Wilson Jones Co., est. 1893 by [[Made-in-Chicago Museum]]

Reviewer #3 (Public review):

Summary:

The authors investigated the role of dopaminergic neurons (dopamine transporter expressing, DAT) in the dorsal raphe nucleus (DRN) in regulating social and affective behavior through projections to the central nucleus of the amygdala (CeA), bed nucleus of the stria terminalis (BNST), and the posterior subdivision of the basolateral amygdala. The largest effect observed was in the DRN-DAT projections to the CeA. Augmenting previously published results from this group (Matthews et al., 2016), the comprehensive behavioral analysis relative to social dominance, gene expression analysis, electrophysiological profiling, and in vivo imaging provides novel insights into how DRN-DAT projections to the CeA influence the engagement of social behavior in the contexts of group housed and socially isolated mice.

Strengths:

Correlational analysis with social dominance is a nice addition to the study. The overall computational analyses performed are well-designed and rigorous.

Weaknesses:

(1) Analysis of dopamine receptor expression did not include Drd3, Drd4, or Drd5 which may provide more insights into how dopamine modulates downstream targets. This is particularly relevant to the BNST projection in which the densest innervation did not robustly co-localize with the expression of either Drd1 or Drd2. It is also possible that dopamine release from DRN-DAT neurons in any or all of these structures in modulating neurotransmitter release from inputs to these regions that contain D2 receptors on their terminals.

(2) Although not the focus of this study, without pharmacological blockade of dopamine receptors, it is not possible to assess what the contribution of dopamine is to the behavioral outcomes. Given the co-release of glutamate and GABA from these neurons it is possible that dopamine plays only a marginal role in the functional connectivity of DRN-DAT neurons.

(3) Photostimulation parameters used during the behavioral studies (8 pulses of light delivered at 30 Hz for several minutes) could lead to confounding results limiting data interpretation. As shown in Figure 6J, 8 pulses of light delivered at 30 Hz results in a significant attenuation of the EPSC amplitude in the BLP and CeA projection. Thus, prolonged stimulation could lead to significant synaptic rundown resulting in an overall suppression of connectivity in the later stages of the behavioral analyses.

Comments on revisions:

No further issues have been identified.

Reviewer #3 (Public review):

Summary:

In microbiology, accurately characterizing microbial populations and communities is essential. One widely used approach is to measure the absolute or relative abundance of microbial species. Recent research in microbial ecology, for instance, has shown that even genetically identical hosts exposed to the same microbial pool can develop very different gut microbiota, largely due to random colonization events. This study builds on that idea but adds a valuable layer: it suggests that some of the observed variability might actually result from experimental noise, specifically the randomness introduced by dilution and plate counting techniques. To address this, the authors introduce REPOP, a new tool designed to improve the quantification of microbial populations by explicitly accounting for the inherent stochasticity in these methods. They test REPOP using both simulated and experimental datasets, showing how it can help recover meaningful trends.

Strengths:

Overall, this paper is a good contribution to the field. The motivation is clear: improving our ability to quantify microbial populations is crucial for many research areas. The authors make a strong case that ignoring experimental noise is no longer acceptable, and they offer a well-argued solution. The manuscript is well-written and easy to follow, and the logic behind REPOP is convincingly laid out. The use of simulated data is especially valuable, as it allows the authors to test whether the method can recover known inputs, an important validation step. Even with experimental data, where true values are unknown, the method seems to behave in a reasonable and expected way, which is reassuring. All in all, this is an important step forward in how we quantify microbial populations.

Weaknesses:

While the study is promising, there are a few areas where the paper could be strengthened to increase its impact and usability. First, the extent to which dilution and plating introduce noise is not fully explored. Could this noise significantly affect experimental conclusions? And under what conditions does it matter most? Does it depend on experimental design or specific parameter values? Clarifying this would help readers appreciate when and why REPOP should be used. Second, more practical details about the tool itself would be very helpful. Simply stating that it is available on GitHub may not be enough. Readers will want to know what programming language it uses, what the input data should look like, and ideally, see a step-by-step diagram of the workflow. Packaging the tool as an easy-to-use resource, perhaps even submitting it to CRAN or including example scripts, would go a long way, especially since microbiologists tend to favor user-friendly, recipe-like solutions. Third, it would be great to see the method tested on existing datasets, such as those from Nic Vega and Jeff Gore (2017), which explore how colonization frequency impacts abundance fluctuation distributions. Even if the general conclusions remain unchanged, showing that REPOP can better match observed patterns would strengthen the paper's real-world relevance. Lastly, it would be helpful for the authors to briefly discuss the limitations of their method, as no approach is without its constraints. Acknowledging these would provide a more balanced and transparent perspective.

Reviewer #3 (Public review):

Why mitochondria are finely maintained in the female germ cell (oocyte), zygotes, and preimplantation embryos? Mitochondrial fusion seems beneficial in somatic cells to compensate for unhealthy mitochondria, for example, mitochondria with mutated mtDNA that potentially defuel the respiratory activity if accumulated above a certain threshold. However, in the germ cells, it may rather increase the risk of transmitting mutated mtDNA to the next generation. Also, finely maintained mitochondria would also be beneficial for efficient removal when damaged, as the authors briefly discussed. Due in part to the limited suitable model, physiological role of mitochondrial fission in embryos were obscure. In this study, authors demonstrated that mitochondrial fission prevents multiple adverse outcomes, especially including the aberrant demixing of parental genome (a clinical phenotype of human embryos) in zygotic stage. Thus, this study would be also of clinical importance that could contribute by proposing a novel mechanism.

The authors have adequately indicated the limitations at each of the specific points. The revisions the authors made have consolidated their conclusion, thus still, making this study an excellent one.

Reviewer #3 (Public review):

Summary:

This study describes a computational model of the rat spinal locomotor circuit and how it could be reconfigured after lateral hemisection or contusion injuries to replicate gaits observed experimentally.

The model suggests the emergence of detour circuits after lateral hemisection, whereas after a midline contusion, the model suggests plasticity of left-right and sensory inputs below the injury.

Strengths:

The model accurately models many known connections within and between forelimb and hindlimb spinal locomotor circuits.

The simulation results mirror closely gait parameters observed experimentally. Many gait parameters were studied, as well as variability in these parameters in intact versus injured conditions.

Weaknesses:

The study could provide some sense of the relative importance of the various modified connectivities after injury in setting the changes in gait seen after the two types of injuries.

Overall, the authors achieved their aims, and the model provides solid support for the changes in connectivity after the two types of injuries were modelled. This work emphasizes specific changes in connectivity after lateral hemisection or after contusion that could be investigated experimentally. The model is available for public use and could serve as a tool to analyze the relative importance of various highlighted or previously undiscovered changes in connectivity that may underlie the recovery of locomotor function in spinalized rats.

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Reviewer #3 (Public review):

This study profiles small intestine NETs and one mixed lung NET at single cell resolution and identifies two subtypes of neuroendocrine cells, as well as explores the proliferation patterns in malignant and nonmalignant cell types, identifying MIF as a potential factor that promotes proliferation of B and plasma cells in siNETs. Furthermore, they explore the single-cell landscape of a mixed LCNEC and squamous cell carcinoma, from which they identify a putative stem cell population with expression of features from both lineages.

Strengths:

This work showcases single-cell profiling of a rare tumor type, which is very informative for the field of NETs. The authors highlight very interesting observations, including the identification of the epithelial and neuronal subtype of siNETs, which they validated with an independent bulk RNA sequencing cohort. Furthermore, the observation of low cycling in malignant cells and high cycling in nonmalignant cells is an interesting one which may be applicable to other NETs.

Weaknesses:

• The authors do not connect their findings to clinical outcome. For example, is the epithelial or neuronal subtype enriched in tumors with worse or better prognosis or high grade vs. low grade siNETs or in patients who metastasize vs. who don't? As the authors show they can identify epithelial vs. neuronal subtypes in bulk RNA seq, perhaps they can take advantage of these other studies with larger sample sizes to investigate this. Additionally, the authors identify that the phenomenon of higher B/plasma cell proliferation is particular to epithelial siNETs and write that "The implications of high B/plasma cell turnover, and of other downstream effects of high MIF expression, are unclear, but raise the possibility that MIF-CD74 interaction may constitute a relevant target for the epithelial-like SiNET subtype." However, if this interaction contributes to survival in these patients, targeting this interaction may not be beneficial. Thus, it is important for the authors to try to connect their finding to clinical outcomes to enhance the translational relevance of this paper.

• The generalizability of this study would be enhanced if the authors analyzed other available single cell studies of NETs and found a similar phenomenon of high proliferating nonmalignant cell types. Although these studies are also very limited in sample size, seeing concordance in findings across independent cohorts and different experimental techniques would help to strengthen the findings. While the authors rationalize that these other studies are too distinct from their own due to enrichment for immune cells, this limitation should be noted but does not prevent such an analysis from being attempted.

• On page 3, the authors claim that "Technical effects (e.g. single cell analysis of fresh samples vs. single nuclei analysis of frozen samples) could also impact the capture of distinct cell types, although we did not observe a clear pattern of such bias." Can the authors show that cell type frequencies are not significantly different between the samples profiled with these two methods?

• Why did siNET3 and siNET9 have much lower recovery of neuroendocrine cells compared to other samples? It would be interesting to see how similar or different the transcriptional profiles are of the samples that were obtained from the same patient, considering that multifocal siNETs are found to derive from distinct clones, although this analysis is understandably not possible in this case due to the lack of neuroendocrine cells in one of two samples from the same patient.

• It should be more clearly stated in the text that these samples were previously treated with somatostatin analogues, as this impacts the interpretation of the findings.

• The identification of a potential progenitor subtype in the miNEN is very intriguing, albeit a case study and represents a distinct cancer from the lowly proliferating siNETs. While the authors mention this in the text, the case study feels rather tangential to the other parts of the paper.

• How the authors compared the DE genes to known signatures for the fibroblast and endothelial cells should be clarified and discussed in the Methods section.

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Reviewer #3 (Public review):

In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field.

Latest comments:

As with my previous assessment, I remain supportive of publication of this manuscript. Though I agree with the other reviewers that additional experimentation would increase the value of this study even further, I feel it will also be a useful contribution to the field as is.

Reviewer #3 (Public review):

Summary:

The authors investigated a possible role of Endophilin A1 in the inhibitory postsynaptic density.

Strengths:

The authors used a broad array of experimental approaches to investigate this, including tests of seizure susceptibility, electrophysiology, biochemistry, neuronal culture and image analysis.

Weaknesses:

Many results are difficult to interpret, and data quality is not always convincing, unfortunately. The basic premise of the study, that gephyrin and endophilin A1 interact, requires more robust analysis to be convincing.

Specific comments:

The authors have made a substantial effort to improve their manuscript. A number of issues, related to numbers of observations mentioned by the reviewers, are clarified in the revised manuscript. The authors have also clarified some of the other questions from the reviewers. The long list of issues brought up by the reviewers and the many corrections needed still raise questions about data quality in this manuscript.<br /> In response to my comments (Point 2), the added experiment with PSD95.FingR and GPN.FingR in cultured neurons (Fig. S5A-D) is a good addition; the in vivo data using FingRs in Figure S3 look less convincing however. In response to my Point 5, the authors have added a cell-free binding assay (Figure 5I). This is a useful addition, but to convincingly make the point of interaction between Gephyrin and EndoA1, more rigorous biophysical quantitation of binding is needed. The legend in Figure 5I states that 4 independent experiments were performed, but the graph only shows 3 dots. This needs to be corrected.

Reviewer #3 (Public review):

Summary:

Ross et al. show that knockdown of zebrafish podocalyxin-like (podxl) by CRISPR/Cas or morpholino injection decreased the number of hepatic stellate cells (HSC). The authors then generated 5 different mutant alleles representing a range of lesions, including premature stop codons, in-frame deletion of the transmembrane domain, and deletions of the promoter region encompassing the transcription start site. However, unlike their knockdown experiment, HSC numbers did not decrease in podxl mutants; in fact, for two of the mutant alleles, the number of HSCs increased compared to the control. Injection of podxl CRISPR/Cas constructs into these mutants had no effect on HSC number, suggesting that the knockdown phenotype is not due to off-target effects but instead that the mutants are somehow compensating for the loss of podxl. The authors then present multiple lines of evidence suggesting that compensation is not exclusively due to transcriptional adaptation - evidence of mRNA instability and nonsense-mediated decay was observed in some but all mutants; expression of the related gene endoglycan (endo) was unchanged in the mutants and endo knockdown had no effect on HSC numbers; and, expression profiling by RNA sequencing did not reveal changes in other genes that share sequence similarity with podxl. Instead, their RNA-seq data showed hundreds of differentially expressed genes, especially ECM-related genes, suggesting that compensation in podxl mutants is complex and multi-genic.

Strengths:

The data presented is impressively thorough, especially in its characterization of the 5 different podxl alleles and exploration of whether these mutants exhibit transcriptional adaptation.

Weaknesses:

RNA sequencing expression profiling was done on adult livers. However, compensation of HSC numbers is apparent by 6 dpf, suggesting compensatory mechanisms would be active at larval or even embryonic stages. Although possible, it's not clear that any compensatory changes in gene expression would persist to adulthood.

Reviewer #3 (Public review):

Summary:

Recent studies have established that trypanocidal drugs, including pentamidine and melarsoprol, enter the trypanosomes via the glyceroaquaporin AQP2 (TbAQP2). Interestingly, drug resistance in trypanosomes is, at least in part, caused by recombination with the neighbouring gene, AQP3, which is unable to permeate pentamidine or melarsoprol. The effect of the drugs on cells expressing chimeric proteins is significantly reduced. In addition, controversy exists regarding whether TbAQP2 permeates drugs like an ion channel, or whether it serves as a receptor that triggers downstream processes upon drug binding. In this study the authors set out to achieve three objectives:<br /> (1) to determine if TbAQP2 acts as a channel or a receptor,<br /> (2) to understand the molecular interactions between TbAQP2 and glycerol, pentamidine, and melarsoprol, and<br /> (3) to determine the mechanism by which mutations that arise from recombination with TbAQP3 result in reduced drug permeation.

Indeed, all three objectives are achieved in this paper. Using MD simulations and cryo-EM, the authors determine that TbAQP2 likely permeates drugs like an ion channel. The cryo-EM structures provide details of glycerol and drug binding, and show that glycerol and the drugs occupy the same space within the pore. Finally, MD simulations and lysis assays are employed to determine how mutations in TbAQP2 result in reduced permeation of drugs by making entry and exit of the drug relatively more energy-expensive. Overall, the strength of evidence used to support the author's claims is solid.

Strengths:

The cryo-EM portion of the study is strong, and while the overall resolution of the structures is in the 3.5Å range, the local resolution within the core of the protein and the drug binding sites is considerably higher (~2.5Å).

I also appreciated the MD simulations on the TbAQP2 mutants and the mechanistic insights that resulted from this data.

Weaknesses:

(1) The authors do not provide any empirical validation of the drug binding sites in TbAQP2. While the discussion mentions that the binding site should not be thought of as a classical fixed site, the MD simulations show that there's an energetically preferred slot (i.e., high occupancy interactions) within the pore for the drugs. For example, mutagenesis and a lysis assay could provide us with some idea of the contribution/importance of the various residues identified in the structures to drug permeation. This data would also likely be very valuable in learning about selectivity for drugs in different AQP proteins.

(2) Given the importance of AQP3 in the shaping of AQP2-mediated drug resistance, I think a figure showing a comparison between the two protein structures/AlphaFold structures would be beneficial and appropriate.

(3) A few additional figures showing cryo-EM density, from both full maps and half maps, would help validate the data.

(4) Finally, this paper might benefit from including more comparisons with and analysis of data published in Chen et al (doi.org/10.1038/s41467-024-48445-4), which focus on similar objectives. Looking at all the data in aggregate might reveal insights that are not obvious from either paper on their own. For example, melarsoprol binds differently in structures reported in the two respective papers, and this may tell us something about the energy of drug-protein interactions within the pore.

Reviewer #3 (Public review):

Summary:

In summary, the scientists used Visium spatial transcriptomics technology to create a thorough spatial transcriptomic atlas of the adult male mouse adrenal gland and the adipose tissues that surround it. Their primary goals were to map the cell communication network, determine the differentiation direction of various cell types, and find marker genes for various adrenal zones.

Strengths:

(1) Undoubtedly, one of the biggest strengths of the manuscript is a spatial transcriptomic o mouse adrenal gland tissue, which, to my knowledge, has not been done before.

(2) Comprehensive Zonal Characterization: Seven distinct clusters were identified, corresponding to known anatomical and functional regions (ZG, ZF, ZX, medulla, connective tissue, brown and white adipose tissue), each with robust marker gene sets.

(3) The authors manage to integrate advanced bioinformatical tools such as CellChatDB, Monocle3, and CARD to study the relationship between cell types and differentiation of the tissue.

(4) The authors manage to identify novel marker genes for some adrenal zones.

Weaknesses:

(1) The study focused only on one adult male CD1 IGS mouse, which is a limiting factor for other strains, ages, or females, especially given the sexual dimorphism of the ZX. Although the authors claim that four slices of the adrenal gland have been processed on Visium and sequenced, for "clarity," they show only one, which might bias the results.

(2) Lack of detailed QC analysis of the Visium slide.

(3) The study misses the functional validation of the novel marker genes - this needs to be addressed.

(4) What worries me a lot is the fact that, actually, there might be more than one cell present within a Visium spot, so the only way to define zones is by anatomical observation rather than cellular composition.

(5) In cell chat analysis, the authors show the strength of the interactions, but miss out on the number of interactions.

Conclusions:

The authors' stated goals were mostly accomplished:

By mapping the mouse adrenal gland's molecular landscape, they were able to clearly establish unique molecular signatures for every anatomical zone.

Pseudotime study of the cell progression from the capsule through ZG, ZF, and ZX demonstrates that the data strongly support the centripetal differentiation concept. Conclusions on the functional importance of newly discovered marker genes are conjectural and need additional experimental support.

Nevertheless, several findings are still tentative and will need more experimental support, especially when it comes to the significance of ZX persistence and the functional involvement of recently discovered marker genes.

DOI: 10.1016/j.devcel.2025.06.015

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:ZFIN_ZDB-ALT-120810-3

What is this?

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Reviewer #3 (Public review):

Summary:

One goal of this paper is to introduce a new approach for highly accurate decoding of finger movements from human magnetoencephalography data via dimension reduction of a "multi-scale, hybrid" feature space. Following this decoding approach, the authors aim to show that early skill learning involves "contextualization" of the neural coding of individual movements, relative to their position in a sequence of consecutive movements. Furthermore, they aim to show that this "contextualization" develops primarily during short rest periods interspersed with skill training, and correlates with a performance metric which the authors interpret as an indicator of offline learning.

Strengths:

A strength of the paper is the innovative decoding approach, which achieves impressive decoding accuracies via dimension reduction of a "multi-scale, hybrid space". This hybrid-space approach follows the neurobiologically plausible idea of concurrent distribution of neural coding across local circuits as well as large-scale networks.

Weaknesses:

A clear weakness of the paper lies in the authors' conclusions regarding "contextualization". Several potential confounds, which partly arise from the experimental design, and which are described below, question the neurobiological implications proposed by the authors, and offer a simpler explanation of the results. Furthermore, the paper follows the assumption that short breaks result in offline skill learning, while recent evidence casts doubt on this assumption.

Specifically:

The authors interpret the ordinal position information captured by their decoding approach as a reflection of neural coding dedicated to the local context of a movement (Figure 4). One way to dissociate ordinal position information from information about the moving effectors is to train a classifier on one sequence, and test the classifier on other sequences that require the same movements, but in different positions (Kornysheva et al., Neuron 2019). In the present study, however, participants trained to repeat a single sequence (4-1-3-2-4). As a result, ordinal position information is potentially confounded by the fixed finger transitions around each of the two critical positions (first and fifth press). Across consecutive correct sequences, the first keypress in a given sequence was always preceded by a movement of the index finger (=last movement of the preceding sequence), and followed by a little finger movement. The last keypress, on the other hand, was always preceded by a ring finger movement, and followed by an index finger movement (=first movement of the next sequence). Figure 3 - supplement 5 shows that finger identity can be decoded with high accuracy (>70%) across a large time window around the time of the keypress, up to at least {plus minus}100 ms (and likely beyond, given that decoding accuracy is still high at the boundaries of the window depicted in that figure). This time window approaches the keypress transition times in this study. Given that distinct finger transitions characterized the first and fifth keypress, the classifier could thus rely on persistent (or "lingering") information from the preceding finger movement, and/or "preparatory" information about the subsequent finger movement, in order to dissociate the first and fifth keypress. Currently, the manuscript provides little evidence that the context information captured by the decoding approach is more than a by-product of temporally extended, and therefore overlapping, but independent neural representations of consecutive keypresses that are executed in close temporal proximity - rather than a neural representation dedicated to context.

During the review process, the authors pointed out that a "mixing" of temporally overlapping information from consecutive keypresses, as described above, should result in systematic misclassifications and therefore be detectable in the confusion matrices in Figures 3C and 4B, which indeed do not provide any evidence that consecutive keypresses are systematically confused. However, such absence of evidence (of systematic misclassification) should be interpreted with caution. The authors also reported that there was only a weak relation between inter-press intervals and "online contextualization" (Figure 5 - figure supplement 6), however, their analysis suprisingly includes a keypress transition that is shared between OP1 and OP5 ("4-4"), rather than focusing solely on the two distinctive transitions ("2-4" and "4-1").

Such temporal overlap of consecutive, independent finger representations may also account for the dynamics of "ordinal coding"/"contextualization", i.e., the increase in 2-class decoding accuracy, across Day 1 (Figure 4C). As learning progresses, both tapping speed and the consistency of keypress transition times increase (Figure 1), i.e., consecutive keypresses are closer in time, and more consistently so. As a result, information related to a given keypress is increasingly overlapping in time with information related to the preceding and subsequent keypresses. Furthermore, learning should increase the number of (consecutively) correct sequences, and, thus, the consistency of finger transitions. Therefore, the increase in 2-class decoding accuracy may simply reflect an increasing overlap in time of increasingly consistent information from consecutive keypresses, which allows the classifier to dissociate the first and fifth keypress more reliably as learning progresses, simply based on the characteristic finger transitions associated with each. In other words, given that the physical context of a given keypress changes as learning progresses - keypresses move closer together in time, and are more consistently correct - it seems problematic to conclude that the mental representation of that context changes. During the review process, authors pointed at absence of evidence of a relation between tapping speed and "ordinal coding" (Figure 5 - figure supplement 7). However, a rigorous test of the idea that the mental representation of context changes would require a task design in which the physical context remains constant.

A similar difference in physical context may explain why neural representation distances ("differentiation") differ between rest and practice (Figure 5). The authors define "offline differentiation" by comparing the hybrid space features of the last index finger movement of a trial (ordinal position 5) and the first index finger movement of the next trial (ordinal position 1). However, the latter is not only the first movement in the sequence, but also the very first movement in that trial (at least in trials that started with a correct sequence), i.e., not preceded by any recent movement. In contrast, the last index finger of the last correct sequence in the preceding trial includes the characteristic finger transition from the fourth to the fifth movement. Thus, there is more overlapping information arising from the consistent, neighbouring keypresses for the last index finger movement, compared to the first index finger movement of the next trial. A strong difference (larger neural representation distance) between these two movements is, therefore, not surprising, given the task design, and this difference is also expected to increase with learning, given the increase in tapping speed, and the consequent stronger overlap in representations for consecutive keypresses.

A further complication in interpreting the results stems from the visual feedback that participants received during the task. Each keypress generated an asterisk shown above the string on the screen. It is not clear why the authors introduced this complicating visual feedback in their task, besides consistency with their previous studies. The resulting systematic link between the pattern of visual stimulation (the number of asterisks on the screen) and the ordinal position of a keypress makes the interpretation of "contextual information" that differentiates between ordinal positions difficult. While the authors report the surprising finding that their eye-tracking data could not predict asterisk position on the task display above chance level, the mean gaze position seemed to vary systematically as a function of ordinal position of a movement - see Figure 4 - figure supplement 3.

The authors report a significant correlation between "offline differentiation" and cumulative micro-offline gains. However, to reach the conclusion that "the degree of representational differentiation -particularly prominent over rest intervals - correlated with skill gains.", the critical question is rather whether "offline differentiation" correlates with micro-offline gains (not with cumulative micro-offline gains). That is, does the degree to which representations differentiate "during" a given rest period correlate with the degree to which performance improves from before to after the same rest period (not: does "offline differentiation" in a given rest period correlate with the degree to which performance has improved "during" all rest periods up to the current rest period - but this is what Figure 5 - figure supplements 1 and 4 show).

The authors follow the assumption that micro-offline gains reflect offline learning. However, there is no compelling evidence in the literature, and no evidence in the present manuscript, that micro-offline gains (during any training phase) reflect offline learning. Instead, emerging evidence in the literature indicates that they do not (Das et al., bioRxiv 2024), and instead reflect transient performance benefits when participants train with breaks, compared to participants who train without breaks, however, these benefits vanish within seconds after training if both groups of participants perform under comparable conditions (Das et al., bioRxiv 2024). During the review process, the authors argued that differences in the design between Das et al. (2024) on the one hand (Experiments 1 and 2), and the study by Bönstrup et al. (2019) on the other hand, may have prevented Das et al. (2024) from finding the assumed (lasting) learning benefit by micro-offline consolidation. However, the Supplementary Material of Das et al. (2024) includes an experiment (Experiment S1) whose design closely follows the early learning phase of Bönstrup et al. (2019), and which, nevertheless, demonstrates that there is no lasting benefit of taking breaks for the acquired skill level, despite the presence of micro-offline gains.

Along these lines, the authors argue that their practice schedule "minimizes reactive inhibition effects", in particular their short practice periods of 10 seconds each. However, 10 seconds are sufficient to result in motor slowing, as report in Bächinger et al., elife 2019, or Rodrigues et al., Exp Brain Res 2009.

An important conceptual problem with the current study is that the authors conclude that performance improves, and representation manifolds differentiate, "during" rest periods. However, micro-offline gains (as well as offline contextualization) are computed from data obtained during practice, not rest, and may, thus, just as well reflect a change that occurs "online", e.g., at the very onset of practice (like pre-planning) or throughout practice (like fatigue, or reactive inhibition).

The authors' conclusion that "low-frequency oscillations (LFOs) result in higher decoding accuracy compared to other narrow-band activity" should be taken with caution, given that the critical decoding analysis for this conclusion was based on data averaged across a time window of 200 ms (Figure 2), essentially smoothing out higher frequency components.

Reviewer #3 (Public review):

Summary:

In this study, the authors have developed a new Ca indicator conjugated to the peptide, which likely recognizes synaptic ribbons and have measured microdomain Ca near synaptic ribbons at retinal bipolar cells. This interesting approach allows one to measure Ca close to transmitter release sites, which may be relevant for synaptic vesicle fusion and replenishment. Though microdomain Ca at the active zone of ribbon synapses has been measured by Hudspeth and Moser, the new study uses the peptide recognizing synaptic ribbons, potentially measuring the Ca concentration relatively proximal to the release sites.

Strengths:

The study is, in principle, technically well done, and the peptide approach is technically interesting, which allows one to image Ca near the particular protein complexes. The approach is potentially applicable to other types of imaging.

Weaknesses:

Peptides may not be entirely specific, and genetic approach tagging particular active zone proteins with fluorescent Ca indicator proteins may well be more specific. Although the authors are aware of this and the peptide approach is generally used for ribbon synapses, the authors should be aware of this, when interpreting the results.

Reviewer #3 (Public review):

Summary:

Ribbon synapses are complex molecular assemblies responsible for synaptic vesicle trafficking in sensory cells of the eye and the inner ear. The Ca2+-dependent exocytosis occurs at the active zone (AZ), however, the molecular mechanisms orchestrating the structure and function of the AZs of ribbon synapses are not well understood. To advance in the understanding of those mechanisms, the authors present a novel and interesting experimental strategy pursuing the reconstitution of a minimal active zone of a ribbon synapse within a synapse-naïve cell line: HEK293 cells. The authors have used stably transfected HEK293 cells that express voltage-gated Ca2+ channels subunits (constitutive -CaV beta3 and CaV alpha2 beta1- and inducible CaV1.3 alpha1). They have expressed in those cells several proteins of the ribbon synapse active zone: (1) RIBEYE, (2) a modified version of Bassoon that binds to the plasma membrane through artificial palmitoylation (Palm-Bassoon) and (3) RIM-binding protein 2 (RBP2) to induce the formation of a minimal active zone that they called SyRibbons. The formation of such structures is convincing, however, the evidence of such structures having a functional impact (for example enhancing Ca2+-currents), as the authors claim, is weak. In conclusion, the novel approach shows that expression of a multiprotein complex partially reproduces properties, especially structural properties, of ribbon-type active zones in a heterologous system. Although the approach opens interesting possibilities for further experiments, the evidence supporting the functional properties of the so called "synthetic ribbon synapses" is incomplete.

Strengths of the study:

(1) The study is carefully carried out using a remarkable combination of (1) superresolution, correlative light microscopy and cryo-electron tomography, to analyze the formation and subcellular distribution of molecular assemblies and (2) functional assessment of voltage-gated Ca2+ channels using patch-clamp recording of Ca2+-currents and fluorometry to correlate Ca2+ influx with the molecular assemblies formed by AZ proteins. The results are of high quality and are in general accompanied of required control experiments.<br /> (2) The method opens new opportunities to further investigate the minimal and basic properties of AZ proteins that are difficult to study using in vivo systems. The cells that operate through ribbon synapses (e.g. photoreceptors and hair cells) are particularly difficult to manipulate, so setting up and validating the use of a heterologous system more suitable for molecular manipulations is highly valuable.<br /> (3) The structures formed by RIBEYE and Palm-Bassoon in HEK293 cells identified by STED nanoscopy and cryo-electron microscopy share relevant similarities similar to the AZs of ribbon synapses found in rat inner hair cells.

Weaknesses of the study:

(1) The evidence of the functional properties of the "synthetic ribbon-type active zones" has been only assessed by its effect on the modulation of Ca2+-channel function, and that effect is rather weak. The authors provide reasonable explanations regarding such a weak effect but, however, it is difficult to conclude that indeed the "synthetic ribbon-type active zones" are bona fide functional multiprotein complexes.

Reviewer #2 (Public review):

Summary

The report by Dalas and colleagues introduces a significant novelty in the field of pentameric ligand-gated ion channels (pLGICs). Within this family of receptors, numerous structures are available, but a widely recognised problem remains in assigning structures to functional states observed in biological membranes. Here, the authors obtain both structural and functional information of a pLGIC in a liposome environment. The model receptor ELIC is captured in the resting, desensitised and open states. Structures in large nanodiscs, possibly biased by receptor-scaffold protein interactions, are also reported. Altogether these results set the stage for the adoption of liposomes as a proxy for the biological membranes, for cryoEM studies of pLGICs and membrane proteins in general.

Strengths

The structural data is comprehensive, with structures in liposomes in the 3 main states (and for each, both inward-facing and outward-facing), and an agonist-bound structure in the large spNW25 nanodisc (and a retreatment of previous data obtained in a smaller disc). It adds up to a series of work from the same team that constitutes a much-needed exploration of various types of environment for the transmembrane domain of pLGICs. The structural analysis is thorough.<br /> The tone of the report is particularly pleasant, in the sense that the authors' claims are not inflated. For instance, a sentence such as "By performing structural and functional characterization under the same reconstitution conditions, we increase our confidence in the functional annotation of these structures." is exemplary.

Weakness

All the details necessary to reproduce the work are present in the Methods. Nevertheless, the biochemistry might have been shown and discussed in greater details. While I do believe that liposomes will be in most cases better than, say, nanodiscs, the process that leads from the protein in its membrane down to the liposome will play a big role in preserving the native structure.

Reviewer #3 (Public review):

A bias in how people infer the amount of control they have over their environment is widely believed to be a key component of several mental illnesses including depression, anxiety, and addiction. Accordingly, this bias has been a major focus in computational models of those disorders. However, all of these models treat control as a unidimensional property, roughly, how strongly outcomes depend on action. This paper proposes---correctly, I think---that the intuitive notion of "control" captures multiple dimensions in the relationship between action and outcome. In particular, the authors identify one key dimension: the degree to which outcome depends on how much *effort* we exert, calling this dimension the "elasticity of control". They additionally argue that this dimension (rather than the more holistic notion of controllability) may be specifically impaired in certain types of psychopathology. This idea has the potential to change how we think about several major mental disorders in a substantial way and can additionally help us better understand how healthy people navigate challenging decision-making problems. More concisely, it is a very good idea.

Unfortunately, my view is that neither the theoretical nor empirical aspects of the paper really deliver on that promise. In particular, most (perhaps all) of the interesting claims in the paper have weak empirical support.

Starting with theory, the authors do not provide a strong formal characterization of the proposed notion of elasticity. There are existing, highly general models of controllability (e.g., Huys & Dayan, 2009; Ligneul, 2021) and the elasticity idea could naturally be embedded within one of these frameworks. The authors gesture at this in the introduction; however, this formalization is not reflected in the implemented model, which is highly task-specific. Moreover, the authors present elasticity as if it is somehow "outside of" the more general notion of controllability. However, effort and investment are just specific dimensions of action; and resources like money, strength, and skill (the "highly trained birke") are just specific dimensions of state. Accordingly, the notion of elasticity is necessarily implicitly captured by the standard model. Personally, I am compelled by the idea that effort and resource (and therefore elasticity) are particularly important dimensions, ones that people are uniquely tuned to. However, by framing elasticity as a property that is different in kind from controllability (rather than just a dimension of controllability), the authors only make it more difficult to integrate this exciting idea into generalizable models.

Turning to experiment, the authors make two key claims: (1) people infer the elasticity of control, and (2) individual differences in how people make this inference are importantly related to psychopathology.

Starting with claim 1, there are three subclaims here; implicitly, the authors make all three. (1A) People's behavior is sensitive to differences in elasticity, (1B) people actually represent/track something like elasticity, and (1C) people do so naturally as they go about their daily lives. The results clearly support 1A. However, 1B and 1C are not strongly supported.

(1B) The experiment cannot support the claim that people represent or track elasticity because effort is the only dimension over which participants can engage in any meaningful decision-making. The other dimension, selecting which destination to visit, simply amounts to selecting the location where you were just told the treasure lies. Thus, any adaptive behavior will necessarily come out in a sensitivity to how outcomes depend on effort.

Notes on rebuttal: The argument that vehicle/destination choice is not trivial because people occasionally didn't choose the instructed location is not compelling to me-if anything, the exclusion rate is unusually low for online studies. The finding that people learn more from non-random outcomes is helpful, but this could easily be cast as standard model-based learning very much like what one measures with the Daw two-step task (nothing specific to control here). Their final argument is the strongest, that to explain behavior the model must assume "a priori that increased effort could enhance control." However, more literally, the necessary assumption is that each attempt increases the probability of success-e.g. you're more likely to get a heads in two flips than one. I suppose you can call that "elasticity inference", but I would call it basic probabilistic reasoning.

For 1C, the claim that people infer elasticity outside of the experimental task cannot be supported because the authors explicitly tell people about the two notions of control as part of the training phase: "To reinforce participants' understanding of how elasticity and controllability were manifested in each planet, [participants] were informed of the planet type they had visited after every 15 trips." (line 384).

Notes on rebuttal: The authors try to retreat, saying "our research question was whether people can distinguish between elastic and inelastic controllability." I struggle to reconcile this with the claim in the abstract "These findings establish the elasticity of control as a distinct cognitive construct guiding adaptive behavior". That claim is the interesting one, and the one I am evaluating the evidence in light of.

Finally, I turn to claim 2, that individual differences in how people infer elasticity are importantly related to psychopathology. There is much to say about the decision to treat psychopathology as a unidimensional construct (the authors claim otherwise, but see Fig 6C). However, I will keep it concrete and simply note that CCA (by design) obscures the relationship between any two variables. Thus, as suggestive as Figure 6B is, we cannot conclude that there is a strong relationship between Sense of Agency (SOA) and the elasticity bias---this result is consistent with any possible relationship (even a negative one). As it turns out, Figure S3 shows that there is effectively no relationship (r=0.03).

Notes on rebuttal: The authors argue for CCA by appeal to the need to "account for the substantial variance that is typically shared among different forms of psychopathology". I agree. A simple correlation would indeed be fairly weak evidence. Strong evidence would show a significant correlation after *controlling for* other factors (e.g. a regression predicting elasticity bias from all subscales simultaneously). CCA effectively does the opposite, asking whether-with the help of all the parameters and all the surveys-one can find any correlation between the two sets of variables. The results are certainly suggestive, but they provide very little statistical evidence that the elasticity parameter is meaningfully related to any particular dimension of psychopathology.

There is also a feature of the task that limits our ability to draw strong conclusions about individual differences about elasticity inference. In the original submission, the authors stated that the study was designed to be "especially sensitive to overestimation of elasticity". A straightforward consequence of this is that the resulting *empirical* estimate of estimation bias (i.e., the gamma_elasticity parameter) is itself biased. This immediately undermines any claim that references the directionality of the elasticity bias (e.g. in the abstract). Concretely, an undirected deficit such as slower learning of elasticity would appear as a directed overestimation bias.

When we further consider that elasticity inference is the only meaningful learning/decision-making problem in the task (argued above), the situation becomes much worse. Many general deficits in learning or decision-making would be captured by the elasticity bias parameter. Thus, a conservative interpretation of the results is simply that psychopathology is associated with impaired learning and decision-making.

Notes on rebuttal: I am very concerned to see that the authors removed the discussion of this limitation in response to my first review. I quote the original explanation here:

- In interpreting the present findings, it needs to be noted that we designed our task to be especially sensitive to overestimation of elasticity. We did so by giving participants free 3 tickets at their initial visits to each planet, which meant that upon success with 3 tickets, people who overestimate elasticity were more likely to continue purchasing extra tickets unnecessarily. Following the same logic, had we first had participants experience 1 ticket trips, this could have increased the sensitivity of our task to underestimation of elasticity in elastic environments. Such underestimation could potentially relate to a distinct psychopathological profile that more heavily loads on depressive symptoms. Thus, by altering the initial exposure, future studies could disambiguate the dissociable contributions of overestimating versus underestimating elasticity to different forms of psychopathology.

The logic of this paragraph makes perfect sense to me. If you assume low elasticity, you will infer that you could catch the train with just one ticket. However, when elasticity is in fact high, you would find that you don't catch the train, leading you to quickly infer high elasticity-eliminating the bias. In contrast, if you assume high elasticity, you will continue purchasing three tickets and will never have the opportunity to learn that you could be purchasing only one-the bias remains.

The authors attempt to argue that this isn't happening using parameter recovery. However, they only report the *correlation* in the parameter, whereas the critical measure is the *bias*. Furthermore, in parameter recovery, the data-generating and data-fitting models are identical-this will yield the best possible recovery results. Although finding no bias in this setting would support the claims, it cannot outweigh the logical argument for the bias that they originally laid out. Finally, parameter recovery should be performed across the full range of plausible parameter values; using fitted parameters (a detail I could only determine by reading the code) yields biased results because the fitted parameters are themselves subject to the bias (if present). That is, if true low elasticity is inferred as high elasticity, then you will not have any examples of low elasticity in the fitted parameters and will not detect the inability to recover them.

Minor comments:

Below are things to keep in mind.

The statistical structure of the task is inconsistent with the framing. In the framing, participants can make either one or two second boarding attempts (jumps) by purchasing extra tickets. The additional attempt(s) will thus succeed with probability p for one ticket and 2p - p^2 for two tickets; the p^2 captures the fact that you only take the second attempt if you fail on the first. A consequence of this is buying more tickets has diminishing returns. In contrast, in the task, participants always jumped twice after purchasing two tickets, and the probability of success with two tickets was exactly double that with one ticket. Thus, if participants are applying an intuitive causal model to the task, they will appear to "underestimate" the elasticity of control. I don't think this seriously jeopardizes the key results, but any follow-up work should ensure that the task's structure is consistent with the intuitive causal model.

The model is heuristically defined and does not reflect Bayesian updating. For example, it over-estimates maximum control by not using losses with less than 3 tickets (intuitively, the inference here depends on what your beliefs about elasticity). Including forced three-ticket trials at the beginning of each round makes this less of an issue; but if you want to remove those trials, you might need to adjust the model. The need to introduce the modified model with kappa is likely another symptom of the heuristic nature of the model updating equations.

Reviewer #3 (Public review):

Summary:

This paper by Meier et al introduces a new optogenetic module for the regulation of bacterial gene expression based on "bathy-BphP" proteins. Their paper begins with a careful characterization of kinetics and pH dependence of a few family members, followed by extensive engineering to produce infrared-regulated transcriptional systems based on the authors' previous design of the pDusk and pDERusk systems, and closing with characterization of the systems in bacterial species relevant for biotechnology.

Strengths:

The paper is important from the perspective of fundamental protein characterization, since bathy-BphPs are relatively poorly characterized compared to their phytochrome and cyanobacteriochrome cousins. It is also important from a technology development perspective: the optogenetic toolbox currently lacks infrared-stimulated transcriptional systems. Infrared light offers two major advantages: it can be multiplexed with additional tools, and it can penetrate into deep tissues with ease relative to the more widely used blue light-activated systems. The experiments are performed carefully, and the manuscript is well written.

Weaknesses:

My major criticism is that some information is difficult to obtain, and some data is presented with limited interpretation, making it difficult to obtain intuition for why certain responses are observed. For example, the changes in red/infrared responses across different figures and cellular contexts are reported but not rationalized. Extensive experiments with variable linker sequences were performed, but the rationale for linker choices was not clearly explained. These are minor weaknesses in an overall very strong paper.

Reviewer #3 (Public review):

Summary:

The paper studies learning rules in a simple sigmoidal recurrent neural network setting. The recurrent network has a single layer of 10 to 40 units. It is first confirmed that feedback alignment (FA) can learn a value function in this setting. Then so-called bio-plausible constraints are added: (1) when value weights (readout) is non-negative, (2) when the activity is non-negative (normal sigmoid rather than downscaled between -0.5 and 0.5), (3) when the feedback weights are non-negative, (4) when the learning rule is revised to be monotic: the weights are not downregulated. In the simple task considered all four biological features do not appear to impair totally the learning.

Strengths:

(1) The learning rules are implemented in a low-level fashion of the form: (pre-synaptic-activity) x (post-synaptic-activity) x feedback x RPE. Which is therefore interpretable in terms of measurable quantities in the wet-lab.

(2) I find that non-negative FA (FA with non negative c and w) is the most valuable theoretical insight of this paper: I understand why the alignment between w and c is automatically better at initialization.

(3) The task choice is relevant, since it connects with experimental settings of reward conditioning with possible plasticity measurements.

Weaknesses:

(4) The task is rather easy, so it's not clear that it really captures the computational gap that exists with FA (gradient-like learning) and simpler learning rule like a delta rule: RPE x (pre-synpatic) x (post-synaptic). To control if the task is not too trivial, I suggest adding a control where the vector c is constant c_i=1.

(5) Related to point 3), the main strength of this paper is to draw potential connection with experimental data. It would be good to highlight more concretely the prediction of the theory for experimental findings. (Ideally, what should be observed with non-negative FA that is not expected with FA or a delta rule (constant global feedback) ?).

(6a) Random feedback with RNN in RL have been studied in the past, so it is maybe worth giving some insights how the results and the analyzes compare to this previous line of work (for instance in this paper [1]). For instance, I am not very surprised that FA also works for value prediction with TD error. It is also expected from the literature that the RL + RNN + FA setting would scale to tasks that are more complex than the conditioning problem proposed here, so is there a more specific take-home message about non-negative FA? or benefits from this simpler toy task?

(6b) Related to task complexity, it is not clear to me if non-negative value and feedback weights would generally scale to harder tasks. If the task in so simple that a global RPE signal is sufficient to learn (see 4 and 5), then it could be good to extend the task to find a substantial gap between: global RPE, non-negative FA, FA, BP. For a well chosen task, I expect to see a performance gap between any pair of these four learning rules. In the context of the present paper, this would be particularly interesting to study the failure mode of non-negative FA and the cases where it does perform as well as FA.

(7) I find that the writing could be improved, it mostly feels more technical and difficult than it should. Here are some recommendations:<br /> 7a) For instance, the technical description of the task (CSC) is not fully described and requires background knowledge from other paper which is not desirable.<br /> 7b) Also the rationale for the added difficulty with the stochastic reward and new state is not well explained.<br /> 7c) In the technical description of the results I find that the text dives into descriptive comments of the figures but high-level take home messages would be helpful to guide the reader. I got a bit lost, although I feel that there is probably a lot of depth in these paragraphs.

(8) Related to the writing issue and 5), I wished that "bio-plausibility" was not the only reason to study positive feedback and value weights. Is it possible to develop a bit more specifically what and why this positivity is interesting? Is there an expected finding with non-negative FA both in the model capability? or maybe there is a simpler and crisp take-home message to communicate the experimental predictions to the community would be useful?

[1] https://www.nature.com/articles/s41467-020-17236-y

Comments on revisions:

Thank you for addressing all my comments in your reply.

Reviewer #3 (Public review):

Summary:

Gouirand et al explore the function of Layilin on Treg in the context of psoriasis using both patient samples and a conditional mutant mouse model. They perform functional analysis in the patient samples using Cas9-mediated deletion. The authors suggest that Layilin works in concert with integrins to bind collagen IV to attenuate cell movement.

The work is well done and built on solid human data. The report is a modest advance from the authors' previous report in 2021 that focused on tumor responses, with this report focusing on psoriasis. There are some experimental concerns that should be considered.

Strengths:

(1) Good complementation of patient and animal model data.

(2) Solid experimentation using state-of-the-art approaches.

(3) There is clearly a biological effect of LAYN deficiency in the mouse model.

(4) The report adds some new information to what was already known from the previous reports.

Weaknesses:

(1) It is not clear that the assays used for functional analysis of the patient samples were optimal.

(2) Several conclusions are not fully substantiated.

(3) The report is lacking some experimental details.

Reviewer #3 (Public review):

This revised paper develops and characterizes a new approach for screening drugs for epilepsy. The idea is to increase the ability to study seizures in animals with epilepsy because most animal models have rare seizures. Thus, the authors use the existing intrahippocampal kainic acid (IHKA) mouse model, which can have very unpredictable seizures with long periods of time between seizures. This approach is of clear utility to researchers who may need to observe many seizure events per mouse during screening of antiseizure medications. A key strength is also that more utility can be derived from each individual mouse. The authors modified the IHKA model to inject KA into CA3 instead of CA1 in order to preserve the CA1 pyramidal cells that they will later stimulate. To express the excitatory opsin channelrhodopsin (ChR2) in area CA1, they use a virus that expresses ChR2 in cells that express the Thy-1 promoter. The authors demonstrate that CA3 delivery of KA can induce a very similar chronic epilepsy phenotype to the injection of KA in CA1 and show that optical excitation of CA1 can reliably induce seizures. The authors evaluate the impact of repeated stimulation on the reliability of seizure induction and show that seizures can be reliably induced by CA1 stimulation, at least for the short term (up to 16 days). These are strengths of the study.

However, there are several limitations: the seizures are evoked, not spontaneous. It is not clear how induced seizures can be used to investigate if antiseizure medication can reduce spontaneous seizures. Although seizure inducibility and severity can be assessed, the lack of spontaneous seizures is a limitation. To their credit, the authors show that electrophysiological signatures of induced vs spontaneous seizures are similar in many ways, but the authors also show several differences. Notably, the induced seizures are robustly inhibited by the antiseizure medication levetiracetam and variably but significantly inhibited by diazepam, similar to many mouse models with chronic recurrent seizure activity. One also wonders if using a mouse model with numerous seizures (such as the pilocarpine model) might be more efficient than using a modified IHKA protocol.

In this revised manuscript, the authors address some previous concerns related to definitions of seizures and events that are trains of spikes, sex as a biological variable, and present new images of ChR2 expression (but these images could be improved to see the cells more clearly). A few key concerns remain unaddressed, however. For example, it is still not clear that evoked seizures triggered by stimulating CA1 are similar to spontaneous seizures, regardless of the idea that CA1 plays a role in seizure disorders. It also remains unclear whether repeated activation of the hippocampal circuit will result in additional alterations to this circuit that affect the seizure phenotype over prolonged intervals (after 16 days). Furthermore, the use of SVM with the number of seizures being used as replicates (instead of number of mice) is inappropriate. Another theoretical concern is whether the authors are correct in suggesting that one will be able to re-use the mice for screening multiple drugs in a row.

Strengths:<br /> - The authors show that the IHKA model of chronic epilepsy can be modified to preserve CA1 pyramidal cells, allowing optogenetic stimulation of CA1 to trigger seizures.<br /> - The authors show that repeated optogenetic stimulation of CA1 in untreated mice can promote kindling and induce seizures, indeed generating two mouse models in total.<br /> - Many electrophysiological signatures are similar between the induced and spontaneous seizures, and induced seizures reliably respond to treatment with antiseizure medications.<br /> - Given that more seizures can be observed per mouse using on-demand optogenetics, this model enhances the utility of each individual mouse.<br /> - Mice of each sex were used.

Weaknesses:<br /> - Evaluation of seizure similarity using the SVM modeling and clustering is not sufficiently justified when using number of seizures as the statistical replicate (vs mice).<br /> - Related to the first concern, the utility of increasing number of seizures for enhancing statistical power is limited because standard practice is for sample size to be numbers of mice.<br /> - The term "seizure burden" usually refers to the number of spontaneous seizures per day, not the severity of the seizures themselves. Because the authors are evoking the seizures being studied, this study design precludes assessment of seizure burden.<br /> - It seems likely that repeatedly inducing seizures will have a long-term effect, especially in light of the downward slope at day 13-16 for induced seizures seen in Figure 4C. A duration of evaluation that is longer than 16 days is warranted.<br /> - Human epilepsy is extensively heterogeneous in both etiology and individual phenotype, and it may be hard to generalize the approach.

Reviewer #3 (Public review):

Summary:

Zawieja et al. aimed to identify the pacemaker cells in the lymphatic collecting vessels. Authors have used various Cre-based expression systems and optogentic tools to identify these cells. Their findings suggest these cells are lymphatic muscle cells that drive the pacemaker activity in the lymphatic collecting vessels.

Strengths:

The authors have used multiple approaches to test their hypothesis. Some findings are presented as qualitative images, while some quantitative measurements are provided.

Weaknesses:<br /> - More quantitative measurements.<br /> - Possible mechanisms associated with the pacemaker activity.<br /> - Membrane potential measurements.

Comments on revisions: I do not have any additional comments.

Reviewer #3 (Public review):

The introduction does a very good job of discussing the issue around whether there is ongoing replication in people with HIV on antiretroviral therapy. Sporadic, non-sustained replication likely occurs in many PWH on ART related to adherence, drug-drug interactions and possibly penetration of antivirals into sanctuary areas of replication and as the authors point out proving it does not occur is likely not possible and proving it does occur is likely very dependent on the population studied and the design of the intervention. Whether the consequences of this replication in the absence of evolution toward resistance have clinical significance challenging question to address.

It is important to note that INSTI-based therapy may have a different impact on HIV replication events that results in differences in virus release for specific cell type (those responsible for "second phase" decay) by blocking integration in cells that have completed reverse transcription prior to ART initiation but have yet to be fully activated. In a PI or NNRTI-based regimen, those cells will release virus, whereas with an INSTI-based regimen, they will not.

Given the very small sample size, there is a substantial risk of imbalance between the groups in important baseline measures. Unfortunately, with the small sample size, a non-significant P value is not helpful when comparing baseline measures between groups. One suggestion would be to provide the full range as opposed to the inter-quartile range (essentially only 5 or 6 values). The authors could also report the proportion of participants with baseline HIV RNA target not detected in the two groups.

A suggestion that there is a critical imbalance between groups is that the control group has significantly lower total HIV DNA in PBMC, despite the small sample size. The control group also has numerically longer time of continuous suppression, lower unspliced RNA, and lower intact proviral DNA. These differences may have biased the ability to see changes in DNA and US RNA in the control group. Notably, there was no significant difference in the change in US RNA/DNA between groups (Figure 2C). The fact that the median relative change appears very similar in Figure 2C, yet there is a substantial difference in P values, is also a comment on the limits of the current sample size. The text should report the median change in US RNA and US RNA/DNA when describing Figures 2A-2C. This statistical comparison of changes in IPDA results between groups should be reported. The presentation of the absolute values of all the comparisons in the supplemental figures is a strength of the manuscript.

In the assessment of ART intensification on immune activation and exhaustion, the fact that none of the comparisons between randomized groups were significant should be noted and discussed.

The changes in CD4:CD8 ratio and sCD14 levels appear counterintuitive to the hypothesis and are commented on in the discussion.

Overall, the discussion highlights the significant changes in the intensified group, which are suggestive. There is limited discussion of the comparisons between group,s where the results are less convincing.

The limitations of the study should be more clearly discussed. The small sample size raises the possibility of imbalance at baseline. The supplemental figures (S3-S5) are helpful in showing the differences between groups at baseline, and the variability of measurements is more apparent. The lack of blinding is also a weakness, though the PK assessments do help (note 3TC levels rise substantially in both groups for most of the time on study (Figure S2).

The many assays and comparisons are listed as a strength. The many comparisons raise the possibility of finding significance by chance. In addition, if there is an imbalance at baseline outcomes, measuring related parameters will move in the same direction.

The limited impact on activation and inflammation should be addressed in the discussion, as they are highlighted as a potentially important consequence of intermittent, not sustained replication in the introduction.

The study is provocative and well executed, with the limitations listed above. Pharmacokinetic analyses help mitigate the lack of blinding. The major impact of this work is if it leads to a much larger randomized, controlled, blinded study of a longer duration, as the authors point out.

Reviewer #3 (Public review):

Summary:

The manuscript by Chang and colleagues provides new insights into how cancer cells adapt their metabolism under nutrient-deprived conditions. They find cells respond differentially to serine and lipid deprivation via oxidising the cell redox state, which enables biomass synthesis and cell proliferation. They identified mitochondrial respiration as the major mechanism that dictates the endogenous NAD+/NADH ratio. By incorporating a dual stress paradigm, serine and lipid deprivation, the study further suggests that the NAD+/NADH ratio can serve as a link to orchestrate the complex interplay between multiple nutrient changes in the tumour microenvironment.

Strengths:

A novel aspect of this study is the idea that cancer cells are not uniformly passive victims of nutrient limitation; some can actively invoke endogenous NAD+ regeneration to combat nutrient stress. The conclusion is well-supported by comparing multiple cell lines from different tissues and genetic backgrounds, which improves generalizability. While most of the smaller conclusions align with common reasoning and expectations, the step-by-step deduction that leads to a novel 'big picture' is commendable. Another notable strength is the integration of dual stress (lipid and serine deprivation), which better mimics the complex tumor microenvironment with multiple nutrient fluctuations, raising the translational potential of these findings. The observation that lipid-deprived cells can stimulate serine synthesis and support proliferation in a subset of cancer cell lines offers a novel perspective on metabolic plasticity under starvation conditions.

Weaknesses:

Although the authors derive a novel and valuable overarching concept, the presentation of this "big picture" is not clearly articulated, making it less accessible to readers outside the immediate field. It would greatly enhance the manuscript to include a clearer summary of the overarching model and its implications. Additionally, discussing the potential clinical significance and applications of the findings would increase the relevance and broader impact of the work. Finally, the manuscript's clarity and credibility are undermined by inconsistent figure labeling and the lack of statistical analysis, particularly for the Western blot data.

While this study identifies changes in serine synthesis, mitochondrial respiration, PHGDH protein levels, and NAD+/NADH ratio in different cell lines, some of these relationships appear correlative rather than causally established (Figure 2; Figure 5; Figure 6). Some claims are thus overinterpreted. For example, the co-occurrence of increased NAD+/NADH ratio and citrate levels under lipid deprivation in A549 cells does not establish causality (Figure 5). Direct perturbation experiments that manipulate NAD+/NADH and assess downstream effects on citrate synthesis would substantially strengthen the conclusions.

The study focuses predominantly on mitochondrial respiration as a source of NAD+ regeneration. However, it will also be interesting to check other significant pathways, such as NAD+ salvage, which have been implicated in supporting serine biosynthesis. In addition, the subcellular distribution of NAD+ may distinguish whether some cells are truly redox-unresponsive. Mitochondrial NAD+ regeneration might counteract the cytosolic NAD+ consumption, rendering a relatively stable intracellular NAD+/NADH ratio. The malate-aspartate shuttle can be an interesting aspect.

The authors should acknowledge the limitations of short-term isotope tracing in their experimental design. Differences in metabolic rates across cell lines can affect the kinetics of metabolite labeling, limiting the direct comparability of metabolic fluxes between them. As a result, observed changes may reflect transient adaptations rather than stable metabolic reprogramming. It is important to clarify that the study primarily captures short-term responses, and the conclusions may not extrapolate to longer-term adaptations or protein-level changes under sustained nutrient stress.

Reviewer #3 (Public review):

Summary:

This work presents a novel neural network-based framework for parameterizing individual differences in human behavior. Using two distinct decision-making experiments, the authors demonstrate the approach's potential and claims it can predict individual behavior (1) within the same task, (2) across different tasks, and (3) across individuals. While the goal of capturing individual variability is compelling and the potential applications are promising, the claims are weakly supported, and I find that the underlying problem is conceptually ill-defined.

Strengths:

The idea of using neural networks for parameterizing individual differences in human behavior is novel, and the potential applications can be impactful.

Weaknesses:

(1) To demonstrate the effectiveness of the approach, the authors compare a Q-learning cognitive model (for the MDP task) and RTNet (for the MNIST task) against the proposed framework. However, as I understand it, neither the cognitive model nor RTNet is designed to fit or account for individual variability. If that is the case, it is unclear why these models serve as appropriate baselines. Isn't it expected that a model explicitly fitted to individual data would outperform models that do not? If so, does the observed superiority of the proposed framework simply reflect the unsurprising benefit of fitting individual variability? I think the authors should either clarify why these models constitute fair control or validate the proposed approach against stronger and more appropriate baselines.

(2) It's not very clear in the results section what it means by having a shorter within-individual distance than between-individual distances. Related to the comment above, is there any control analysis performed for this? Also, this analysis appears to have nothing to do with predicting individual behavior. Is this evidence toward successfully parameterizing individual differences? Could this be task-dependent, especially since the transfer is evaluated on exceedingly similar tasks in both experiments? I think a bit more discussion of the motivation and implications of these results will help the reader in making sense of this analysis.

(3) The authors have to better define what exactly he meant by transferring across different "tasks" and testing the framework in "more distinctive tasks". All presented evidence, taken at face value, demonstrated transferring across different "conditions" of the same task within the same experiment. It is unclear to me how generalizable the framework will be when applied to different tasks.

(4) Conceptually, it is also unclear to me how plausible it is that the framework could generalize across tasks spanning multiple cognitive domains (if that's what is meant by more distinctive). For instance, how can an individual's task performance on a Posner task predict task performance on the Cambridge face memory test? Which part of the framework could have enabled such a cross-domain prediction of task performance? I think these have to be at least discussed to some extent, since without it the future direction is meaningless.

(5) How is the negative log-likelihood, which seems to be the main metric for comparison, computed? Is this based on trial-by-trial response prediction or probability of responses, as what usually performed in cognitive modelling?

(6) None of the presented evidence is cross-validated. The authors should consider performing K-fold cross-validation on the train, test, and evaluation split of subjects to ensure robustness of the findings.

(7) The authors excluded 25 subjects (20% of the data) for different reasons. This is a substantial proportion, especially by the standards of what is typically observed in behavioral experiments. The authors should provide a clear justification for these exclusion criteria and, if possible, cite relevant studies that support the use of such stringent thresholds.

(8) The authors should do a better job of creating the figures and writing the figure captions. It is unclear which specific claim the authors are addressing with the figure. For example, what is the key message of Figure 2C regarding transfer within and across participants? Why are the stats presentation different between the Cognitive model and the EIDT framework plots? In Figure 3, it's unclear what these dots and clusters represent and how they support the authors' claim that the same individual forms clusters. And isn't this experiment have 98 subjects after exclusion, this plot has way less than 98 dots as far as I can tell. Furthermore, I find Figure 5 particularly confusing, as the underlying claim it is meant to illustrate is unclear. Clearer figures and more informative captions are needed to guide the reader effectively.

(9) I also find the writing somewhat difficult to follow. The subheadings are confusing, and it's often unclear which specific claim the authors are addressing. The presentation of results feels disorganized, making it hard to trace the evidence supporting each claim. Also, the excessive use of acronyms (e.g., SX, SY, CG, EA, ES, DA, DS) makes the text harder to parse. I recommend restructuring the results section to be clearer and significantly reducing the use of unnecessary acronyms.

Reviewer #3 (Public review):

Summary:

This manuscript investigates the conditions under which representational distances estimated from brain-activity measurements accurately mirror the true geometry of the underlying neural representations. Using a theoretical framework and simulations, the authors show that (i) random weighted sampling of individual neurons preserves representational distances; (ii) the non-negative pooling characteristic of fMRI stretches the geometry along the population-mean dimension; and (iii) subtracting the across-channel mean from each activity pattern removes this distortion, explaining the well-known success of correlation-based RSA. They further argue that a mean-centred, squared Euclidean (or Mahalanobis) distance retains this corrective benefit while avoiding some pitfalls of variance normalisation.

Strengths:

(1) Theoretical clarity and novelty:<br /> The paper offers an elegant and convincing proof of how linear measurement models affect representational geometry and pinpoints the specific condition (non-zero-mean sampling weights) under which voxel pooling introduces a systematic bias. This quantitative explanation of why mean removal is effective in RSA is new and valuable.

(2) Simulations:<br /> Experiments on both synthetic high-dimensional fMRI data and macaque-IT-inspired embeddings corroborate the mathematics, providing practical insights into the theoretical reasoning outlined by the authors.

(3) Actionable recommendations:<br /> The work summarises the results into clear guidelines: random single-unit sampling is "safe" (the estimated geometry is undistorted); fMRI voxel data with unstructured or single-scale codes should be mean-centred; and multi-scale cortical maps require explicit forward modelling. These guidelines are clear, and useful for future research.

Weaknesses:

(1) Simplistic assumptions:<br /> The assumption that measurement-channel weights are drawn independently and identically distributed (i.i.d.) from a univariate distribution is a significant idealisation for fMRI data. Voxels have spatially structured responses (and noise), meaning they do not sample neurons with i.i.d. weights. The extent to which the conclusions (especially the "exact recovery" with mean centring) hold when this assumption is violated needs more discussion. While the paper states that the non-negative IWLCS model is a best-case scenario, the implications of deviations from this best case could be elaborated.

(2) Random-subpopulation model for electrophysiology:<br /> Similarly, the "random subpopulation model" is presented as an idealisation of single-cell recordings. In reality, electrophysiological sampling is often biased (e.g., towards larger, more active neurons or neurons in accessible locations). The paper acknowledges biased sampling as a challenge that requires separate modelling, but the gap between this idealised model and actual practice should be highlighted more strongly when interpreting the optimistic results.

(3) Noise as an "orthogonal issue":<br /> The theoretical derivations largely ignore measurement noise, treating it as an orthogonal problem solvable by cross-validation. Although bias from noise is a well-known problem, interactions between noise and sampling-induced distortions (especially the down-scaling of orthogonal dimensions) could complicate the picture. For instance, if a dimension is already heavily down-scaled by averaging, it might become more susceptible to being obscured by noise. Addressing or highlighting these points more explicitly would make the limitations of this theoretical framework more transparent.

(4) Simulation parameters and generalizability:<br /> The random ground-truth geometries were generated from a Gaussian mixture in 5-D and then embedded into 1,024-D, with ≈25 % of the variance coming from the mean dimension. The sensitivity of the findings to these specific parameters (initial dimensionality, geometry complexity, proportion of mean variance, and sample size) could be discussed. How would the results change if the true neural geometry had a much higher or lower intrinsic dimensionality, or if the population-mean component were substantially smaller or larger? If the authors' claims are to generalise, more scenarios should be considered.

(5) Mean addition to the neural-data simulation:<br /> In simulations based on neural data from Kiani et al., a random mean was added to each pattern to introduce variation along the mean dimension. This was necessary because the original patterns had identical mean activation. However, the procedure might oversimplify how population means vary naturally and could influence the conclusions, particularly regarding the impact of the population-mean dimension. While precisely modelling how the mean varies across conditions is beyond the manuscript's scope, this point should be stated and discussed more clearly.

(6) Effect of mean removal on representational geometry:<br /> As noted, the benefits of mean removal hold "under ideal conditions". Real data often violates these assumptions. A critical reader might ask: What if conditions differ in overall activation and in more complex ways (e.g., differing correlation structures across neurons)? Is it always desirable to remove population-mean differences? For example, if a stimulus truly causes a global increase in firing across the entire population (perhaps reflecting arousal or salience), subtracting the mean would treat this genuine effect as a nuisance and eliminate it from the geometry. Prior literature has cautioned that one should interpret RSA results after demeaning carefully. For instance, Ramírez (2017) dubbed this problem "representational confusion", showing that subtracting the mean pattern can change the relationships between conditions in non-intuitive ways. These potential issues and previous results should be discussed and properly referenced by the authors.

Appraisal, Impact, and Utility:

The authors set out to identify principled conditions under which measured representational distances faithfully reflect the underlying neural geometry and to provide practical guidance for RSA across modalities. Overall, I believe they achieved their goals. Theoretical derivations identify the bias-inducing factors in linear measurement models, and the simulations verify the analytic claims, demonstrating that mean-pattern subtraction can indeed correct some mean-related geometric distortions. These conclusions strongly rely on idealised assumptions (e.g., i.i.d. sampling weights and negligible noise), but the manuscript is explicit about them, and the reasoning from evidence to claim is sound. A deeper exploration of how robust each conclusion is to violations of these assumptions, particularly correlated voxel weights and realistic noise, would make the argument even stronger.

Beyond their immediate aims, the authors offer contributions likely to shape future work. Its influence is likely to influence both analysis decisions and the design of future studies exploring the geometry of brain representations. By clarifying why correlation-based RSA seems to work so robustly, they help demystify a practice that has so far been adopted heuristically. Their proposal to adopt mean-centred Euclidean or Mahalanobis distances promises a straightforward alternative that better aligns representational geometry with decoding-based interpretations.

In sum, I see this manuscript as a significant and insightful contribution to the field. The theoretical work clarifying the impact of sampling schemes and the role of mean removal is highly valuable. However, the identified concerns, primarily regarding the idealized nature of the models (especially for fMRI), the treatment of noise, and the need for more nuanced claims, suggest that some revisions are necessary. Addressing these points would substantially strengthen the paper's conclusions and enhance its impact on the neuroscience community by ensuring the proposed methods are robustly understood and appropriately applied in real-world research settings.

Reviewer #3 (Public review):

Summary:

This study aimed to investigate the effect of environmental enrichment (EE) during the critical perinatal period on the developing brain structure and compare it with other periods. Different datasets of mice with EE or standard housing (SH) were compared with post-mortem MRI: dataset A (MRI at P96; 13 animals in EE during adulthood P53-P96, 14 animals in SH), dataset P (MRI at P43; 24 animals in EE during perinatal period and adulthood E17-P43, 25 animals in SH) and dataset N (MRI at P7; 52 animals in EE during perinatal period E13-P7, 67 animals in SH / resulting from 5 dams with 2 litters: 4 dams in EE and 6 dams in SH). The study replicated the effects observed during adulthood (main neuroanatomical EE/SH difference in datasets A and P: increase in the hippocampus volume) but also showed that volumetric changes for some regions differ between datasets A and P, suggesting different mechanisms of brain responses to enrichment depending on the period when EE was applied. Results on dataset N further showed that EE leads to lower brain size and differences for various regions: volume reduction in striatum, frontal, parietal, and occipital regions, hippocampus; volume increase for a few thalamic nuclei and hindbrain, suggesting different patterns of perinatal EE effects in datasets P and N. Since mice at P7 show little engagement with their environment, the authors further explored the hypothesis that the dams' behavior and interaction with neonates could be a mediator of brain differences observed at P7 between EE and SH animals. Maternal contact time was related to the P7 volumes for some regions (striatum, brainstem), but the variability and low sample size prevented a clear separation between EE and SH in terms of maternal behaviors.

Strengths:

(1) The question raised by this article is important at a fundamental level for our understanding of the complex interactions between the brain, behavior, and the environment.

(2) This study replicates previous observations on the effects of EE in adult mice.

(3) While some studies have been performed on neonates of dams exposed to EE during gestation, it is the first time that the effects of perinatal EE are investigated, in both the developing and mature brains with MRI. From a translational perspective, this is crucial for our understanding of human neurodevelopment in interaction with the environment.

(4) The analyses carried out are numerous and detailed.

Weaknesses:

(1) The analyses carried out do not allow us to fully assess whether differences in maternal care mediate the effects of EE on brain structure during development. The observations support this causal hypothesis, but a complete mediation analysis would be useful if permitted by the sample size and the variability observed between litters.

(2) The article is quite dense to read, given the number of analyses carried out. It is difficult at first reading to get a global view of the results. Figure 4 could be highlighted earlier to present the hypotheses and tests carried out.

(3) The figures could be more explicit in terms of legends (particularly the supplementary figures).

Reviewer #3 (Public review):

Summary:

The paper by Saito et al. studies the properties of anthozoan-specific opsins (ASO-II) from organisms found in reef-building coral. Their goal was to test if ASO-II opsins can absorb visible light, and if so, what are they key factors involved.

The most exciting aspect of this work is their discovery that ASO-II opsins do not have a counterion residue (Asp or Glu) located at any of the previously known sites found in other animal opsins.

This is very surprising. Opsins are only able to absorb visible (long wavelength light) if the retinal Schiff base is protonated, and the latter requires (as the name implies) a "counter ion". However, the authors clearly show that some ASO-II opsins do absorb visible light.

To address this conundrum, they tested if the counterion could be provided by exogenous chloride ions (Cl-). Their results find compelling evidence supporting this idea, and their studies of ASO-II mutant E292A suggests E292 also plays a role in G protein activation and is a counterion for a protonated Schiff base in the light-activated form.

Strengths:

Overall, the methods are well described and carefully executed, and the results very compelling.

Their analysis of seven ASO-II opsin sequences undoubtedly shows they all lack a Glu or Asp residue at "normal" (previously established) counter-ion sites in mammalian opsins (typically found at positions 94, 113 or 181). The experimental studies clearly demonstrate the necessity of Cl- for visible light absorbance, as do their studies of the effect of altering the pH.

Importantly, the authors also carried out careful QM/MM computational analysis (and corresponding calculation of the expected absorbance effects), thus providing compelling support for the Cl- acting directly as a counterion to the protonated retinal Schiff base, and thus limiting the possibility that the Cl- is simply altering the absorbance of ASO-II opsins through some indirect effect on the protein.

Altogether, the authors clearly achieved their aims, and the results support their conclusions. The manuscript is carefully written, and refreshingly, the results and conclusions not overstated.

This study is impactful for several reasons. There is increasing interest in optogenetic tools, especially those that leverage G protein coupled receptor systems. Thus, the authors demonstration that ASO-II opsins could be useful for such studies is of interest.

Moreover, the finding that visible light absorbance by an opsin does not absolutely require a negatively charged amino acid be placed at one of the expected sites (94, 113 or 181) typically found in animal opsins is very intriguing and will help future protein engineering efforts. The argument that the Cl- counterion system they discover here might have been a preliminary step in the evolution of amino acid based counterions used in animal opsins is also interesting.

Finally, given the ongoing degradation of coral reefs worldwide, the focus on these curious opsins is very timely, as is the authors proposal that the lower Schiff base pKa they discovered here for ASO-II opsins may cause them to change their spectral sensitivity and G protein activation due to changes in their environmental pH.

Reviewer #3 (Public review):

Summary:

This is a lucidly written manuscript describing the use of transition-metal FRET to assess distance changes during functional conformational changes in a CNG channel. The experiments were performed on an isolated C-terminal nucleotide binding domain (CNBD) and on a purified full-length channel, with FRET partners placed at two positions in the CNBD.

The data and quantitative analysis are exemplary, and they provide a roadmap for the use of this powerful approach in other proteins. In particular, the use of the fluorescence-lifetime decay histograms to learn not just the mean distance reported by the FRET, but also the distribution of states with different distances, allows better refinement of hypotheses for the gating motions.

Reviewer #3 (Public review):

In this manuscript, Nishi et al. propose a new model to explain the previously reported myeloid-biased hematopoiesis associated with aging. Traditionally, this phenotype has been explained by the expansion of myeloid-biased hematopoietic stem cell (HSC) clones during aging. Here, the authors question this idea and show how their Hoxb5 reporter model can discriminate long-term (LT) and short-term (ST) HSC and characterized their lineage output after transplant. From these analyses, the authors conclude that changes during aging in the LT/ST HSC proportion explain the myeloid bias observed.

Comments on revisions:

I appreciate the authors' reply to some of my comments. However, there are some key aspects that remain unresolved. Please see below.

- The authors propose a critical change in the way we consider the mechanisms leading to lineage biased hematopoiesis during aging. As Reviewer 2 mentioned, such a strong claim needs to be supported by solid experimental data. Unfortunately, the level of variability in key in vivo experiments (Figure 2 and 3) diminishes the robustness of these results.

The authors argue that even with the low number of mice used in some of these experiments and the high level of variability, differences still reach (or not) statistical significance according to their analysis. I am not an expert on statistics but the only test that is mentioned is their methodology is a Welch's t test, which is only appropriate for data following a normal distribution. A more rigorous statistical analysis should be performed to sustain the claims included in the current manuscript.

- The chosen irradiation regiment might contribute to the uncertainty of the data and influence their interpretation. As the authors show in their response to my "comment to our #3-4 response", there is a considerable (and variable) amount of "radioresistant" CD45.1+CD45.2- cells in their primary recipients, which become concerningly high in the secondary transplant. This is not found in previous publications focused on this topic and, therefore, it makes it difficult to compare those studies with the present manuscript. The inclusion of this aspect in the text is appreciated but definitely reduces the impact of their claims.

- The correction introduced in the main text as an answer to the original comment #3-6 is still misleading. There is an assumption for GMP, CMP and MEP to increase with age if myeloid-biased HSC clones increase with age ("in contrast to what we anticipated"). Again, the link between these two changes could be more complex than just a direct correlation.

Reviewer #3 (Public review):

Summary:

This is an interesting and clinically relevant in vitro study by Taber et al., exploring how mutations in PHD2 contribute to erythrocytosis and/or neuroendocrine tumors. PHD2 regulates HIFα degradation through prolyl-hydroxylation, a key step in the cellular oxygen-sensing pathway.

Using a time-resolved NMR-based assay, the authors systematically analyze seven patient-derived PHD2 mutants and demonstrate that all exhibit structural and/or catalytic defects. Strikingly, the P317R variant retains normal activity toward the C-terminal proline but fails to hydroxylate the N-terminal site. This provides the first direct evidence that N-terminal prolyl-hydroxylation is not dispensable, as previously thought.

The findings offer valuable mechanistic insight into PHD2-driven effects and refine our understanding of HIF regulation in hypoxia-related diseases.

Strengths:

The manuscript has several notable strengths. By applying a novel time-resolved NMR approach, the authors directly assess hydroxylation at both HIF1α ODD sites, offering a clear functional readout. This method allows them to identify the P317R variant as uniquely defective in NODD hydroxylation, despite retaining normal activity toward CODD, thereby challenging the long-held view that the N-terminal proline is biologically dispensable. The work significantly advances our understanding of PHD2 function and its role in oxygen sensing, and might help in the future interpretation and clinical management of associated erythrocytosis.

Weaknesses:

There is a lack of in vivo/ex vivo validation. This is actually required to confirm whether the observed defects in hydroxylation-especially the selective NODD impairment in P317R-are sufficient to drive disease phenotypes such as erythrocytosis.

The reliance on HRE-luciferase reporter assays may not reliably reflect the PHD2 function and highlights a limitation in the assessment of downstream hypoxic signaling.

The study clearly documents the selective defect of the P317R mutant, but the structural basis for this selectivity is not addressed through high-resolution structural analysis (e.g., cryo-EM).

Given the proposed central role of HIF2α in erythrocytosis, direct assessment of HIF2α hydroxylation by the mutants would have strengthened the conclusions.

Reviewer #3 (Public review):

Summary: Li et al describe a set of experiments to probe the role of FMRP in ribosome stalling and RNA granule composition. The authors are able to recapitulate findings from a previous study performed in rats (this one is in mice).

Strengths:

1) The work addresses an important and challenging issue, investigating mechanisms that regulate stalled ribosomes, focusing on the role of FMRP. This is a complicated problem, given the heterogeneity of the granules and the challenges related to their purification. This work is a solid attempt at addressing this issue, which is widely understudied.

2) The interpretation of the results could be interesting, if supported by solid data. The idea that FMRP could control the formation and release of RNA granules, rather than the elongation by stalled ribosomes is of high importance to the field, offering a fresh perspective into translational regulation by FMRP.

3) The authors focused on recapitulating previous findings, published elsewhere (Anadolu et al., 2023) by the same group, but using rat tissue, rather than mouse tissue. Overall, they succeeded in doing so, demonstrating, among other findings, that stalled ribosomes are enriched in consensus mRNA motifs that are linked to FMRP. These interesting findings reinforce the role of FMRP in formation and stabilization of RNA granules. It would be nice to see extensive characterization of the mouse granules as performed in Figure 1 of Anadolu and colleagues, 2023.

4) Some of the techniques incorporated aid in creating novel hypotheses, such as the ribopuromycilation assay and the cryo-EM of granule ribosomes.

Weaknesses:

1) The RNA granule characterization needs to be more rigorous. Coomassie is not proper for this type of characterization, simply because protein weight says little about its nature. The enrichment of key proteins is not robust and seems to not reach significance in multiple instances, including S6 and UPF1. Furthermore, S6 is the only proxy used for ribosome quantification. Could the authors include at least 3 other ribosomal proteins (2 from small, 2 from large subunit)?

2) Page 12-13 - The Gene Ontology analysis is performed incorrectly. First, one should not rank genes by their RPKM levels. It is well known that housekeeping genes such as those related to actin dynamics, molecular transport and translation are highly enriched in sequencing datasets. It is usually more informative when significantly different genes are ranked by p adjust or log2 Fold Change, then compared against a background to verify enrichment of specific processes. However, the authors found no DEGs. I would suggest the removal of this analysis, incorporation of a gene set enrichment analyses (ranked by p adjust). I further suggest that the authors incorporate a dimensionality reduction analysis to demonstrate that the lack of significance stems from biology and not experimental artifacts, such as poor reproducibility across biological replicates.

Reviewer #3 (Public review):

Summary:

In their manuscript, Kumar et al. investigate the mechanisms underlying the tumor suppressive function of the RNA binding protein ZMAT3, a previously described tumor suppressor in the p53 pathway. To this end, they use RNA-sequencing and proteomics to characterize changes in ZMAT3-deficient cells, leading them to identify the hexokinase HKDC1 as upregulated with ZMAT3 deficiency first in colorectal cancer cells, then in other cell types of both mouse and human origin. This increase in HKDC1 is associated with increased mitochondrial respiration. As ZMAT3 has been reported as an RNA-binding and DNA-binding protein, the authors investigated this via PAR-CLIP and ChIP-seq but did not observe ZMAT3 binding to HKDC1 pre-mRNA or DNA. Thus, to better understand how ZMAT3 regulates HKDC1, the authors used quantitative proteomics to identify ZMAT3-interacting proteins. They identified the transcription factor JUN as a ZMAT3-interacting protein and showed that JUN promotes the increased HKDC1 RNA expression seen with ZMAT3 inactivation. They propose that ZMAT3 inhibits JUN-mediated transcriptional induction of HKDC1 as a mechanism of tumor suppression. This work uncovers novel aspects of the p53 tumor suppressor pathway.

Strengths:

This novel work sheds light on one of the most well-established yet understudied p53 target genes, ZMAT3, and how it contributes to p53's tumor suppressive functions. Overall, this story establishes a p53-ZMAT3-HKDC1 tumor suppressive axis, which has been strongly substantiated using a variety of orthogonal approaches, in different cell lines and with different data sets.

Weaknesses:

While the role of p53 and ZMAT3 in repressing HKDC1 is well substantiated, there is a gap in understanding how ZMAT3 acts to repress JUN-driven activation of the HKDC1 locus. How does ZMAT3 inhibit JUN binding to HKDC1? Can targeted ChIP experiments or RIP experiments be used to make a more definitive model? Can ZMAT3 mutants help to understand the mechanisms? Future work can further establish the mechanisms underlying how ZMAT3 represses JUN activity.

Reviewer #3 (Public review):

Summary:

BicD2 is a motor adapter protein that facilitates cellular transport pathways, which are impacted by human disease mutations of BicD2, causing spinal muscular atrophy with lower extremity dominance (SMALED2). The authors provide evidence that some of these mutations result in interactome changes, which may be the underlying cause of the disease. This is supported by proximity biotin ligation screens, immunoprecipitation, and cell biology assays. The authors identify several novel BicD2 interactions, such as the HOPS complex that participates in the fusion of late endosomes and autophagosomes with lysosomes, which could have important functions. Three BicD2 disease mutants studied had changes in the interactome, which could be an underlying cause for SMALED2. The study extends our understanding of the BicD2 interactome under physiological conditions, as well as of the changes in cellular transport pathways that result in SMALED2. It will be of great interest for the BicD2 and dynein fields.

Strengths:

Extensive interactomes are presented for both WT BicD2 as well as the disease mutants, which will be valuable for the community. The HOPS complex was identified as a novel interactor of BicD2, which is important for fusion of late endosomes and lysosomes, which is of interest, since some of the BicD2 disease mutations result in Golgi-fragmentation phenotypes. The interaction with the HOPS complex is affected by the R747C mutation, which also results in a gain-of-function interaction with GRAMD1A.

Weaknesses:

The manuscript should be strengthened by further evidence of the BicD2/HOPS complex interaction and the functional implications for spinal muscular atrophy by changes in the interactome through mutations. Which functional implications does the loss of the BicD2/HOPS complex interaction and the gain of function interaction with GRAMD1A have in the context of the R747C mutant?

Major points:

(1) In the biotin proximity ligation assay, a large number of targets were identified, but it is not clear why only the HOPS complex was chosen for further verification. Immunoprecipitation was used for target verification, but due to the very high number of targets identified in the screen, and the fact that the HOPS complex is a membrane protein that could potentially be immunoprecipitated along with lysosomes or dynein, additional experiments to verify the interaction of BicD2 with the HOPS complex (reconstitution of a complex in vitro, GST-pull down of a complex from cell extracts or other approaches) are needed to strengthen the manuscript.

(2) In the biotin proximity ligation assay, a large number of BicD2 interactions were identified that are distinct between the mutant and the WT, but it was not clear why, particularly GRAMD1A was chosen as a gain-of-function interaction, and what the functional role of a BicD2/GRAMD1A interaction may be. A Western blot shows a strengthened interaction with the R747C mutant, but GRAMD1A also interacts with WT BicD2.

(3) Furthermore, the functional implications of changed interactions with HOPS and GRAMD1A in the R747C mutant are unclear. Additional experiments are needed to establish the functional implication of the loss of the BicD2/HOPS interaction in the BicD2/R747C mutant. For the GRAMD1A gain of function interaction, according to the authors, a significant amount of the protein localized with BicD2/R747C at the centrosomal region. This changed localization is not very clear from the presented images (no centrosomal or other markers were used, and the changed localization could also be an effect of dynein hyperactivation in the mutant). Furthermore, the functional implication of a changed localization of GRAMD1A is unclear from the presented data.

Reviewer #3 (Public review):

This paper investigates the role of Chi3l1 in regulating the fate of liver macrophages in the context of metabolic dysfunction leading to the development of MASLD. I do see value in this work, but some issues exist that should be addressed as well as possible.

Here are my comments:

(1) Chi3l1 has been linked to macrophage functions in MASLD/MASH, acute liver injury, and fibrosis models before (e.g., PMID: 37166517), which limits the novelty of the current work. It has even been linked to macrophage cell death/survival (PMID: 31250532) in the context of fibrosis, which is a main observation from the current study.

(2) The LysCre-experiments differ from experiments conducted by Ariel Feldstein's team (PMID: 37166517). What is the explanation for this difference? - The LysCre system is neither specific to macrophages (it also depletes in neutrophils, etc), nor is this system necessarily efficient in all myeloid cells (e.g., Kupffer cells vs other macrophages). The authors need to show the efficacy and specificity of the conditional KO regarding Chi3l1 in the different myeloid populations in the liver and the circulation.

(3) The conclusions are exclusively based on one MASLD model. I recommend confirming the key findings in a second, ideally a more fibrotic, MASH model.

(4) Very few human data are being provided (e.g., no work with own human liver samples, work with primary human cells). Thus, the translational relevance of the observations remains unclear.

Reviewer #3 (Public review):

Smit and Robbins' manuscript investigates the dynamics of aggression among female groupmates across five gorilla groups. The authors utilize longitudinal data to examine how reproductive state, group size, presence of males, and resource availability influence patterns of aggression and overall dominance rankings as measured by Elo scores. The findings underscore the important role of group composition and reproductive status, particularly pregnancy, in shaping dominance relationships in wild gorillas. While the study addresses a compelling and understudied topic, I have several comments and suggestions that may enhance clarity and improve the reader's experience.

(1) Clarification of longitudinal data - The manuscript states that 25 years of behavioral data were used, but this number appears unclear. Based on my calculations, the maximum duration of behavioral observation for any one group appears to be 18 years. Specifically: - ATA: 6 years - BIT: 8 years - KYA: 18 years - MUK: 6 years - ORU: 8 years I recommend that the authors clarify how the 25-year duration was derived.

(2) Consideration of group size - The authors mention that group size was excluded from analyses to avoid conflating the opposing effects of female and male group members. While this is understandable, it may still be beneficial to explore group size effects in supplementary analyses. I suggest reporting statistics related to group size and potentially including a supplementary figure. Additionally, given that the study includes both mountain and wild gorillas, it would be helpful to examine whether any interspecies differences are apparent.

(3) Behavioral measures clarification - Lines 112-116 describe the types of aggressive behaviors observed. It would be helpful to clarify how these behaviors differ from those used to calculate Elo scores, or whether they overlap. A brief explanation would improve transparency regarding the methodology.

(4) Aggression rates versus Elo scores - The manuscript uses aggression rates rather than dominance rank (as measured by Elo scores) as the main outcome variable, but there is no explanation on why. How would the results differ if aggression rates were replaced or supplemented with Elo scores? The current justification for prioritizing aggression rates over dominance rank needs to be more clearly supported.

Reviewer #3 (Public review):

This paper addresses an important question about how changes in wing morphology vs. wing kinematics change with body size across an important group of high-performance insects, the hoverflies. The biomechanics and morphology convincingly support the conclusions that there is no significant correlation between wing kinematics and size across the eight specific species analyzed in depth and that instead wing morphology changes allometrically. The morphological analysis is enhanced with phylogenetically appropriate tests across a larger data set incorporating museum specimens.

The authors have made very extensive revisions that have significantly improved the manuscript and brought the strength of conclusions in line with the excellent data. Most significantly, they have expanded their morphological analysis to include museum specimens and removed the conclusions about evolutionary drivers of miniaturization. As a result, the conclusion about morphological changes scaling with body size rather than kinematic properties is strongly supported and very nicely presented with a strong complementary set of data. I only have minor textual edits for them to consider.

Reviewer #3 (Public review):

Summary:

In this delightful study, the authors use local indentation of the cell surface combined with out-of-focus microscopy to measure the rates of pressure spread in the cell and to argue that the results can be explained with the poroelastic model. Osmotic shock that decreases cytoskeletal mesh size supports this notion. Experiments with water injection and water suction further support it, and also, together with a mechanical model and elegant measurements of decreasing fluorescence in the cell 'flashed' by external flow, demonstrate that the membrane is permeable, and that steady flow and pressure gradient can exist in a cell with water source/sink in different locations. Use of blebs as indicators of the internal pressure further supports the notion of differential cytoplasmic pressure.

Strengths:

The study is very imaginative, interesting, novel and important.

Weaknesses: I have two broad critical comments:

(1) I sense that the authors are correct that the best explanation of their results is the passive poroelastic model. Yet, to be thorough, they have to try to explain the experiments with other models and show why their explanation is parsimonious. For example, one potential explanation could be some mechanosensitive mechanism that does not involve cytoplasmic flow; another could be viscoelastic cytoskeletal mesh, again not involving poroelasticity. I can imagine more possibilities. Basically, be more thorough in the critical evaluation of your results. Besides, discuss the potential effect of significant heterogeneity of the cell.

(2) The study is rich in biophysics but a bit light on chemical/genetic perturbations. It could be good to use low levels of chemical inhibitors for, for example, Arp2/3, PI3K, myosin etc, and see the effect and try to interpret it. Another interesting question - how adhesive strength affects the results. A different interesting avenue - one can perturb aquaporins. Etc. At least one perturbation experiment would be good.

Reviewer #3 (Public review):

Summary:

Replication through DNA lesions such as UV-induced pyrimidine dimers is mainly performed by Y-family pols. These translesion synthesis (TLS) pols are intrinsically error-prone. However, in living cells, TLS must be conducted in an error-free manner. This manuscript demonstrated that WRN and WRNIP1 ATPases play an important role in addition to WRN 3'>5' exonuclease in human cells.

Strengths:

The authors made use of WT human fibroblasts and WRN-deficient cell line for TLS assays in human cells and siRNA knock-down experiments to analyze TLS efficiency. For the cII mutation assay, the big blue mouse embryonic fibroblasts were used. These materials, as well as other Materials and Methods, had already been well established by this group or other groups. The authors used Pol eta, iota, kappa, and theta as TLS pols, and used UV-induced CPD, (6-4)PP, epsilon dA, and thymine glycol as DNA lesions. Thus, the authors examined the generality of their results in terms of TLS pols and DNA lesions.

Weaknesses:

Although the main part of this manuscript is the impact of the deficiencies of WRN and WRNIP1 ATPases on TLS by Y-family DNA polymerases, especially on TLS efficiency and mutation spectrum, many readers would be interested in how these ATPases could change molecular structure of Pol eta, because the structure of it have been studied for some time.

Reviewer #3 (Public review):

The authors have significantly improved the paper in revising to make its contributions distinct from their prior paper. They have also responded to my concerns about quantification and parameter dependency of the integration conclusion. While I think there is still more that could be done in this capacity, especially in terms of the temporal statistics and quantification of the conflict responses, they have a made a case for the conclusions as stated. The paper still stands as an important paper with solid evidence a bit limited by these concerns.

[Editors' note: Due to very minor revisions, the paper was not sent to reviewers for an additional round of review.]

Reviewer #3 (Public review):

This paper discusses how non-sensory and latent, sensory-like attentional templates are represented during attentional preparation. Using multivariate pattern analysis, they found that visual impulses can enhance the decoding generalization from perception to attention tasks in the preparatory stage in the visual cortex. Furthermore, the emergence of the sensory-like template coincided with enhanced information connectivity between V1 and frontoparietal areas and was associated with improved behavioral performance. It is an interesting paper with supporting evidence for the latent, sensory-like attentional template.

(1) The authors addressed most of my previous concerns and provided additional data analysis. They conducted further analyses to demonstrate that the observed changes in network communication are associated with behavioral RTs, supporting the idea that the impulse-driven sensory-like template enhances informational connectivity between sensory and frontoparietal areas, and relates to behavior.

(2) I would like to further clarify my previous points regarding the definition of the two types of templates and the evidence for their coexistence. The authors stated that the sensory-like template likely existed in a latent state and was reactivated by visual pings, proposing that sensory and non-sensory templates coexist. However, it remains unclear whether this reflects a dynamic switch between formats or true coexistence. If the templates are non-sensory in nature, what exactly do they represent? Are they meant to be abstract or conceptual representations, or, put simply, just "top-down attentional information"? If so, why did the generalization analyses-training classifiers on activity during the stimulus selection period and testing on preparatory activity-fail to yield significant results? While the stimulus selection period necessarily encodes both target and distractor information, it should still contain attentional information. I would appreciate more discussion from this perspective.

Reviewer #3 (Public review):

Summary

This study by Wang et al. explores a compelling link between two fundamental innate behaviors in Drosophila melanogaster, mating and feeding, demonstrating that repeated sexual failure in male flies leads to a transient yet reversible decrease in sweet taste perception. The authors show that this modulation is mediated by dopamine signaling from a specific subset of dopaminergic neurons in the subesophageal zone (SEZ) that directly influence Gr5a⁺ sweet-sensing neurons.

Aims of the Study

The authors aimed to understand whether unsuccessful mating attempts could affect sensory processing of sweet stimuli and thus feeding behavior in male fruit flies. They further sought to dissect the neural circuitry and molecular pathways underlying this behavioral plasticity, with a particular focus on dopaminergic modulation.

Major Strengths and Weaknesses

Strengths:

• Novelty: The idea that reproductive experience modulates gustatory perception adds a new dimension to our understanding of cross-modal behavioral integration.

• Experimental approach: The study uses a broad array of genetic, pharmacological, imaging, and behavioral assays to demonstrate a causal relationship between sexual failure and reduced sweet perception, mediated by specific dopaminergic pathways.

• Methodological design: The authors link behavioral outcomes (reduced proboscis extension reflex) with neural activity (calcium imaging of Gr5a⁺ neurons) and molecular specificity (dopamine receptor subtype roles), providing a robust multi-level framework.

Weaknesses:

• Ecological relevance: While the laboratory conditions are well controlled, the adaptive value or natural context of this taste modulation following mating failure remains speculative.

Achievement of Aims and Support for Conclusions

The authors have convincingly achieved their central aim. The results support the conclusion that sexual failure reduces sweet taste sensitivity through dopamine signaling. The reduced activity in Gr5a⁺ neuron after courtship rejection, its rescue by dopamine or successful copulation, and the requirement of specific dopamine receptors support the proposed model.

Impact and Utility

This work advances the field's understanding of how motivational states shaped by social experiences can directly influence sensory perception and behavior. It underscores the role of the dopaminergic system not only in reward but in integrating internal states across distinct behavioral responses. The experimental approach, including courtship conditioning paradigms and in vivo imaging methods, provides a valuable foundation for related studies in sensory modulation and behavioral plasticity.

Additional Context

This study supports a growing body of literature suggesting that insects possess emotion-like internal states that influence their behavior across contexts. The findings resonate with prior work on how stressors like social isolation or courtship failure lead to compensatory changes in other reward-seeking behaviors (e.g., ethanol consumption). Moreover, the concept that neural systems underlying basic drives like hunger and mating are dynamically interconnected may be conserved across phyla, suggesting broader relevance to understanding internal state-dependent modulation of behavior.

The authors addressed all the comments of previous reviews. The changes increased the clarity of the manuscript, the interpretation of the results and reinforce the conclusion.

Searching printables.com, thingaverse.com, and makerworld.com might unearth other printable rubber typewriter parts like feet. (Be sure to search for singular and plural versions: i.e. "foot" and "feet".) Etsy and Ebay searches might uncover others who make and sell the feet you're looking for.

edited reply for: https://www.facebook.com/groups/705152958470148/posts/1013945174257590/

Reviewer #3 (Public review):

The study by Fallah et al. provides a thorough characterization of the effects of two basal ganglia output pathways, the SNr and the GPe, on cholinergic, glutamatergic, and GABAergic neurons of the PPN. Using a combination of optogenetics-assisted electrophysiology and behavioral assays in genetically defined mouse lines, the authors show that SNr projections broadly inhibit all PPN subtypes along the rostrocaudal axis, whereas GPe projections are mostly restricted to the caudal PPN and predominantly target glutamatergic neurons, with a lesser effect on GABAergic neurons. Activation of these inputs in vivo revealed opposing behavioral effects: SNr stimulation increased locomotion and caused avoidance in the real-time place preference (RTPP) task, while GPe stimulation reduced locomotion and increased time spent in the stimulation zone.

Strengths:

The evidence for functional connectivity between SNr and GPe inputs and specific PPN cell types is solid and highlights a prominent influence of SNr across the PPN. The identification of a GPe projection that selectively targets caudal glutamatergic PPN neurons is unexpected and highly relevant to understanding basal ganglia-brainstem interactions. The study stands out for its systematic cell-type-specific approach and the combination of electrophysiological and behavioral data. Importantly, the authors addressed key concerns from the initial review by performing new analyses and adding important controls:

Motor activity was re-analyzed at higher temporal resolution, revealing more nuanced effects of stimulation (Fig. S2).

The concern that motor effects might confound RTPP performance was mitigated by analyzing unstimulated test sessions, which showed that place preference or aversion persisted in the absence of stimulation (Fig. 7G).

The potential recruitment of SNc dopaminergic projections was directly tested using DAT-Cre mice, confirming that dopaminergic axon stimulation drives locomotion and reward but does not explain the aversive effect seen with broader SNr activation (Fig. S3).

Weaknesses:

While the revised analyses and added data strengthen the conclusions, the interpretation of the behavioral effects remains somewhat limited by the use of RTPP, which can be influenced by motor changes, even with unilateral stimulation. Nonetheless, the additional controls and thorough discussion now acknowledge and address these caveats appropriately.

Some minor clarifying edits would enhance the manuscript's precision and readability, including improvements to terminology, data presentation, figure referencing, and the organization of behavioral and statistical reporting.

Conclusion:

This is a strong and compelling study that provides a detailed and novel characterization of basal ganglia inputs to the PPN and their behavioral relevance. The authors were responsive to reviewer feedback, and the revised manuscript is significantly improved. The findings advance our understanding of how basal ganglia output pathways engage brainstem circuits to modulate locomotion and valence.

Reviewer #3 (Public review):

Summary:

The authors used recurrent neural network modelling of spatial navigation tasks to investigate border and place cell behaviour during remapping phenomena.

Strengths:

The neural network training seemed for the most part (see comments later) well-performed, and the analyses used to make the points were thorough.

The paper and ideas were well-explained.

Figure 4 contained some interesting and strong evidence for map-like generalisation as environmental geometry was warped.

Figure 7 was striking and potentially very interesting.

It was impressive that the RNN path-integration error stayed low for so long (Fig A1), given that normally networks that only work with dead-reckoning have errors that compound. I would have loved to know how the network was doing this, given that borders did not provide sensory input to the network. I could not think of many other plausible explanations... It would be even more impressive if it was preserved when the network was slightly noisy.

Update:

The analysis of how the RNN remapped, using a context signal to switch between largely independent maps, and the examination of the border like tuning in the recurrent units of the RNN, were both thorough and interesting. Further, in the updated response I appreciated the additional appendix E which helped substantiate the claim that the RNN neurons were border cells.

Reviewer #3 (Public review):

Summary:

Chemical communication is essential for the organization of eusocial insect societies. It is used in various important contexts, such as foraging and recruiting colony members to food sources. While such pheromones have been chemically identified and their function demonstrated in bioassays, little is known about their perception. Excellent candidates are the odorant receptors that have been shown to be involved in pheromone perception in other insects including ants and bees but not termites. The authors investigated the function of the odorant receptor PsimOR14, which was one of four target odorant receptors based on gene sequences and phylogenetic analyses. They used the Drosophila empty neuron system to demonstrate that the receptor was narrowly tuned to the trail pheromone neocembrene. Similar responses to the odor panel and neocembrene in antennal recordings suggested that one specific antennal sensillum expresses PsimOR14. Additional protein modeling approaches characterized the properties of the ligand binding pocket in the receptor. Finally, PsimOR14 transcripts were found to be significantly higher in worker antennae compared to soldier antennae, which corresponds to the worker's higher sensitivity to neocembrene.

Strengths:

The study presents an excellent characterization of a trail pheromone receptor in a termite species. The integration of receptor phylogeny, receptor functional characterization, antennal sensilla responses, receptor structure modeling, and transcriptomic analysis is especially powerful. All parts build on each other and are well supported with a good sample size. (I cannot comment on protein modeling and docking due to a lack of expertise in this area)

Weaknesses:

None.

Reviewer #3 (Public review):

Summary:

In the current work, Howell et al studied the connectivity between cortex and thalamus using DTI tractography per parcel to all voxels in the thalamus. Following they performed various dimensional reduction techniques to uncover how differences in connectivity to the thalamus vary across cortical parcels. Following they explore the spatial correlation of these variations with cortical myelin and functional organization, thalamic nuclei, gene expression derived core-matrix cell differentiation, and extend the model towards macaques. Overall, the authors find a differentiation between sensory and association areas in terms of the association with the thalamus, which reflects differences in cortical microstructure and function, and links to core-matrix differences and can be replicated in macaques.

Strengths:

A clear strength of the current work is the combination of different models and approaches to study the link between the cortex and the thalamus. This approach nicely bridges different approaches to describe the role of the thalamus in cortical organisation using a diffusion-based approach. Especially the extension of the model to the macaque is quite nice.

Appraisal:

The aim of the study: 'to investigate the spatial extent of anatomical connectivity patterns within the thalamus in both humans and non-human primates and determine if such patterns differ between sensorimotor and association cortical areas' has been met. Further work may continue to investigate other implications of this finding.

Discussion:

Overall, I think the study is an intriguing addition to a growing literature studying the anatomical connectivity between thalamus and cortex and its functional implications.

Comments on revised version:

Thank you for the responses.

Reviewer #3 (Public review):

This paper reports a considerable technical achievement: the optogenetic activation of single retinal ganglion cells in vivo in monkeys. As clearly specified in the paper, this is an important step towards causal tests of the role of specific ganglion cell types in visual perception. The paper is brief, and it will be important to follow this work with a more detailed methodological description to guide related work, to explore limitations, and to build confidence in the specificity of the approach.

Reviewer #3 (Public review):

Summary:

This study by the Boddy and Otomo laboratories further characterizes the roles of SMC5/6 loader proteins and related factors in SMC5/6-mediated repression of extrachromosomal circular DNA. The work shows that mutations engineered at an AlphaFold-predicted protein-protein interface formed between the loader SLF2/SIMC1 and SMC6 (similar to the interface in the yeast counterparts observed by cryo-EM) prevent co-IP of the respective proteins. The mutations in SLF2 also hinder plasmid DNA silencing when expressed in SLF2-/- cell lines, suggesting that this interface is needed for silencing. SIMC1 is dispensable for recruitment of SMC5/6 to sites of DNA damage, while SLF1 is required, thus separating the functions of the two loader complexes. Preventing SUMOylation (with a chemical inhibitor) increases transcription from plasmids but does not in SLF2-deleted cell lines, indicating the SMC5/6 silences plasmids in a SUMOylation dependent manner. Expression of LT is sufficient for increased expression, and again, not additive or synergistic with SIMC1 or SLF2 deletion, indicating that LT prevents silencing by directly inhibiting 5/6. In contrast, PML bodies appear dispensable for plasmid silencing.

Strengths:

The manuscript defines the requirements for plasmid silencing by SMC5/6 (an interaction of Smc6 with the loader complex SLF2/SIMC1, SUMOylation activity) and shows that SLF1 and PML bodies are dispensable for silencing. Furthermore, the authors show that LT can overcome silencing, likely by directly binding to (but not degrading) SMC5/6.

Weaknesses:

(1) Many of the findings were expected based on recent publications.

(2) While the data are consistent with SIMC1 playing the main function in plasmid silencing, it is possible that SLF1 contributes to silencing, especially in the absence of SIMC1. This would potentially explain the discrepancy with the data reported in ref. 50. SLF2 deletion has a stronger effect on expression than SIMC1 deletion in many but not all experiments reported in this manuscript. A double mutant/deletion experiments would be useful to explore this possibility.

(3) SLF2 is part of both types of loaders, while SLF1 and SIMC1 are specific to their respective loaders. Did the authors observe differences in phenotypes (growth, sensitivities to DNA damage) when comparing the mutant cell lines or their construction? This should be stated in the manuscript.

(4) It would be desirable to have control reporter constructs located on the chromosome for several experiments, including the SUMOylation inhibition (Figures 5A and 5-S2) and LT expression (Figure 5D) to exclude more general effects on gene expression.

(5) Figure 5A: There appears to be an increase in GFP in the SLF2-/- cells with SUMOi? Is this a significant increase?

(6) The expression level of SFL2 mut1 should be tested (Figure 3B).

Reviewer #3 (Public review):

Summary:

The authors set out to determine how GABAergic inhibitory premotor circuits contribute to the rhythmic alternation of leg flexion and extension during Drosophila grooming. To do this, they first mapped the ~120 13A and 13B hemilineage inhibitory neurons in the prothoracic segment of the VNC and clustered them by morphology and synaptic partners. They then tested the contribution of these cells to flexion and extension using optogenetic activation and inhibition and kinematic analyses of limb joints. Finally, they produced a computational model representing an abstract version of the circuit to determine how the connectivity identified in EM might relate to functional output. The study, in its current form, makes an important but overclaimed contribution to the literature due to a mismatch between the claims in the paper and the data presented.

Strengths:

The authors have identified an interesting question and use a strong set of complementary tools to address it:

(1) They analysed serial‐section TEM data to obtain reconstructions of every 13A and 13B neuron in the prothoracic segment. They manually proofread over 60 13A neurons and 64 13B neurons, then used automated synapse detection to build detailed connectivity maps and cluster neurons into functional motifs.

(2) They used optogenetic tools with a range of genetic driver lines in freely behaving flies to test the contribution of subsets of 13A and 13B neurons.

(3) They used a connectome-constrained computational model to determine how the mapped connectivity relates to the rhythmic output of the behavior.

Weaknesses:

The manuscript aims to reveal an instructive, rhythm-generating role for premotor inhibition in coordinating the multi-joint leg synergies underlying grooming. It makes a valuable contribution, but currently, the main claims in the paper are not well-supported by the presented evidence.

Major points

(1) Starting with the title of this manuscript, "Inhibitory circuits generate rhythms for leg movements during Drosophila grooming", the authors raise the expectation that they will show that the 13A and 13B hemilineages produce rhythmic output that underlies grooming. This manuscript does not show that. For instance, to test how they drive the rhythmic leg movements that underlie grooming requires the authors to test whether these neurons produce the rhythmic output underlying behavior in the absence of rhythmic input. Because the optogenetic pulses used for stimulation were rhythmic, the authors cannot make this point, and the modelling uses a "black box" excitatory network, the output of which might be rhythmic (this is not shown). Therefore, the evidence (behavioral entrainment; perturbation effects; computational model) is all indirect, meaning that the paper's claim that "inhibitory circuits generate rhythms" rests on inferred sufficiency. A direct recording (e.g., calcium imaging or patch-clamp) from 13A/13B during grooming - outside the scope of the study - would be needed to show intrinsic rhythmogenesis. The conclusions drawn from the data should therefore be tempered. Moreover, the "black box" needs to be opened. What output does it produce? How exactly is it connected to the 13A-13B circuit? The context in which the 13A and 13B hemilineages sit also needs to be explained. What do we know about the other inputs to the motorneurons studied? What excitatory circuits are there? Furthermore, the introduction ignores many decades of work in other species on the role of inhibitory cell types in motor systems. There is some mention of this in the discussion, but even previous work in Drosophila larvae is not mentioned, nor crustacean STG, nor any other cell types previously studied. This manuscript makes a valuable contribution, but it is not the first to study inhibition in motor systems, and this should be made clear to the reader.

(2) The experimental evidence is not always presented convincingly, at times lacking data, quantification, explanation, appropriate rationales, or sufficient interpretation.

(3) The statistics used are unlike any I remember having seen, essentially one big t-test followed by correction for multiple comparisons. I wonder whether this approach is optimal for these nested, high‐dimensional behavioral data. For instance, the authors do not report any formal test of normality. This might be an issue given the often skewed distributions of kinematic variables that are reported. Moreover, each fly contributes many video segments, and each segment results in multiple measurements. By treating every segment as an independent observation, the non‐independence of measurements within the same animal is ignored. I think a linear mixed‐effects model (LMM) or generalized linear mixed model (GLMM) might be more appropriate.

(4) The manuscript mentions that legs are used for walking as well as grooming. While this is welcome, the authors then do not discuss the implications of this in sufficient detail. For instance, how should we interpret that pulsed stimulation of a subset of 13A neurons produces grooming and walking behaviours? How does neural control of grooming interact with that of walking?

(5) The manuscript needs to be proofread and edited as there are inconsistencies in labelling in figures, phrasing errors, missing citations of figures in the text, or citations that are not in the correct order, and referencing errors (examples: 81 and 83 are identical; 94 is missing in text).

Reviewer #3 (Public review):

Summary:

This is a very focused and well-performed study that uses a somewhat less common approach in the field of tissue mechanics, a deformable particle model, to propose a solution to some important phenomenological inconsistencies between the standard vertex- and SPV-model approaches and experiments. The authors' focus in their study is on the role of adhesion in glassy dynamics and solid-fluid transition of epithelia.

Strengths:

It is a carefully performed study with an important technical edge compared to "mainstream" vertex and SPV models: the ability to describe cell-cell boundaries with two distinct membranes. This may have an important implication for the phenomenology, like the role of adhesion in solid-fluid transition.

Weaknesses:

Apart from some specific suggestions for improvement and clarification, I believe the authors could do a better job in comparing their results and their approach to other similar models, such as the one by Kim et al (Reference 7).

Reviewer #3 (Public review):

Summary:

This study examines how synaptic endocytic zones are positioned using a combination of cultured neurons and the Drosophila neuromuscular junction. The authors test whether neuronal activity, active zone assembly, or liprin-α function is required to localize endocytic zone markers, including Dynamin, Amphiphysin, Nervous Wreck, PIPK1γ, and AP-180. None of the manipulations tested caused a coordinated disruption in the localization or abundance of these markers, leading to the conclusion that endocytic zones form independently of synaptic activity and active zone scaffolds.

Strengths:

The work is systematic and carefully executed, using multiple manipulations and two complementary model systems. The authors consistently examine multiple molecular markers, strengthening the interpretation that endocytic zone positioning is robust to changes in activity and structural assembly.

Weaknesses:

The main limitation is that the study does not test whether the methods used are sensitive enough to detect subtle functional disruption, and no condition tested produces clear disorganization of the endocytic zone. As a result, the conclusion that these zones assemble independently is supported by negative data, without a strong positive control for disassembly or mislocalization.

This paper addresses a longstanding question in synaptic biology and provides a well-supported boundary on the types of mechanisms that are likely to govern endocytic zone localization. The conclusions are well justified by the data, though additional evidence would be needed to define the assembly mechanism itself.

Reviewer #3 (Public review):

Summary:

The manuscript Kroon et al. described two algorithms, which when combined achieve high throughput automation of "martinizing" protein structures with selected protonation states and post-translational modifications.

The authors have addressed all of my concerns as provided previously. Specifically, Figure S2 will be a very useful guideline for future improvement (e.g., parallelization) of the code.

for - 3 major worldviews - citation

for - worldviews - major 3 contemporary - traditional - religion - modern - science - rational - materialism - 'clockwork universe' - postmodern - postindustrial - information society - critique of - progress - inequality of capitalism - ecological destruction - advocates for - justice - egalitarianism - pluralism - relativism - constructivism

Reviewer #3 (Public review):

Summary:

Kaldun et al. investigated the role of Dopamine Receptor Dop1R2 in different types and stages of olfactory associative memory in Drosophila melanogaster. Dop1R2 is a type 1 Dopamine receptor that can act both through Gs-cAMP and Gq-ERCa2+ pathways. The authors first developed a sophisticated tool where tissue-specific knock-out mutants can be generated using Crispr/Cas9 technology in combination with the Gal4/UAS gene-expression toolkit. They direct the K.O. mutation to intrinsic neurons of the main associative memory centre fly brain: the mushroom body (MB). There are three main types of MB-neurons, or Kenyon cells, according to their axonal projections: a/b; a'/b' and g neurons.

Kaldun et al. found that, while not required for short-term memory, dop1R2 is necessary in a/b and a'/b' but not in gamma neurons to display normal appetitive and aversive middle-term (2h) and long-term (24h) memory. These results showcase a compartmentalized role of Dop1R2 in specific neuronal subtypes of the main memory centre of the fly brain for the expression of middle and long-term memories.

The conclusions of this paper are very well supported by the data, and the authors systematically addressed the requirement of a very interesting type of dopamine receptor in both appetitive and aversive memories. These findings are important for the fields of learning and memory and dopaminergic neuromodulation, among others.

Importantly, the authors of this paper produced a tool to generate tissue-specific knock out mutants of dop1R2. Although reports on the requirement of this gene in different memory phases exist, the genetic tools used here represent the most sophisticated approach to induce a loss of function phenotypes in neurons of interest.

Overall, the authors generated a very useful tool to study dopamine neuromodulation in any given circuit when used in combination with the powerful genetic toolkit available in Drosophila. The reports on this paper confirmed a previously described role of Dop1R2 in the expression of aversive and appetitive LTM providing spatio-temporal resolution and additionally, they mapped these effects to two types of memory neurons in the fly brain, shedding light into the intricate modulation of dopamine in memory circuits.

Reviewer #2 (Public review):

Summary:

The study aims to probe the neural correlates of visual serial dependence - the phenomenon that estimates of a visual feature (here motion direction) are attracted towards the recent history of encoded and reported stimuli. The authors utilize an established retro-cue working memory task together with magnetoencephalography, which allows to probe neural representations of motion direction during encoding and retrieval (retro-cue) periods of each trial. The main finding is that neural representations of motion direction are not systematically biased during the encoding of motion stimuli, but are attracted towards the motion direction of the previous trial's target during the retrieval (retro-cue period), just prior to the behavioral response. By demonstrating a neural signature of attractive biases in working memory representations, which align with attractive behavioral biases, this study highlights the importance of post-encoding memory processes in visual serial dependence.

Strengths:

The main strength of the study is its elegant use of a retro-cue working memory task together with high temporal resolution MEG, enabling to probe neural representations related to stimulus encoding and working memory. The behavioral task elicits robust behavioral serial dependence and replicates previous behavioral findings by the same research group. The careful neural decoding analysis benefits from a large number of trials per participant, considering the slow-paced nature of the working memory paradigm. This is crucial in a paradigm with considerable trial-by-trial behavioral variability (serial dependence biases are typically small, relative to the overall variability in response errors). While the current study is broadly consistent with previous studies showing that attractive biases in neural responses are absent during stimulus encoding (prev. studies reported repulsive biases), to my knowledge, it is the first study showing attractive biases in current stimulus representations during working memory. The study also connects to previous literature showing reactivations of previous stimulus representations, although the link between reactivations and biases remains somewhat vague in the current manuscript. Together, the study reveals an interesting avenue for future studies investigating the neural basis of visual serial dependence.

Weaknesses:

The main weakness of the current manuscript is that the authors could have done more analyses to address the concern that their neural decoding results are driven by signals related to eye movements. The authors show that participants' gaze position systematically depended on the current stimuli's motion directions, which, together with previous studies on eye movement-related confounds in neural decoding, justifies such a concern. The authors seek to rule out this confound by showing that the consistency of stimulus-dependent gaze position does not correlate with (a) the neural reconstruction fidelity and (b) the attractive shift in reconstructed motion direction. However, the authors' approach of quantifying stimulus-dependent eye movements only considers gaze angle and not gaze amplitude, and thus potentially misses important features of eye movements that could manifest in the MEG data. Moreover, it is unclear whether the gaze consistency metric should correlate with attractive history biases in neural decoding, if there were a confound. These two concerns could be potentially addressed by (1) directly decoding stimulus motion direction from x-y gaze coordinates and relating this decoding performance to neural reconstruction fidelity, and (2) investigating whether gaze coordinates themselves are history-dependent and are attracted to the average gaze position associated with the previous trials' target stimulus. If the authors could show that (2) is not the case, I would be much more convinced that their main finding is not driven by eye movement confounds.

The sample size (n = 10) is definitely at the lower end of sample sizes in this field. The authors collected two sessions per participant, which partly alleviates the concern. However, given that serial dependencies can be very variable across participants, I believe that future studies should aim for larger sample sizes.

It would have been great to see an analysis in source space. As the authors mention in their introduction, different brain areas, such as PPC, mPFC and dlPFC have been implicated in serial biases. This begs the question which brain areas contribute to the serial dependencies observed in the current study? For instance, it would be interesting to see whether attractive shifts in current representations and pre-stimulus reactivations of previous stimuli are evident in the same or different brain areas.

Reviewer #3 (Public review):

Summary:

Conditioned analgesia refers to the ability of a learned fear cue to suppress pain-related behavior and neural activity. Understudied, the authors developed a novel conditioned analgesia procedure in which a cue that had been paired or unpaired with shock was played while a hot plate increased temperature. Compared to several control conditions, the authors found increased latency to a nociceptive response (paw licking). The authors identified somatostatin neurons in the periaqueductal gray as a likely mediator of the behavior. They then showed that: (1) stimulating vlPAG-SST neurons blocked nociceptive response latency increases to the CS+, (2) stimulating vlPAG-SST neurons suppressed fear retrieval freezing, (3) stimulating vs. inhibiting vlPAG-SST neurons drove opposing modulation of c-fibers and Aδ-fibers, (4) direct-projecting vlPAG SST neurons modulate freezing while RVM-projecting vlPAG SST neurons modulate conditioned analgesia.

Strengths:

These experiments have many strengths. The behavioral assay is chief among them. The assay is robust and controls for confounding factors to reveal a repeatable effect of a shock-paired cue to delay nociceptive responding. The optogenetic experiments provide the correct level of temporal precision, given the authors' time-specific interest in cued responding. Combining neuronal manipulations with spinal recordings is particularly innovative, especially in the context of more behavioral neuroscience-based assays. All-in-all, I found this to be an exceptionally strong set of experiments.

Weaknesses:

No obvious weaknesses were identified by this Reviewer.

Reviewer #3 (Public review):

Summary:

The authors investigated fibroblasts' communication with key cell types in developing and neonatal hearts, with focus on critical roles of fibroblast-cardiomyocyte and fibroblast-endothelial cells network in cardiac morphogenesis. They tried to map the spatial distribution of these cell types and reported the major pathways and signaling molecules driving the communication. They also used Cre-DTA system to ablate Pdgfra labeled cells and observed myocardial and endothelial cell defects at development. They screened the pathways and genes using sequencing data of ablated heart. Lastly they reported a compensatory collagen expression in long term ablated neonate heart. Overall, this study provides us with important insight on fibroblasts' roles in cardiac development and will be a powerful resource for collagens and ECM focused research.

Strengths:

The authors utilized good analyzing tools to investigate on multiple database of single cell sequencing and Multi-seq. They identified significant pathways, cellular and molecular interactions of fibroblasts. Additionally, they compared some of their analytic findings with human database, and identified several groups of ECM genes with varying roles in mice.

Weaknesses:

This study is majorly based on sequencing data analysis. At the bench, they used very strident technique to study fibroblast functions by ablating one of the major cell population of heart. Also, experimental validation of their analyzed downstream pathways will be required eventually.

Comments on Revised Version (from BRE):

The authors did a good job addressing the questions asked at first review. However, I have some minor concerns.

(1) The paper notes that collagen signaling is observed in FB-VasEC in humans, but not in FB-VenCM, unlike mice. Did the authors analyze predictive ligand receptor interaction as they did with control and ablated mice heart? This could add valuable new insights that how FB regulate ventricular CM in human heart.

(2) The authors provided data on Defect in CD31 expression in several models. Did they observe any other phenotypes associated with defective endothelial or vascular system? Such as, blood accumulation in pericardium, larger/smaller capillaries? Did they also examine percentage of Cdh5+ cells?

(3) Please mention the sample age of Figure 2A-C.

(4) Please follow the same style to describe X axis in graphs in Figure 3D (and all similar graphs in the manuscript) as followed in 3G.

(5) It is important to provide echocardiographic M mode images with a comparable number of cardiac cycles in control and ablated (Fig. 6H).

(6) In the long-term neonatal ablation experiments, collagen expressions return to normal. The manuscript attributes this to possible "compensatory expression," Do they have any thoughts how this is regulated? Are other cell types stepping in, or are surviving FBs proliferating?

(7) While collagen is shown to be a dominant signaling molecule, its centrality is inferred primarily from scRNA-seq and ligand-receptor predictions. Did authors try any functional rescue experiment (e.g., exogenous collagen supplementation or receptor blockade) to directly validate this pathway's role in vivo?

Reviewer #3 (Public review):

Summary:

The authors investigated the assembly and polar localization of the chemosensory cluster in P. aeruginosa. They discovered that a certain protein (FlhF) is required for the polar localization of the chemosensory cluster while a fully-assembled motor is necessary for the assembly of the cluster. They found that flagella and chemosensory clusters always co-localize in the cell; either at the cell pole in wild type cells or randomly-located in the cell in FlhF mutant cells. They hypothesize that this co-localization is required to keep the level of another protein (CheY-P), which controls motor switching, at low levels as the presence of high-levels of this protein (if the flagella and chemosensory clusters were not co-localized) is associated with high-levels of c-di-GMP and cell aggregations.

Strengths:

The manuscript is clearly written and straightforward. The authors applied multiple techniques to study the bacterial motility system including fluorescence light microscopy and gene editing. In general, the work enhances our understanding of the subtlety of interaction between the chemosensory cluster and the flagellar motor to regulate cell motility.

Weaknesses:

The major weakness for me in this paper is that the authors never discussed how the flagellar genes expression is controlled in P. aeruginosa. For example, in E. coli there is a transcriptional hierarchy for the flagellar genes (early, middle, and late genes, see Chilcott and Hughes, 2000). Similarly, Campylobacter and Helicobacter have a different regulatory cascade for their flagellar genes (See Lertsethtakarn, Ottemann, and Hendrixson, 2011). How does the expression of flagellar genes in P. aeruginosa compare to other species? how many classes are there for these genes? is there a hierarchy in their expression and how does this affect the results of the FliF and FliG mutants? In other words, if FliF and FliG are in class I (as in E. coli) then their absence might affect the expression of other later flagellar genes in subsequent classes (i.e., chemosensory genes). Also, in both FliF and FliG mutants no assembly intermediates of the flagellar motor are present in the cell as FliG is required for the assembly of FliF (see Hiroyuki Terashima et al. 2020, Kaplan et al. 2019, Kaplan et al. 2022). It could be argued that when the motor is not assembled then this will affect the expression of the other genes (e.g., those of the chemosensory cluster) which might play a role in the decreased level of chemosensory clusters the authors find in these mutants.

Comments on revisions:

I believe the authors have performed additional experiments that improved their manuscript and they have answered many of my comments and those of the other reviewers. I am supportive of publishing this manuscript, but I still find the following points that are not clear to me (probably I am misunderstanding some points; the authors can clarify).

(1) In response to reviewer 1, the authors say that they "analyzed and categorized the distribution of the chemotaxis complex in both wild-type and flhF mutant strains into three patterns: precise-polar, near-polar, and mid-cell localization." I can see what they mean by polar and mid-cell, but near-polar sounds a bit elusive? Can they provide examples of this stage and mention how accurately they can identify it? Also, do the pie charts they show in Figure S4 really show "significant alterations"? There is a difference between 98% and 85% as they mention in their response to reviewer 1, but I am not sure that this is significant? Probably they can explain/change the language in the text? Also, the number of cells they counted for FlhF mutant is more than the double of other strains (WT and FlhF FliF mutant)?

(2) One thing that also confused me is the following: One point that the authors stress is that FlhF localizes both the flagellum and the chemoreceptors to the pole. However, if I look at Figure 2B, the flagellum and the chemoreceptors still co-localize together (although not at the pole). If FlhF was responsible for co-localizing both of them to the pole, then wouldn't one expect them to be randomly localized in this mutant and by that I mean that they do not co-localize but that each of them (the flagellum and the chemoreceptors) are located in a different random location of the cell (not co-localized). The fact that they are still co-localized together in this mutant could also be interpreted by, for example, that FlhF localizes the flagellum to the pole and another mechanism localizes the chemoreceptors to the flagellum, hence, they still co-localize in this mutant because the chemoreceptors follow the flagellum by another mechanism to wherever it goes?

(3) In the response to reviewers, the authors mention "suggesting that the assembly of the receptor complex is likely influenced mainly by the C-ring and MS-ring structures rather than by the P ring" . However, in the article, they still write "The complete assembly of the motor serves as a partial prerequisite for the assembly of the chemotaxis complex, and its assembly site is also regulated by the polar anchor protein FlhF" despite their FlgI results which is not in accordance with this statement? Also, As I mentioned in my previous report, in FliG and FliF mutant the motor does not assemble (see Hiroyuki Terashima et al. 2020., and Kaplan et al., 2022).

(4) The authors have said in their response to my point "and currently, there is no evidence that FliA activity is influenced by proteins like FliG". I just want to clarify what I meant in my previous report: In E. coli, FliA binds to FlgM, and when the hook is assembled FlgM is secreted outside the cell allowing FliA to trigger the transcription of class III genes, which include the chemosensory genes (see Figure 5 in Beeby et al, 2020 in FEMS Microbiology, and Chilcott and Hughes, 2000). This implies that if the hook is not built, then late genes (including the chemoreceptors) should not be present. However, in Kaplan et al., 2019, the authors imaged a FliF mutant in Shewanella oneidensis (Figure S3) and still saw that chemoreceptors are present (I believe the authors must highlight this). This suggests that species such as Shewanella and Pseudomonas have a different assembly process than that E. coli, and although the authors say that in the text, I believe they still can refine this part more in the spirit of what I wrote here.

I do not like to ask for additional experiments in the second round of review, so for me if the authors modify the text to tackle these points and allow for probable alternative explanations/ highlight gaps/ modify language used for some claims, then that is fine with me.

Reviewer #3 (Public review):

Summary:

Activity of serotonin (5-HT) releasing neurons as well as 5-HT levels in brain structures targeted by serotoninergic axons are known to fluctuate substantially across the animal's sleep/wake cycle, with high 5-HT during wakefulness (WAKE), intermediate 5-HT levels during non-REM sleep (NREM) and very low 5-HT levels during REM sleep. Recent studies have shown that during NREM, activity of 5-HT neurons in raphe nuclei oscillates at very low frequencies (0.01 - 0.05 Hz) and this ultraslow oscillation is negatively coupled to broadband EEG power. However, how exactly this 5-HT oscillation affects neural activity in downstream structures is unclear.

The present study addresses this gap by replicating the observation of the ultraslow oscillation in the 5-HT system, and further observing that hippocampal sharp wave-ripples (SWRs), biomarkers of offline memory processing, occur preferentially in barrages on the falling phase of the 5-HT oscillation during both wakefulness and NREM sleep. In contrast, the study found that the raising phase of the 5-HT oscillation is associated with microarousals during NREM and increased muscular activity during WAKE. Finally, the raising 5-HT phase was also found to be associated with increased synchrony between the hippocampus and neocortex.

In vivo findings are further supported by an ex vivo demonstration of dose-dependent serotonergic SWR modulation, lends support to the potential causal relationship between 5-HT slow oscillation and hippocampal dynamics.

Overall, the study constitutes a valuable contribution to the field by reporting a close association between, on one hand, raising 5-HT and arousal and, on the other hand, falling 5-HT and offline memory processes.

Strengths:

The study makes a compelling use of the state-of-the art methodology to address its aims: the genetically encoded 5-HT sensor used in the study is ideal for capturing the ultraslow 5-HT dynamics and the novel detection method for SWRs outperforms current state-of-the-art algorithms and will be useful to many scientists in the field. Explicit validation of both of these methods is a particular strength of this study.

The analytical methods used in the article are appropriate and are convincingly applied, the use of a general linear mixed model for statistical analysis is a particularly welcome choice as it guards against pseudoreplication while preserving statistical power.

Pharmacological demonstration of serotonergic SWR modulation in brain slices adds further weight to the possible direct role of 5-HT in hippocampal dynamics in vivo.

Overall, the manuscript makes a strong case for distinct sub-states across WAKE and NREM, associated with different phases of the 5-HT oscillation.

Weaknesses:

All in vivo evidence presented in the study is correlational, although the ex vivo results do suggest a possibility of a causal relationship between 5HT levels and hippocampal dynamics in the intact brain.

Reviewer #3 (Public review):

Summary:

This important study shows that stationary phase bacteria survive antimicrobial peptide treatment by switching on efflux pumps, generating low accumulating subpopulations that evade killing-a finding with clear implications for the design of peptide based antibiotics and for researchers studying antimicrobial resistance. The evidence is solid and frequently convincing, as diverse single cell assays, genetics and chemical inhibition coherently link reduced intracellular peptide to survival, even though a few mechanistic details warrant further exploration.

Strengths:

The authors investigate how Escherichia coli (and, to a lesser extent, Pseudomonas aeruginosa) survive exposure to the antimicrobial peptide (AMP) tachyplesin. Because resistance to AMPs is thought to rely heavily on non genetic adaptations rather than on classical mutation based mechanisms, the study focuses on phenotypic heterogeneity and seeks to pinpoint the cellular processes that protect a subset of cells. Using fluorescently labelled tachyplesin, single cell imaging, flow cytometry, transcriptomics, targeted genetics, and chemical perturbations, the authors report that stationary phase cultures harbor two phenotypic states: high accumulating cells that die and low accumulating cells that survive. They further propose and show that inducible efflux activity is the primary driver of survival and show that either efflux inhibition (sertraline, verapamil) or nutrient supplementation prevents the emergence of low accumulators and boosts killing.

The experiments unambiguously reveal that the cells respond to stress heterogeneously, with two distinct subpopulations - one with better survival than the other. This primary phenotype is convincingly shown across various E. coli strains, including clinical isolates. The authors probed the underlying mechanism from several angles, with important additional experiments in the revised version that strengthens the original conclusions in several ways. Newly added efflux assays with ethidium bromide, together with proteinase treatment experiments and ΔacrAΔtolC and ΔqseB/qseC mutant data, illustrate that the low accumulating subpopulation can actively export intracellular compounds. The authors took great care to temper their language to acknowledge other potential alternatives that could explain some of the data such as altered influx, vesicle release or proteolysis, metabolic activity of the cells, indirect effects of sertraline treatment, etc. Additional metabolic dye measurements confirm that low accumulators are less metabolically active, and a new data on nutrient supplementation shows that forcing growth increases peptide uptake and lethality. The authors clarify the crucial point of where antimicrobial peptides actually bind on the cell within the broader survival mechanism and present their conclusions, along with potential caveats, with commendable clarity.

Weaknesses:

Despite these advances, the contribution of efflux may require more direct evidence to further dissect whether efflux is necessary, sufficient, or contributory. The facts that the key low-efflux mutant still retains a small fraction of survivors and that the inhibitors used may cause other physiological changes leading to higher efflux are still unaccounted for. The lipidomic and vesicle findings, while intriguing, remain descriptive, and direct tests of their functional relevance would further solidify the mechanistic models.

Conclusion:

Even with these limitations, the study provides valuable insight into non genetic resistance mechanisms to AMPs and highlights inducible heterogeneity as a critical obstacle to peptide therapeutics. In a much broader context, this study also underscores the importance of efflux physiology even for those antimicrobials that seemingly would not have intracellular targets.

Reviewer #3 (Public review):

Summary:

The paper presents a novel contractile gut organoid system that allows for in vitro studying of rudimentary peristaltic motions in embryonic tissues by facilitating GCaMP-live imaging of Ca2+ dynamics, while highlighting the importance and sufficiency of ICC and SMC interactions in generating consistent contractions reminiscent of peristalsis. It also argues that ENS at later embryonic stages might not be necessary for coordination of peristalsis.

Strengths:

The manuscript by Yagasaki, Takahashi, and colleagues represents an exciting new addition to the toolkit available for studying fundamental questions in the development and physiology of the hindgut. The authors carefully lay out the protocol for generating contractile gut organoids from chick embryonic hindgut and perform a series of experiments that illustrate the broader utility of these organoids for studying the gut. This reviewer is highly supportive of the manuscript following highly responsive revisions in response to prior reviewer feedback.

Reviewer #3 (Public review):

Summary:

The authors performed time-resolved proteomics and phospho-proteomics in Xenopus oocytes from prophase I through the MII arrest of the unfertilized egg. The data contains protein abundance and phosphorylation sites of a large number set of proteins at different stages of oocyte maturation. The large sets of data are of high quality. In addition, the authors discussed several key pathways critical for the maturation. The data is very useful for researchers, not only researchers in Xenopus oocytes but also those in oocyte biology in other organisms.

Strengths:

The data of proteomics and phospho-proteomics in Xenopus oocyte maturation is very useful for future studies to understand molecular networks in oocyte maturation.

Weaknesses:

Although the authors offered molecular pathways of the phosphorylation in translation, protein degradation, cell cycle regulation, and chromosome segregation. The authors did not check the validity of the molecular pathways based on their proteomic data by experimentation. But this is not essential since this is a resource paper.

Reviewer #3 (Public review):

Summary:

This manuscript aims to determine cultural biases and misconceptions in inclusive sex research and evaluate the efficacy of interventions to improve knowledge and shift perceptions to decrease perceived barriers for including both sexes in basic research.

Overall, this study demonstrates that despite the intention to include both sexes and a general belief in the importance of doing so, relatively few people routinely include both sexes. Further, the perceptions of barriers to doing so are high, including misconceptions surrounding sample size, disaggregation, and variability of females. There was also a substantial number of individuals without the statistical knowledge to appropriately analyze data in studies inclusive of sex. Interventions increased knowledge and decreased perception of barriers.

Strengths:

(1) This manuscript provides evidence for the efficacy of interventions for changing attitudes and perceptions of research.

(2) This manuscript also provides a training manual for expanding this intervention to broader groups of researchers.

Weaknesses:

The major weakness here is that the post-workshop assessment is a single time point, soon after the intervention. As this paper shows, intention for these individuals is already high, so does decreasing perception of barriers and increasing knowledge change behavior, and increase the number of studies that include both sexes?

Similarly, does the intervention start to shift cultural factors? Do these contribute to a change in behavior?

Reviewer #3 (Public review):

Summary:

This study investigates the role of the host protein RBMX2 in regulating the response to Mycobacterium bovis infection and its connection to epithelial-mesenchymal transition (EMT), a key pathway in cancer progression. Using bovine and human cell models, the authors have wisely shown that RBMX2 expression is upregulated following M. bovis infection and promotes bacterial adhesion, invasion, and survival by disrupting epithelial tight junctions via the p65/MMP-9 signaling pathway. They also demonstrate that RBMX2 facilitates EMT and is overexpressed in human lung cancers, suggesting a potential link between chronic infection and tumor progression. The study highlights RBMX2 as a novel host factor that could serve as a therapeutic target for both TB pathogenesis and infection-related cancer risk.

Strengths:

The major strengths lie in its multi-omics integration (transcriptomics, proteomics, metabolomics) to map RBMX2's impact on host pathways, combined with rigorous functional assays (knockout/knockdown, adhesion/invasion, barrier tests) that establish causality through the p65/MMP-9 axis. Validation across bovine and human cell models and in clinical tissue samples enhances translational relevance. Finally, identifying RBMX2 as a novel regulator linking mycobacterial infection to EMT and cancer progression opens exciting therapeutic avenues.

Weaknesses:

Although it's a solid study, there are a few weaknesses noted below.

(1) In the transcriptomics analysis, the authors performed (GO/KEGG) to explore biological functions. Did they perform the search locally or globally? If the search was performed with a global reference, then I would recommend doing a local search. That would give more relevant results. What is the logic behind highlighting some of the enriched pathways (in red), and how are they relevant to the current study?

(2) While the authors show that RBMX2 expression correlates with EMT-related gene expression and barrier dysfunction, the evidence for direct association remains limited in this study. How does RBMX2 activate p65? Does it bind directly to p65 or modulate any upstream kinases? Could ChIP-seq or CLIP-seq provide further evidence for direct RNA or DNA targets of RBMX2 that drive EMT or NF-κB signaling?

(3) The manuscript suggests that RBMX2 enhances adhesion/invasion of several bacterial species (e.g., E. coli, Salmonella), not just M. bovis. This raises questions about the specificity of RBMX2's role in Mycobacterium-specific pathogenesis. Is RBMX2 a general epithelial barrier regulator or does it exhibit preferential effects in mycobacterial infection contexts? How does this generality affect its potential as a TB-specific therapeutic target?

(4) The quality of the figures is very poor. High-resolution images should be provided.

(5) The methods are not very descriptive, particularly the omics section.

(6) The manuscript is too dense, with extensive multi-omics data (transcriptomics, proteomics, metabolomics) but relatively little mechanistic integration. The authors should have focused on the key mechanistic pathways in the figures. Improving the narratives in the Results and Discussion section could help readers follow the logic of the experimental design and conclusions.

Reviewer #3 (Public review):

The superfamily I 3'-5' DNA helicase Srs2 is well known for its role as an anti-recombinase, stripping Rad51 from ssDNA, as well as an anti-crossover factor, dissociating extended D-loops and favoring non-crossover outcome during recombination. In addition, Srs2 plays a key role in in ribonucleotide excision repair. Besides DNA repair defects, srs2 mutants also show a reduced recovery after DNA damage that is related to its role in downregulating the DNA damage signaling or checkpoint response. Recent work from the Zhao laboratory (PMID: 33602817) identified a role of Srs2 in downregulating the DNA damage signaling response by removing RPA from ssDNA. This manuscript reports further mechanistic insights into the signaling downregulation function of Srs2.

Using the genetic interaction with mutations in RPA1, mainly rfa1-zm2, the authors test a panel of mutations in Srs2 that affect CDK sites (srs2-7AV), potential Mec1 sites (srs2-2SA), known sumoylation sites (srs2-3KR), Rad51 binding (delta 875-902), PCNA interaction (delta 1159-1163), and SUMO interaction (srs2-SIMmut). All mutants were generated by genomic replacement and the expression level of the mutant proteins was found to be unchanged. This alleviates some concern about the use of deletion mutants compared to point mutations. Double mutant analysis identified that PCNA interaction and SUMO sites were required for the Srs2 checkpoint dampening function, at least in the context of the rfa1-zm2 mutant. There was no effect of this mutants in a RFA1 wild type background. This latter result is likely explained by the activity of the parallel pathway of checkpoint dampening mediated by Slx4, and genetic data with an Slx4 point mutation affecting Rtt107 interaction and checkpoint downregulation support this notion. Further analysis of Srs2 sumoylation showed that Srs2 sumoylation depended on PCNA interaction, suggesting sequential events of Srs2 recruitment by PCNA and subsequent sumoylation. Kinetic analysis showed that sumoylation peaks after maximal Mec1 induction by DNA damage (using the Top1 poison camptothecin (CPT)) and depended on Mec1. This data are consistent with a model that Mec1 hyperactivation is ultimately leading to signaling downregulation by Srs2 through Srs2 sumoylation. Mec1-S1964 phosphorylation, a marker for Mec1 hyperactivation and a site found to be needed for checkpoint downregulation after DSB induction, did not appear to be involved in checkpoint downregulation after CPT damage. The data are in support of the model that Mec1 hyperactivation when targeted to RPA-covered ssDNA by its Ddc2 (human ATRIP) targeting factor, favors Srs2 sumoylation after Srs2 recruitment to PCNA to disrupt the RPA-Ddc2-Mec1 signaling complex. Presumably, this allows gap filling and disappearance of long-lived ssDNA as the initiator of checkpoint signaling, although the study does not extend to this step.

Strengths:

(1) The manuscript focuses on the novel function of Srs2 to downregulate the DNA damage signaling response and provide new mechanistic insights.

(2) The conclusions that PCNA interaction and ensuing Srs2-sumoylation are involved in checkpoint downregulation are well supported by the data.

Reviewer #3 (Public review):

Summary:

This study by Tetenborg S et al. identifies proteins that are physically closely associated with gap junctions in retinal neurons of mice and zebrafish using BioID, a technique that labels and isolates proteins in proximal to a protein of interest. These proteins include scaffold proteins, adhesion molecules, chemical synapse proteins, components of the endocytic machinery, and cytoskeleton-associated proteins. Using a combination of genetic tools and meticulously executed immunostaining, the authors further verified the colocalizations of some of the identified proteins with connexin-positive gap junctions. The findings in this study highlight the complexity of gap junctions. Electrical synapses are abundant in the nervous system, yet their regulatory mechanisms are far less understood than those of chemical synapses. This work will provide valuable information for future studies aiming to elucidate the regulatory mechanisms essential for the function of neural circuits.

Strengths:

A key strength of this work is the identification of novel gap junction-associated proteins in AII amacrine cells and photoreceptors using BioID in combination with various genetic tools. The well-studied functions of gap junctions in these neurons will facilitate future research into the functions of the identified proteins in regulating electrical synapses.

The authors have addressed my concerns in the revised manuscript.

Reviewer #3 (Public review):

Summary:

This manuscript introduces a high-resolution, open-source light-sheet fluorescence microscope optimized for sub-cellular imaging.

The system is designed for ease of assembly and use, incorporating a custom-machined baseplate and in silico optimized optical paths to ensure robust alignment and performance. The authors demonstrate lateral and axial resolutions of ~235 nm and ~350 nm after deconvolution, enabling imaging of sub-diffraction structures in mammalian cells.

The important feature of the microscope is the clever and elegant adaptation of simple gaussian beams, smart beam shaping, galvo pivoting and high NA objectives to ensure a uniform thin light-sheet of around 400 nm in thickness, over a 266 micron wide Field of view, pushing the axial resolution of the system beyond the regular diffraction limited-based tradeoffs of light-sheet fluorescence microscopy.

Compelling validation using fluorescent beads and multicolor cellular imaging highlights the system's performance and accessibility. Moreover, a very extensive and comprehensive manual of operation is provided in the form of supplementary materials. This provides a DIY blueprint for researchers who want to implement such a system.

Strengths:

(1) Strong and accessible technical innovation:

With an elegant combination of beam shaping and optical modelling, the authors provide a high-resolution light-sheet system that overcomes the classical light-sheet tradeoff limit of a thin light-sheet and a small field of view. In addition, the integration of in silico modelling with a custom-machined baseplate is very practical and allows for ease of alignment procedures. Combining these features with the solid and super-extensive guide provided in the supplementary information, this provides a protocol for replicating the microscope in any other lab.

(2) Impeccable optical performance and ease of mounting of samples:

The system takes advantage of the same sample-holding method seen already in other implementations, but reduces the optical complexity. At the same time, the authors claim to achieve similar lateral and axial resolution to Lattice-light-sheet microscopy (although without a direct comparison (see below in the "weaknesses" section). The optical characterization of the system is comprehensive and well-detailed. Additionally, the authors validate the system imaging sub-cellular structures in mammalian cells.

(3) Transparency and comprehensiveness of documentation and resources:

A very detailed protocol provides detailed documentation about the setup, the optical modeling, and the total cost.

Weaknesses:

(1) Limited quantitative comparisons:

Although some qualitative comparison with previously published systems (diSPIM, lattice light-sheet) is provided throughout the manuscript, some side-by-side comparison would be of great benefit for the manuscript, even in the form of a theoretical simulation. While having a direct imaging comparison would be ideal, it's understandable that this goes beyond the interest of the paper; however, a table referencing image quality parameters (taken from the literature), such as signal-to-noise ratio, light-sheet thickness, and resolutions, would really enhance the features of the setup presented. Moreover, based also on the necessity for optical simplification, an additional comment on the importance/difference of dual objective/single objective light-sheet systems could really benefit the discussion.

(2) Limitation to a fixed sample:

In the manuscript, there is no mention of incubation temperature, CO₂ regulation, Humidity control, or possible integration of commercial environmental control systems. This is a major limitation for an imaging technique that owes its popularity to fast, volumetric, live-cell imaging of biological samples.

(3) System cost and data storage cost:

While the system presented has the advantage of being open-source, it remains relatively expensive (considering the 150k without laser source and optical table, for example). The manuscript could benefit from a more direct comparison of the performance/cost ratio of existing systems, considering academic settings with budgets that most of the time would not allow for expensive architectures. Moreover, it would also be beneficial to discuss the adaptability of the system, in case a 30k objective could not be feasible. Will this system work with different optics (with the obvious limitations coming with the lower NA objective)? This could be an interesting point of discussion. Adaptability of the system in case of lower budgets or more cost-effective choices, depending on the needs.

Last, not much is said about the need for data storage. Light-sheet microscopy's bottleneck is the creation of increasingly large datasets, and it could be beneficial to discuss more about the storage needs and the quantity of data generated.

Conclusion:

Altair-LSFM represents a well-engineered and accessible light-sheet system that addresses a longstanding need for high-resolution, reproducible, and affordable sub-cellular light-sheet imaging. While some aspects-comparative benchmarking and validation, limitation for fixed samples-would benefit from further development, the manuscript makes a compelling case for Altair-LSFM as a valuable contribution to the open microscopy scientific community.

Reviewer #3 (Public review):

Summary:

Mitochondrial oxphos causes lipid accumulation, leading to MASH, although the mechanism has been poorly understood. In this study, Funai and colleagues identify that reductions in cardiolipin in the mitochondria cause disruptions in the electron transport chain. Knockout of cardiolipin synthase was sufficient to drive MASH phenotypes, increase respiratory capacity, and cause electron leak at complexes II and III. It is well established that loss of cardiolipin increases ROS. Studies to date have been performed on whole tissue lysates, but to rule out which changes in mitochondrial lipids are driven by changes in mitochondrial number versus lipid synthesis/turnover, the authors uniquely purified mitochondria from human and mouse livers in MASH and NASH models for this study. This study provides critical information to the field that will inevitably help us better understand the mechanisms underlying MASH and NASH onset. The evidence provided is both convincing and compelling. With further suggested revision experiments, this study has the potential to change our understanding of MASH and NASH pathogenesis.

Strengths:

The authors use a unique approach of lipidomics on purified mitochondria. They also analyze many distinct MASH models and provide a unique resource for the field of comprehensive lipidomics analysis of the different ways in which MASH can be induced. The use of human tissue elevates the impact/significance of the findings.

Weaknesses:

The data on the super complexes was the least compelling, and frankly, I do not think the authors needed those data to make a compelling argument! The authors should shift their focus more to the compelling electron leak data they have collected. If possible, it would also strengthen the work to include cardiolipin rescues on more of the experiments. Finally, expanding their explanations of the model systems would be very helpful for the readership.

Reviewer #3 (Public review):

Summary:

In this paper, the authors made some interesting observations that EPB41L4A-AS1 lncRNA can regulate the transcription of both the nearby coding gene and genes on other chromosomes. They started by computationally examining lncRNA-gene pairs by analyzing co-expression, chromatin features of enhancers, TF binding, HiC connectome, and eQTLs. They then zoomed in on four pairs of lncRNA-gene pairs and used LNA antisense oligonucleotides to knock down these lncRNAs. This revealed EPB41L4A-AS1 as the only one that can regulate the expression of its cis-gene target EPB41L4A. By RNA-FISH, the authors found this lncRNA to be located in all three parts of a cell: chromatin, nucleoplasm, and cytoplasm. RNA-seq after LNA knockdown of EPB41L4A-AS1 showed that this increased >1100 genes and decreased >1250 genes, including both nearby genes and genes on other chromosomes. They later found that EPB41L4A-AS1 may interact with SUB1 protein (an RNA-binding protein) to impact the target genes of SUB1. EPB41L4A-AS1 knockdown reduced the mRNA level of SUB1 and altered the nuclear location of SUB1. Later, the authors observed that EPB41L4A-AS1 knockdown caused an increase of snRNAs and snoRNAs, likely via disrupted SUB1 function. In the last part of the paper, the authors conducted rescue experiments that suggested that the full-length, intron- and SNORA13-containing EPB41L4A-AS1 is required to partially rescue snoRNA expression. They also conducted SLAM-Seq and showed that the increased abundance of snoRNAs is primarily due to their hosts' increased transcription and stability. They end with data showing that EPB41L4A-AS1 knockdown reduced MCF7 cell proliferation but increased its migration, suggesting a link to breast cancer progression and/or metastasis.

Strengths:

Overall, the paper is well-written, and the results are presented with good technical rigor and appropriate interpretation. The observation that a complex lncRNA EPB41L4A-AS1 regulates both cis and trans target genes, if fully proven, is interesting and important.

Weaknesses:

The paper is a bit disjointed as it started from cis and trans gene regulation, but later it switched to a partially relevant topic of snoRNA metabolism via SUB1. The paper did not follow up on the interesting observation that there are many potential trans target genes affected by EPB41L4A-AS1 knockdown and there was limited study of the mechanisms as to how these trans genes (including SUB1 or NPM1 genes themselves) are affected by EPB41L4A-AS1 knockdown. There are discrepancies in the results upon EPB41L4A-AS1 knockdown by LNA versus by CRISPR activation, or by plasmid overexpression of this lncRNA.

Reviewer #3 (Public review):

Summary:

To make feeding decisions, animals need to process three types of information: positive cues like sweetness, negative cues like bitterness, and internal states such as hunger or satiety. This study aims to identify where the information is integrated in the fruit fly brain. The authors applied RNA sequencing on second-order gustatory neurons responsible for sweet and bitter processing, under fed and starved conditions. The sequencing data reveal significant changes in gene expression across sweet vs. bitter pathways and fed vs. starved states. The authors focus on the neuropeptide Leucokinin (Lk), whose expression is dependent on the starvation state. They identify a pair of neurons, named SELK neurons, which express Lk and receive direct input from both sweet and bitter gustatory neurons. These SELK neurons are ideal candidates to integrate gustatory and internal state information. Behavioral experiments show that blocking these neurons in starved flies alters their tolerance to bitter substances during feeding.

Strengths:

(1) The study employs a well-designed approach, targeting specific neuronal populations, which is more efficient and precise compared to traditional large-scale genetic screening methods.

(2) The RNAseq results provide valuable data that can be utilized in future studies to explore other molecules beyond Lk.

(3) The identification of SELK neurons offers a promising avenue for future research into how these neurons integrate conflicting gustatory signals and internal state information.

Weaknesses:

Unfortunately, due to technical challenges, the authors were unable to directly image the functional activity of SELK neurons.

Reviewer #3 (Public review):

Summary:

This study investigates the functional differences between barrel and septal columns in the mouse somatosensory cortex, focusing on how local inhibitory dynamics, particularly involving Elfn1-expressing SST⁺ interneurons, may mediate temporal integration of multi-whisker (MW) stimuli in septa. Using a combination of in vivo multi-unit recordings, calcium imaging, and anatomical tracing, the authors propose that septa integrate MW input in an Elfn1-dependent manner, enabling functional segregation from barrel columns.

Strengths:

The core hypothesis is interesting and potentially impactful. While barrels have been extensively characterized, septa remain less understood, especially in mice, and this study's focus on septal integration of MW stimuli offers valuable insights into this underexplored area. If septa indeed act as selective integrators of distributed sensory input, this would add a novel computational role to cortical microcircuits beyond what is currently attributed to barrels alone. The narrative of this paper is intellectually stimulating.

Weaknesses:

The methods used in the current study lack the spatial and cellular resolution needed to conclusively support the central claims. The main physiological findings are based on unsorted multi-unit activity (MUA) recorded via low-channel-count silicon probes. MUA inherently pools signals from multiple neurons across different distances and cell types, making it difficult to assign activity to specific columns (barrel vs. septa) or neuron classes (e.g., SST⁺ vs. excitatory). The recording radius (~50-100 µm or more) and the narrow width of septa (~50-100 µm or less) make it likely that MUA from "septal" electrodes includes spikes from adjacent barrel neurons. The authors do not provide spike sorting, unit isolation, or anatomical validation that would strengthen spatial attribution. Calcium imaging is restricted to SST⁺ and VIP⁺ interneurons in superficial layers (L2/3), while the main MUA recordings are from layer 4, creating a mismatch in laminar relevance.

Furthermore, while the role of Elfn1 in mediating short-term facilitation is supported by prior studies, no new evidence is presented in this paper to confirm that this synaptic mechanism is indeed disrupted in the knockout mice used here. Additionally, since Elfn1 is constitutively knocked out from development, the possibility of altered circuit formation-including changes in barrel structure and interneuron distribution, cannot be excluded and is not addressed.

Reviewer #3 (Public review):

Summary:

In this manuscript, Charonitakis, Pasadaki et al. investigated the neural circuits underlying homosensory/within-modal and heterosensory/cross-modal dishabituation of the olfactory avoidance response in Drosophila. Taking advantage of the accessible and sophisticated gene expression manipulation tools in the flies, this study traced neural pathways underlying response facilitation caused by different types of sensory stimuli and revealed both distinct and convergent neural components underlying these different forms of behavioral plasticity. The study first demonstrated that olfactory habituation of the octanol avoidance response can be facilitated by either a different odor (homosensory stimulus) or a foot shock (heterosensory stimulus). Then, the flies' nervous system was manipulated with gene expression tools to identify key neural components involved in mediating the behavioral facilitation caused by different types of sensory stimuli. It was found that different sensory stimuli are input into different parts of the nervous system, and signals converge in the mushroom bodies to generate response facilitation. It was also found that these facilitatory pathways are different from the olfactory habituation pathway in the lateral horns.

Strengths:

The authors took full advantage of the advanced genetic tools in flies and performed a series of experiments to pinpoint neural components in each pathway.

Weaknesses:

The key issue is that the main concepts of this manuscript appear to be based on a misunderstanding/misinterpretation of the literature. As the authors set out to settle the debate "whether the novel dishabituating stimulus elicits sensitization of the habituated circuits, or it engages distinct neuronal routes to bypass habituation reinstating the naïve response", it seems that the authors based their investigation on the premise that "sensitization" is mediated by a facilitatory process within the S-R pathway, and "dishabituation" by a facilitatory process outside the S-R pathway. This is not the status quo in the field, particularly with the prevailing theory like the Dual-Process Theory.

The original version of Dual-Process Theory (Groves and Thompson 1970, but also see Thompson 2008, Neurobiol Learn Mem) already hypothesized that habituation happens within the specific S-R pathway, and sensitization occurs separately in an "organism-wide" state system that modulates the output of all S-R pathways. Dishabituation is recognized by the Dual-Process Theory as sensitization (organism-wide facilitation) manifested on top of existing habituation (depressed S-R pathway). This notion has been supported by a wide range of studies, including cat spinal cord reflex (e.g. Spencer et al. 1966) and work in Aplysia on heterosynaptic facilitation for both sensitization and dishabituation. Therefore, simply showing that the newly identified facilitatory pathways are outside the S-R habituation pathway is insufficient to demonstrate dishabituation.

As behavioral facilitation of a habituated response can be achieved by dishabituating (specific recovery of the S-R pathway) and/or superimposed sensitizing (organism-wide) processes, dishabituation and sensitization of this olfactory response must be first dissociated; however, the study provided no evidence for the dissociation. Without this piece of evidence, the claim of this paper that the newly identified pathways mediate dishabituation is not fully supported.

The literature review of this manuscript has some discrepancies. In the introduction, the authors wrote "initial studies in Aplysia were consistent with the "dual-process theory" (Groves and Thompson 1979), where response recovery due to dishabituation appeared to result from sensitization superimposed on habituation, thus driving reversal of the attenuated response (Carew, Castellucci et al. 1971, Hochner, Klein et al. 1986, Marcus, Nolen et al. 1988, Ghirardi, Braha et al. 1992, Cohen, Kaplan et al. 1997, Antonov, Kandel et al. 1999, Hawkins, Cohen et al. 2006)." Hochner 1986 and Marcus 1988 in fact indicated otherwise. Hochner 1986 suggests that dishabituation and sensitization involve different molecular processes, while Marcus 1988 showed that dishabituation and sensitization have different behavioral characteristics. Therefore, the authors' statement is not supported by the cited literature.

Reviewer #3 (Public review):

Summary:

This is a valuable study providing solid evidence that the putative non-canonical initiation factor eIF2A has little or no role in the translation of any expressed mRNAs in cultured human (primarily HeLa) cells. Previous studies have implicated eIF2A in GTP-independent recruitment of initiator tRNA to the small (40S) ribosomal subunit, a function analogous to canonical initiation factor eIF2, and in supporting initiation on mRNAs that do not require scanning to select the AUG codon or that contain near-cognate start codons, especially upstream ORFs with non-AUG start codons, and may use the cognate elongator tRNA for initiation. Moreover, the detected functions for eIF2A were limited to, or enhanced by, stress conditions where canonical eIF2 is phosphorylated and inactivated, suggesting that eIF2A provides a back-up function for eIF2 in such stress conditions. CRISPR gene editing was used to construct two different knock-out cell lines that were compared to the parental cell line in a large battery of assays for bulk or gene-specific translation in both unstressed conditions and when cells were treated with inhibitors that induce eIF2 phosphorylation. None of these assays identified any effects of eIF2A KO on translation in unstressed or stressed cells, indicating little or no role for eIF2A as a back-up to eIF2 and in translation initiation at near-cognate start codons, in these cultured cells.

The study is very thorough and generally well executed, examining bulk translation by puromycin labeling and polysome analysis and translational efficiencies of all expressed mRNAs by ribosome profiling, with extensive utilization of reporters equipped with the 5'UTRs of many different native transcripts to follow up on the limited number of genes whose transcripts showed significant differences in translational efficiencies (TEs) in the profiling experiments. They also looked for differences in translation of uORFs in the profiling data and examined reporters of uORF-containing mRNAs known to be translationally regulated by their uORFs in response to stress, going so far as to monitor peptide production from a uORF itself. The high precision and reproducibility of the replicate measurements instil strong confidence that the myriad of negative results they obtained reflects the lack of eIF2A function in these cells rather than data that would be too noisy to detect small effects on the eIF2A mutations. They also tested and found no evidence for a recent claim that eIF2A localizes to the cytoplasm in stress and exerts a global inhibition of translation. Given the numerous papers that have been published reporting functions of eIF2A in specific and general translational control, this study is important in providing abundant, high-quality data to the contrary, at least in these cultured cells.

Strengths:

The paper employed two CRISPR knock-out cell lines and subjected them to a combination of high-quality ribosome profiling experiments, interrogating both main coding sequences and uORFs throughout the translatome, which was complemented by extensive reporter analysis, and cell imaging in cells both unstressed and subjected to conditions of eIF2 phosphorylation, all in an effort to test previous conclusions about eIF2A functioning as an alternative to eIF2.

Weaknesses:

No major issues were observed as the authors have provided additional evidence of the extent of ISR induction by tunicamycin. The discussion was also expanded to address concerns stemming from the previous version of the manuscript.

[Editors note: Reviewers and editors concluded that the authors revised the article in a satisfactory manner and no further concerns were raised]

Reviewer #3 (Public review):

Summary:

The authors seek to place archaeological maize samples (2 kernels) from Bolivia into genetic and geographical context and to assess signatures of selection. The kernels were dated to the end of the Incan empire, just prior to European colonization. Genetic data and analyses were used to characterize the distance from other ancient and modern maize samples and to predict the origin of the sample, which was discovered in a tomb near La Paz, Bolivia. Given the conquest of this region by the Incan empire, it is possible that the sample could be genetically similar to populations of maize in Peru, the center of the Incan empire. Signatures of selection in the sample could help reveal various environmental variables and cultural preferences that shaped maize genetic diversity in this region at that time.

Strengths:

The authors have generated substantial genetic data from these archaeological samples and have assembled a data set of published archaeological and modern maize samples that should help to place these samples in context. The samples are dated to an interesting time in the history of South America during a period of expansion of the Incan empire and just prior to European colonization. Much could be learned from even this small set of samples.

Weaknesses:

(1) Sample preparation and sequencing:<br /> Details of the quality of the samples, including the percentage of endogenous DN,A are missing from the methods. The low percentage of mapped reads suggests endogenous DNA was low, and this would be useful to characterize more fully. Morphological assessment of the samples and comparison to morphological data from other maize varieties is also missing. It appears that the two kernels were ground separately and that DNA was isolated separately, but data were ultimately pooled across these genetically distinct individuals for analysis. Pooling would violate assumptions of downstream analysis, which included genetic comparison to single archaeological and modern individuals.

(2) Genetic comparison to other samples:<br /> The authors did not meaningfully address the varying ages of the other archaeological samples and modern maize when comparing the genetic distance of their samples. The archaeological samples were as old as >5000 BP to as young as 70 BP and therefore have experienced varying extents of genetic drift from ancestral allele frequencies. For this reason, age should explicitly be included in their analysis of genetic relatedness.

(3) Assessment of selection in their ancient Bolivian sample:<br /> This analysis relied on the identification of alleles that were unique to the ancient sample and inferred selection based on a large number of unique SNPs in two genes related to internode length. This could be a technical artifact due to poor alignment of sequence data, evidence supporting pseudogenization, or within an expected range of genetic differentiation based on population structure and the age of the samples. More rigor is needed to indicate that these genetic patterns are consistent with selection. This analysis may also be affected by the pooling of the Bolivian archaeological samples.

(4) Evidence of selection in modern vs. ancient maize: In this analysis, samples were pooled into modern and ancient samples and compared using the XP-EHH statistic. One gene related to ovule development was identified as being targeted by selection, likely during modern improvement. Once again, ancient samples span many millennia and both South, Central, and North America. These, and the modern samples included, do not represent meaningfully cohesive populations, likely explaining the extremely small number of loci differentiating the groups. This analysis is also complicated by the pooling of the Bolivian archaeological samples.

https://clockhistory.com/westclox/products/ben/index.html

Reviewer #3 (Public review):

Summary:<br /> Bone morphogenetic protein (BMP) signaling instructs multiple processes during development including cell proliferation and differentiation. The authors set out to understand the role of PRDM16 in these various functions of BMP signaling. They find that PRDM16 and BMP co-operate to repress stem cell proliferation by regulating the genomic distribution of BMP pathway transcription factors. They additionally show that PRDM16 impacts choroid plexus epithelial cell specification. The authors provide evidence for a regulatory circuit (constituting of BMP, PRDM16 and Wnt) that influences stem cell proliferation/differentiation.

Strengths:<br /> I find the topics studied by the authors in this study of general interest to the field, the experiments well-controlled and the analysis in the paper sound. I have no major scientific concerns.

Weaknesses:<br /> I have some minor recommendations which will help improve the paper (regarding the discussion).

Comments on revised version:

The authors have addressed my concerns in the revised version of the manuscript.

Reviewer #3 (Public review):

Summary:

The tissue regeneration enhancer elements (TREEs) identified in zebrafish have been shown to drive injury-activated temporal-spatial gene expression in mice and large animals. These findings increase the translational potential of findings in zebrafish to mammals. In this manuscript, the authors tested TREEs in combination with different adeno-associated viral (AAV) vectors using in vivo luciferase bioluminescent imaging that allows for longitudinal tracking. The TREE-driven luciferase delivered by a liver de-targeted AAV.cc84 decreased off-target transduction in the liver. They further screened an AAV library to identify capsid variants that display enhanced transduction for myocardium post-myocardial infarction. A new capsid variant, AAV.IR41, was found to show increased transduction at the infarct border zones.

Strengths:

The authors injected AAV-cargo several days after ischemia/reperfusion (I/R) injury as a clinically relevant approach. Overall, this study is significant in that it identifies new AAV vectors for potential new gene therapies in the future. The manuscript is well-written, and their data are also of high quality.

Weaknesses:

The authors might be using MI (myocardial infarction) and I/R injury interchangeably in their text and labels. For instance, "We systemically transduced mice at 4 days after permanent left coronary artery ligation with either AAV9 or IR41 harboring a 2ankrd1aEN-Hsp68::fLuc transgene. IVIS imaging revealed higher expression levels in animals transduced with IR41 compared to AAV9, in both sham and I/R groups (Fig. 5A)". They should keep it consistent. There is also no description for the MI model.

Reviewer #3 (Public review):

Summary

This short paper aims to provide an independent validation of the transgenerational inheritance of learned behaviour (avoidance) that has been published by the Murphy lab. The robustness of the phenotype has been questioned by the Hunter lab. In this paper, the authors present one figure showing that transgenerational inheritance can be replicated in their hands. Overall, it helps to shed some light on a controversial topic.

Strengths

The authors clearly outline their methods, particularly regarding the choice of assay, so that attempting to reproduce the results should be straightforward. It is nice to see these results repeated in an independent laboratory.

Weaknesses

Previous reports on this topic have provided raw data, which is helpful when assessing sample sizes. The authors provided a spreadsheet containing the choice assay results for individual assays, but not the raw data. In the methods, it is stated that F2 animals were produced from F1 animals by bleaching, but there are many more F2 assays than F1. Were multiple F2 assays performed on the offspring from one F1 plate? If so, they do not represent independent assays.

I think that the introduction somewhat overstates their findings - do they really "address potential methodological variations that might influence results"? This makes it sound as though they test different conditions, whereas they only use one assay setup throughout.

Reviewer #3 (Public review):

Summary:

This paper aims to address the problem of exploring potentially rewarding environments that contain danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so.

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards.

Strengths:

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally there is little to criticise about the study.

Weaknesses:

The methods are robust, and generally there is little to criticise about the study. The extent of the testing in human participants is fairly limited, but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later), although this could simply reflect a lengthy evolutionary process.

Reviewer #3 (Public review):

In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field.

Comments on latest version:

With these revisions, the authors have addressed my main concerns.

Reviewer #3 (Public review):

Summary:

The authors suggest a mechanism that explains the preference of<br /> viral protein 35 (VP35) homologs to bind the backbone of double stranded RNA versus blunt ends. These preferences have a biological impact in terms of the ability of different viruses to escape the immune response of the host.<br /> The proposed mechanism involves the existence of a cryptic pocket, where VP35 binds the blunt ends of dsRNA when the cryptic pocket is closed and preferentially binds the RNA double stranded backbone when the pocket is open.<br /> The authors performed MD simulation results, thiol labelling experiments, fluorescence polarization assays, as well as point mutations to support their hypothesis.

Strengths:

This is a genuinely interesting scientific questions, which is approached through multiple complementary experiments as well as extensive MD simulations. Moreover, structural biology studies focused on RNA-protein interactions are particularly rare, highlighting the importance of further research in this area.

Weaknesses:

- Sequence similarity between Ebola-Zaire (94% similarity) explains their similar behaviour in simulations and experimental assays. Marburg instead is a more distant homolog (~80% similarity relative to Ebola/Zaire). This difference is sequence and structure can explain the propensities, without the need to involve the existence of a cryptic pocket.<br /> - No real evidence for the presence of a cryptic pocket is presented, but rather a distance probability distribution between two residues obtained from extensive MD simulations. It would be interesting to characterise the modelled RNA-protein interface in more detail

Comments on revisions:

-I still think that the term cryptic pocket is misleading here, unless the cryptic pocket is more thoroughly characterised. I would find it more appropriate to use the term open/closed state.

- Mg ions are known to be crucial in stabilising RNA structure both in vitro and in MD simulations (see e.g. Draper BJ 2008 and many others). While I understand that the authors cannot repeat simulations in presence of ions, I believe that this detail should be more clearly detailed in the manuscript.

Reviewer #3 (Public review):

The manuscript by Tie et al. provides a quantitative assessment of intra-Golgi transport of diverse cargos. Quantitative approaches using fluorescence microscopy of RUSH synchronized cargos, namely GLIM and measurement of Golgi residence time, previously developed by the author's team (publications from 20216 to 2022), are being used here.

Most of the results have been already published by the same team in 2016, 2017, 2020 and 2021. In this manuscript, the authors have put together measurement of intra-Golgi transport kinetics and Golgi residence time of many cargos. The quantitative results are supported by a large number of Golgi mini-stacks/cells analyzed. They are discussed with regard to the intra-Golgi transport models being debated in the field, namely the cisternal maturation/progression model and the stable compartments model.

The authors show that different cargos have distinct intra-Golgi transport kinetics and that the Golgi residence time of glycosyltransferases is high. From this and experiment using brefeldinA, the authors suggest that the rim progression model, adapted from the stable compartments model, fits with their experimental data.

Strengths:<br /> The major strength of this manuscript is to put together many quantitative results that the authors previously obtained and to discuss them to advance our understanding of the intra-Golgi transport mechanisms.<br /> The analysis by fluorescence microscopy of intra-Golgi transport is tough and this is a tour de force of the authors even though their approach shows limitations, which are clearly stated. Their work is remarkable in regards of the numbers of Golgi markers and secretory cargos which have been analyzed.

Weaknesses:<br /> Most of the data provided here were already published and thus accessible for the community. The tubular connections between cisternae and the diffusion/biochemical properties of cargos are not taken into account to interpret the results. Indeed, tubular connections and biochemical properties of the cargos may affect their transit through the Golgi and the kinetics with which they reach the TGN for Golgi exit.

The use of nocodazole might affect cellular homeostasis but this is clearly stated by the authors and is acceptable as we need to perturb the system to conduct this analysis.

The manual selection of the Golgi mini-stack being analyzed (where the cargo and the Golgi reference markers are clearly detectable ) might introduce a bias in the analysis.

Reviewer #3 (Public review):

Summary:

In this important work, the authors use extensive MD simulations to study how the IRE1 protein can detect unfolded peptides. Their study consolidates contradicting experimental results and offers a unique view of the different sensing models that have been proposed in the literature. Overall, it is an excellent study that is quite extensive. The research is solid, meticulous, and carefully performed, leading to convincing conclusions.

Strengths:

The strength of this work is the extensive and meticulous molecular dynamics simulations. The authors use and investigate different structural models, for example, carefully comparing a model based on a PDB structure with reconstructed loops with an AlphaFold 2 Multimer model. The author also investigates a wide range of different protein structural models that probe different aspects of the peptide sensing process. These solid and meticulous MD simulations allow the authors to obtain convincing conclusions concerning the peptide sensing process of the IRE1 protein.

Weaknesses:

A potential weakness of the study is the usage of equilibrium (unbiased) molecular dynamics simulations, so that processes and conformational changes on the microsecond time scale can be probed. Furthermore, there can be inaccuracies and biases in the description of unfolded peptides and protein segments due to the protein force fields. Here, it should be noted that the authors do acknowledge these possible limitations of their study in the conclusions.

Reviewer #3 (Public review):

Summary:

The study adapts CRISPR-based detection toolkit (SHERLOCK assay) using conserved and species-specific targets for the detection of some members of the Trypanosomatidae family of veterinary importance and species-specific assays to differentiate between the six most common animal trypanosome species responsible for AAT (SHERLOCK4AAT). The assays were able to discriminate between Trypanozoon (T. b. brucei, T. evansi, and T. equiperdum), T. congolense (Savanah, Forest Kilifi, and Dzanga sangha), T. vivax, T. theileri, T. simiae, and T. suis. The design of both broad and species-specific assays was based primarily on sequences of the 18S rRNA, GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), and invariant flagellum antigen (IFX) genes for species identification. Most importantly, the authors showed varying limits of detection for the different SHERLOCK assays, which is somewhat comparable to PCR-derived molecular techniques currently used for detecting animal trypanosomes, even though some of these methodologies have used other primers that target genes such as ITS1 and 7SL sRNA.

The data presented in the study are particularly useful and of significant interest for the diagnosis of AAT in affected areas.

Strengths:

The assays convincingly allow for the analysis and detection of most trypanosomes in AAT.

Weaknesses:

Inability for the assay to distinguish T. b. brucei, T. evansi, and T. equiperdum using the 18S rRNA gene, as well as the IFX gene, not achieving the sensitivity requirements for detection of T. vivax. Both T. brucei brucei and T. vivax are the most predominant infective species in animals (in addition to T. congolense), therefore, a reliable assay should be able to convincingly detect these to allow for proper use of the diagnostic assay.

DOI: 10.1186/s12943-025-02361-3

Resource: RRID:Addgene_48138

Curator: @dhovakimyan1

SciCrunch record: RRID:Addgene_48138

What is this?

DOI: 10.1038/s41440-025-02247-3

Resource: ggpubr (RRID:SCR_021139)

Curator: @dhovakimyan1

SciCrunch record: RRID:SCR_021139

What is this?

Reviewer #3 (Public review):

Summary:

The paper investigates the TMEM16 family of membrane proteins, which play roles in lipid scrambling and ion transport. A total of 27 experimental structures from five TMEM16 family members were analyzed, including mammalian and fungal homologs (e.g., TMEM16A, TMEM16F, TMEM16K, nhTMEM16, afTMEM16). The identified structures were in both Ca²⁺-bound (open) and Ca²⁺-free (closed) states to compare conformations and were preprocessed (e.g., modeling missing loops) and equilibrated. Coarse-grain simulations were performed in DOPC membranes for 10 microseconds to capture the scrambling events. These events were identified by tracking lipids transitioning between the two membrane leaflets and they analysed correlation between scrambling rates, in addition, structural properties such as groove dilation and membrane thinning were calculated. They report 700 scrambling events across structures and the figure 2 elaborates on how open structures show higher activity, also as expected. The authors also address how structures may require open groove, this and other mechanisms around scrambling is a bit controversial in the field.

Strengths:

The strength of this study emerges from comparative analysis of multiple structural starting points and understand global/local motions of the protein with respect to lipid movement. Although the protein is well-studied, both experimentally and computationally, the understanding of conformational events in different family members, especially membrane thickness less compared to fungal scramblases offers good insights.

Weaknesses:

The weakness of the work is to fully reconcile with experimental evidence of Ca²⁺-independent scrambling rates observed in prior studies, but this part is also challenging using coarse-grain molecular simulations. Previous reports have identified lipid crossing, packing defects and other associated events, so it is difficult to place this paper in that context. However, the absence of validation leaves certain claims, like alternative scrambling pathways, speculative.

Reviewer #3 (Public review):

Summary:

Nestor and colleagues identify genes escaping X chromosome inactivation (XCI) in rare individuals with non-mosaic XCI (nmXCI) whose tissue-specific RNA-seq datasets were obtained from the GTEX database. Because XCI is non-mosaic, read counts representing a second allele are tested for statistical significant escape, in this case > 2.5% of active X expression. Whereas a prior GTEX analysis found only one nmXCI female, this study finds two additional donors in GTEX, therefore expanding the number of assessed X-linked genes to 380. Although this is fewer than half of X-linked genes, the study demonstrates that although rare, nmXCI females are represented in RNA-seq databases such as GTEX. Therefore this analytical approach is worthwhile pursuing in other (larger) databases as well, to provide deeper insight into escape from XCI which is relevant to X-linked diseases and sex differences.

Strengths:

The analysis is well-documented, straight-forward and valuable. The supplementary tables are useful, and the claims in the main text well-supported.

Weaknesses:

There are very few, except that this escape catalogue is limited to 3 donors, based on a single (representative) tissue screen in 285 female donors, mostly using muscle samples. However, if only pituitary samples had been screened, nmXCI-1 would have been missed. Additional donors in the 285 representative samples cross a lower threshold of AE = 0.4. It would be worthwhile to query all tissues of the 285 donors to discover more nmXCI cases, as currently fewer than half of X-linked genes received a call using this very worthwhile approach.

Comments on revised version:

The authors incorporated some textual changes, but deferred any new analysis, or expansion from these two new skewed donors to include more individuals/tissues, or going more in depth for individual genes to future manuscripts. They appear to have that option at eLife.

Reviewer #3 (Public review):

Summary:

This study investigates the role of BICC1 in the regulation of PKD1 and PKD2 and its impact on cytogenesis in ADPKD. By utilizing co-IP and functional assays, the authors demonstrate physical, functional, and regulatory interactions between these three proteins.

Strengths:

(1) The scientific principles and methodology adopted in this study are excellent, logical, and reveal important insights into the molecular basis of cystogenesis.

(2) The functional studies in animal models provide tantalizing data that may lead to a further understanding and may consequently lead to the ultimate goal of finding a molecular therapy for this incurable condition.

(3) In describing the patients from the Arab cohort, the authors have provided excellent human data for further investigation in large ADPKD cohorts. Even though there was no patient material available, such as HUREC, the authors have studied the effects of BICC1 mutations and demonstrated its functional importance in a Xenopus model.

Weaknesses:

This is a well-conducted study and could have been even more impactful if primary patient material was available to the authors. A further study in HUREC cells investigating the critical regulatory role of BICC1 and potential interaction with mir-17 may yet lead to a modifiable therapeutic target.

Conclusion:<br /> The authors achieve their aims. The results reliably demonstrate the physical and functional interaction between BICC1 and PKD1/PKD2 genes and their products.

The impact is hopefully going to be manifold:

(1) Progressing the understanding of the regulation of the expression of PKD1/PKD2 genes.

(2) Role of BiCC1 in mir/PKD1/2 complex should be the next step in the quest for a modifiable therapeutic target.

Reviewer #3 (Public review):

Summary:

The authors seek to determine the underlying traits that support the exceptional capacity of Aspergillus oryzae to secrete enzymes and heterologous proteins. To do so, they leverage the availability of multiple domesticated isolates of A. oryzae along with other Aspergillus species to perform comparative imaging and genomic analysis.

Strengths:

The strength of this study lies in the use of multifaceted approaches to identify significant differences in hyphal morphology that correlate with enzyme secretion, which is then followed by the use of genomics to identify candidate functions that underlie these differences.

Weaknesses:

There are aspects of the methods that would benefit from the inclusion of more detail on how experiments were performed and data interpreted.

Overall, the authors have achieved their aims in that they are able to clearly document the presence of two distinct hyphal forms in A. oryzae and other Aspergillus species, and to correlate the presence of the thicker, rapidly growing form with enhanced enzyme secretion. The image analysis is convincing. The discovery that the addition of yeast extract and specific amino acids can stimulate the formation of the novel hyphal form is also notable. Although the conclusions are generally supported by the results, this is perhaps less so for the genetic analysis as it remains unclear how direct the role of RseA and the calcium transporters might be in supporting the formation of the thicker hyphae.

The results presented here will impact the field. The complexity of hyphal morphology and how it affects secretion is not well understood despite the importance of these processes for the fungal lifestyle. In addition, the description of approaches that can be used to facilitate the study of these different hyphal forms (i.e., stimulation using yeast extract or specific amino acids) will benefit future efforts to understand the molecular basis of their formation.

Reviewer #3 (Public review):

In this manuscript, Yang et al. characterize the endocytic accessory protein CCDC32, which has implications in cardio-facio-neuro-developmental syndrome (CFNDS). The authors clearly demonstrate that the protein CCDC32 has a role in the early stages of endocytosis, mainly through the interaction with the major endocytic adaptor protein AP2, and they identify regions taking part in this recognition. Through live cell fluorescence imaging and electron microscopy of endocytic pits, the authors characterize the lifetimes of endocytic sites, the formation rate of endocytic sites and pits and the invagination depth, in addition to transferrin receptor (TfnR) uptake experiments. Binding between CCDC32 and CCDC32 mutants to the AP2 alpha appendage domain is assessed by pull down experiments. While interaction between CCDC32 and the alpha appendage domain of AP2 is clearly described, a discussion of potential association with other AP2 domains would be beneficial to understand the impact of CCDC32 in endocytosis.

Together, these experiments allow deriving a phenotype of CCDC32 knock-down and CCDC32 mutants within endocytosis, which is a very robust system, in which defects are not so easily detected. A mutation of CCDC32, mimicking CFNDS mutations, is also addressed in this study and shown to have endocytic defects.

In summary, the authors present a strong combination of techniques, assessing the impact of CCDC32 in clathrin mediated endocytosis and its binding to AP2.

斯巴达借联盟间接控制盟邦Historical Context: The league allowed Sparta to control allies without direct annexation, using collective security to suppress helot revolts and deter rivals like Athens.

Reviewer #3 (Public review):

Summary:<br /> The article explores the role of mother-child interactions in the development of children's social cognition, focusing on Theory of Mind (ToM) and Social Pain Matrix (SPM) networks. Using a naturalistic fMRI paradigm involving movie viewing, the study examines relationships among children's neural development, mother-child neural synchronization, and interaction quality. The authors identified a developmental pattern in these networks, showing that they become more functionally distinct with age. Additionally, they found stronger neural synchronization between child-mother pairs compared to child-stranger pairs, with this synchronization and neural maturation of the networks associated with the mother-child relationship and parenting quality.

Strengths:<br /> This is a well-written paper, and using dyadic fMRI and naturalistic stimuli enhances its ecological validity, providing valuable insights into the dynamic interplay between brain development and social interactions.

Weaknesses:<br /> The current sample size (N = 34 dyads) is a limitation, particularly given the use of SEM, which generally requires larger samples for stable results. Although the model fit appears adequate, this does not guarantee reliability with the current sample size.

Reviewer #3 (Public review):

Summary:

Mora et al employ published ChIP-seq and RNA-seq from embryonic tissues to nominate transcription factors that work combinatorially during development. This manuscript addresses an important gap in knowledge regarding the complexities of gene regulation. However, as written, the manuscript is focused on confirming mostly known associations and does not unveil principles that can be broadly applied, given multiple technical caveats that are outlined below.

Strengths:

(1) Instead of focusing on a single transcription factor motif enriched within peaks, the authors search the flanking regions of enriched motifs to nominate additional transcription factors that may work cooperatively to provide organ specificity. This type of analysis is a crucial next step in the gene regulation field, as transcription factors rarely work independently.

(2) Figure 6 is a good demonstration of the preliminary experiments that can be done to test the activity of co-occurring motifs.

(3) This is a really nice resource of organ-specific motif associations that can be used to generate many testable hypotheses.

(4) The rationale and writing are very clear and easy to read.

Weaknesses:

(1) Much of this manuscript focuses on confirming transcription factor relationships that have been reported previously. For example, it is well known that GATA4 interacts with MEF2 in the ventricle. There are limited new or unexpected associations discussed and tested.

(2) Embryonic tissues are highly heterogeneous, limiting the utility of the bulk ChIP-seq employed in these analyses. Does the cellular heterogeneity explain the discrepancy between TEAD binding and histone acetylation? Similarly, how does conservation between species affect the TF predictions?

(3) Some of the interpretations should also be fleshed out a bit more to clarify the advantage of the analyses presented here. For example, if Gata4 and Foxa2 transcripts are expressed during different stages of development, then it's likely that (as stated by the authors) these motifs are not used during the same stage of development. But examining the flanking regions wasn't necessary to make that statement. This type of conclusion seems tangential to the benefit of this analysis, which is to understand which TFs work together in a single organ at a single time point.

(4) This manuscript hinges on luciferase assays whose results can be difficult to translate to complex gene regulation networks. Many motifs are often clustered together, which makes designing experiments at endogenous loci important in studies such as this one.

Reviewer #3 (Public review):

This manuscript is a continuation of past work by the last author where they looked at stochasticity in developmental processes leading to inter-individual behavioural differences. In that work, the focus was on a specific behaviour under specific conditions while probing the neural basis of the variability. In this work, the authors set out to describe in detail how stable individuality of animal behaviours is in the context of various external and internal influences. They identify a few behaviours to monitor (read outs of attention, exploration, and 'anxiety'); some external stimuli (temperature, contrast, nature of visual cues, and spatial environment); and two internal states (walking and flying).

They then use high-throughput behavioural arenas - most of which they have built and made plans available for others to replicate - to quantify and compare combinations of these behaviours, stimuli, and internal states. This detailed analysis reveals that:

(1) Many individualistic behaviours remain stable over the course of many days.<br /> (2) That some of these (walking speed) remain stable over changing visual cues. Others (walking speed and centrophobicity) remain stable at different temperatures.<br /> (3) All the behaviours they tested fail to remain stable over spatially varying environment (arena shape).<br /> (4) and only angular velocity (a read out of attention) remains stable across varying internal states (walking and flying)

Thus, the authors conclude that there is a hierarchy in the influence of external stimuli and internal states on the stability of individual behaviours.

The manuscript is a technical feat with the authors having built many new high-throughput assays. The number of animals are large and many variables have been tested - different types of behavioural paradigms, flying vs walking, varying visual stimuli, different temperature among others.

Comments on revisions:'

The authors have addressed my previous concerns.

Reviewer #3 (Public review):

Summary:

The study investigates the development of reinforcement learning across the lifespan with a large sample of participants recruited for an online game. It finds that children gradually develop their abilities to learn reward probability, possibly hindered by their immature spatial processing and probabilistic reasoning abilities. Motor noise and exploration after a failure all contribute to children's subpar performance.  

Strengths:

Experimental manipulations of both the continuity of movement options and the probabilistic nature of the reward function enable the inference of what cognitive factors differ between age groups. <br /> A large sample of participants is studied.<br /> The model-based analysis provides further insights into the development of reinforcement learning ability. 

Weaknesses:

The conclusion that immature spatial processing and probabilistic reasoning abilities limit reinforcement learning here still needs more direct evidence.

Reviewer #3 (Public review):

Nucleus HVC is critical both for song production as well as learning and arguably, sitting at the top of the song control system, is the most critical node in this circuit receiving a multitude of inputs and sending precisely timed commands that determine the temporal structure of song. The complexity of this structure and its underlying organization seem to become more apparent with each experimental manipulation, and yet our understanding of the underlying circuit organization remains relatively poorly understood. In this study, Trusel and Roberts use classic whole-cell patch clamp techniques in brain slices coupled with optogenetic stimulation of select inputs to provide a careful characterization and quantification of synaptic inputs into HVC. By identifying individual projections neurons using retrograde tracer injections combined with pharmacological manipulations, they classify monosynaptic inputs onto each of the three main classes of glutamatergic projection neurons in HVC (RA-, Area X- and Av-projecting neurons). This study is remarkable in the amount of information that it generates, and the tremendous labor involved for each experiment, from the expression of opsins in each of the target inputs (Uva, NIf, mMAN and Av), the retrograde labelling of each type of projection neuron, and ultimately the optical stimulation of infected axons while recording from identified projection neurons. Taken together, this study makes an important contribution to increasing our identification, and ultimately understanding, of the basic synaptic elements that make up the circuit organization of HVC, and how external inputs, which we know to be critical for song production and learning, contribute to the intrinsic computations within this critic circuit.

This study is impressive in its scope, rigorous in its implementation and thoughtful regarding its limitations. The manuscript is well written, and I appreciate the clarity with which the authors use our latest understanding of the evolutionary origins of this circuit to place these studies within a larger context and their relevance to the study of vocal control, including human speech. My comments are minor and primarily about legibility, clarification of certain manipulations and organization of some of the summary figures.

Comments on revisions:

The authors have done a very nice job addressing the reviewers' comments.