true p value is .5). Thus, if indeed random slopes have been omitted, then it is possible that significant effects are significant only due to inflated Type 1 error. Without more information about the models, this cannot be ruled out.

(8) Related to the previous point: The authors report, for example, on the first results page, line 420, an F-test as F(1, 269). This means the test has 269 residual degrees of freedom despite a sample size of about 50 participants. This likely suggests that relevant random slopes for this test were omitted, meaning that this statistical test likely suffers from inflated Type 1 error, and the reported p-value < .001 might be severely inflated. If that is the case, each observation was treated as independent instead of accounting for the nestedness of data within participants. The authors should check this carefully for this and all other statistical tests using mixed-effects models.

(9) Many of the statistical procedures seem quite complex and hard to follow. If the results are indeed so robust as they are presented to be, would it make sense to use simpler analysis approaches (perhaps in addition to the complex ones) that are easier for the average reader to understand and comprehend?

(10) As was noted by an earlier reviewer, the paper reports nearly exclusively about the role of the left DLPFC, while there is also work that demonstrates the role of the right DLPFC in self-control. A more balanced presentation of the relevant scientific literature would be desirable.

(11) Active stimulation reduced procrastination, reduced task aversiveness, and increased the outcome value. If I am not mistaken, the authors claim based on these results that the brain stimulation effect operates via self-control, but - unless I missed it - the authors do not have any direct evidence (such as measures or specific task measures) that actually capture self-control. Thus, that self-control is involved seems speculation, but there is no empirical evidence for this; or am I mistaken about this? If that is indeed correct, I think it needs to be made explicit that it is an untested assumption (which might be very plausible, but it is still in the current study not empirically tested) that self-control plays any role in the reported results.

(12) Figures 3F and 3H show that procrastination rates in the active modulation group go to 0 in all participants by sessions 6 and 7. This seems surprising and, to be honest, rather unlikely that there is absolutely no individual variation in this group anymore. In any case, this is quite extraordinary and should be explicitly discussed, if this is indeed correct: What might be the reasons that this is such an extreme pattern? Just a random fluctuation? Are the results robust if these extreme cells are ignored? The authors remove other cells in their design due to unusual patterns, so perhaps the same should be done here, at least as a robustness check.

(13) The supplemental materials, unfortunately, do not give more information, which would be needed to understand the analyses the authors actually conducted. I had hoped I would find the missing information there, but it's not there.

In sum, the reported/cited/discussed literature gives the impression of being incomplete/selectively reported; the analyses are not reported sufficiently transparently/fully to evaluate whether they are appropriate and thus whether the results are trustworthy or not. At least some of the patterns in the results seem highly unlikely (0 procrastination in the verum group in the last 2 observation periods), and the sample size seems very small for a between-subjects design.

Reviewer #1 (Public review):

Summary:

This paper is a comprehensive review of perturbation studies and the state-dependence of the brain's response to perturbation at the circuit, mesoscale, and macroscale levels.

Strengths:

The strengths of the paper are the thorough description of many perturbation studies at different levels of organization, and the integration of both experimental and modeling studies. The review clearly communicates the need to consider (1) brain or local-population state, and (2) multiple levels of organization, in order to understand perturbation responses. Another major strength is the ability for the reader to reproduce figures using the EBRAINS platform.

Weaknesses:

Two major points of improvement should be resolved with the review, in order to make it useful for a broad audience.

The first is that the review does not include a significant integration across scales, and as a result, reads like three separate (though comprehensive) reviews. Currently, the only integration across the scales is in the brief conclusion paragraph. I would recommend adding an additional section, in which the overarching picture is discussed. (i.e. a unifying view of state dependence, and what is learned by considering across scales). This need not be too long, but it should be longer than a single conclusion paragraph.

The second major weakness is that there is a lack of clarity on many points throughout, which is needed for the reader to fully understand the results described.

Reviewer #1 (Public review):

Summary:

In the paper, the authors review literature on synchronous activity, its relationship to brain state, and the multi-scale mechanisms underlying it.

Strengths:

The overall strength of the paper is the wide range of information reviewed, and the diversity of perspectives/approaches it brings together.

Weaknesses:

However, this strength is also the source of its major weaknesses - namely, that the overall structure lacks clarity, and there are inconsistencies throughout. Overall, in the opinion of this reviewer, the manuscript reads as disorganized and incomplete. Major and minor points are delineated below.

Major points:

(1) Most of the text in many figures was too small to read.

(2) Terminology is inconsistent throughout the manuscript. What is the difference between slow oscillations and delta waves? Sometimes the term slow waves is used instead. For sleep state, sometimes the term SWS is used, sometimes non-REM. Similarly, "spindle activity" is not defined, but simply stated as if the reader knows. This brings up two issues: (a) the manuscript should be clearer and more consistent about its terminology, and (b) it's unclear who is the intended readership of the review - is it a pedagogical review for people outside the field of sleep and slow oscillations, or is it meant to be a consensus statement for readers who are already in the field in which a pressing concern has been addressed? It seems part way between these two, and as a result, is ineffective at either goal.

(3) I suggest the authors look again at the overall structure and flow of the review... many sections feel redundant, and it's unclear how they fit together into a single review.

(4) There are many speculative statements in the review that are not justified or explained sufficiently for the reader. For example: "While highly regular slow waves in vivo suggest a single mechanism of generation, namely local cortical circuits, irregular cycles are compatible with a larger role of subcortical nuclei, ..."; "The involvement of different cortical areas and subcortical nuclei can form the basis of these different roles in memory.". For these statements, I assume the relationship between slow wave statistics, subcortical nuclei, and memory either has been written about before, and then should be cited and summarized, or is a novel claim of the authors, which then should be explained and defended rather than stated. There are other similar examples, and I suggest the authors go through the manuscript and make sure that it's clear what is a novel claim of the authors vs a cited claim, and make sure that both are sufficiently justified for the reader.

(5) An especially notable example can be found in the section on the role of the thalamus, where the authors state that they "hold that slow oscillations are fundamentally cortical". However, this section is far too short, and very little evidence is provided to back up this claim. Please review the ways in which the thalamus modulates, and, e.g., ways in which up-down is similar/different without the thalamus.

Reviewer #1 (Public review):

Summary:

This report demonstrates that the gene expression output of the Wnt pathway, when controlled precisely by a synthetic light-based input, depends substantially on the frequency of stimulation. The particular frequency-dependent trend that is observed - anti-resonance, a suppression of target gene expression at intermediate frequencies given a constant duty cycle - is a novel aspect that has not been clearly shown before for this or other signaling pathways. The paper provides both clear experimental evidence of the phenomenon with engineered cellular systems and a model-based analysis of how the pairing of rate constants in pathway activation/deactivation could result in such a trend.

Strengths:

This report couples in vitro experimental data with an abstracted mathematical model. Both of these approaches appear to be technically sound and to provide consistent and strong support for the main conclusion. The experimental data are particularly clear, and the demonstration that Brachyury expression is subject to anti-resonance in ESCs is particularly compelling. The modeling approach is reasonably scaled for the system at the level of detail that is needed in this case, and the hidden variable analysis provides some insight into how the anti-resonance works.

In this revised manuscript, the authors have addressed issues in presentation and in discussing the broader relevance of their study to other pathways. Other limitations of the paper, including the fact that the anti-resonance phenomenon has not yet been demonstrated using physiological Wnt ligands and that the model has not been validated using experimental manipulations to establish that the mechanisms of the cell system and the model are the same, were deemed out of the scope of this initial demonstration by both the reviewers and authors. These questions will provide an interesting basis for further studies.

Reviewer #1 (Public review):

Summary:

This study presents a valuable contribution of NO signaling in zebrafish retinal regeneration in larval animals. The data on NO signaling are solid. There are multiple limitations to the study, but these are largely acknowledged by the authors in the revised text.

Strengths:

New data on NO signaling is valuable to the field but may be limited to larval "regeneration".

Weaknesses:

A weakness of the approach is testing cone ablation and regeneration in early larval animals. A near identical study was already done by Hoang et al 2020 in the adult zebrafish, a more relevant biological timepoint.