Reviewer #3 (Public review):

Summary:

The authors aimed to overcome the challenges associated with complex, conventional prokaryotic cell-free protein synthesis (CFPS) systems, which require up to thirty-five components, by developing a streamlined and efficient E. coli CFPS platform to encourage broader adoption. The main objective was to reduce the number of reaction components from thirty-five to seven, while also developing an accessible 'fast lysate' preparation protocol that eliminates time-consuming runoff and dialysis steps. The authors also sought to demonstrate the robustness and translational quality of this streamlined system by efficiently synthesising challenging functional proteins, including the cytotoxic restriction endonuclease BsaI and the self-assembling intermediate filament protein vimentin.

Strengths:

This study presents several key strengths of the optimised E. coli cell-free protein synthesis system in terms of its design, performance and accessibility.

- The reaction mixture has been dramatically simplified, with the number of essential core components successfully reduced from up to thirty-five in conventional systems to just seven.<br /> - The "fast lysate" protocol is a significant advance in terms of procedure.<br /> - The system's ability to synthesise challenging, functional proteins is evidence of its robustness.

Weaknesses:

(1) Title: "A simplified and highly efficient cell-free protein synthesis system for prokaryotes".<br /> - This title is misleading since one would expect a simplified and highly efficient cell-free protein synthesis system to yield similar protein levels compared to current cell-free protein synthesis systems. What this study shows is that the composition of cell-free protein synthesis systems can be simplified while maintaining a certain level of protein synthesis. Here, optimisation does not involve maintaining protein synthesis yield while simplifying the cell-free protein synthesis system; rather, it involves developing a simplified cell-free protein synthesis system. As mentioned in my comments below, this study lacks a comparison of protein levels with a typical cell-free protein synthesis system.<br /> - What do the authors mean by "highly efficient"? Highly efficient compared to what experimental conditions? If one is interested by the yield of protein synthesis, is this simplified system highly efficient compared to current systems?

(2) Figure 1, 3-5 :<br /> - What do relative luciferase units represent? How are these units calculated?<br /> - In this system, the level of expression depends mainly on the level of NLuc transcripts and the efficiency of NLuc translation. How did the authors ensure that the chemical composition of the different eCFPS buffers only affected protein translation and not transcript levels? In other words, are luciferase units solely an indicator of protein synthesis efficiency, or do they also depend on transcription efficiency, which could vary depending on the experimental conditions?<br /> - How long were the eCFPS reactions allowed to proceed before performing the luciferase activity measurement? Depending on the reaction time, the absence or presence of certain compounds may or may not impact NLuc expression. For example, it can be assumed that tRNA does not significantly affect NLuc levels over a short period of time, and that endogenous tRNA in the lysate is present at sufficient concentrations. However, over a longer period of time, the addition of tRNA could essential to achieve optimal NLuc levels.<br /> - The authors show that tRNA and amino acids are not strictly essential for the expression of NLuc, likely due to residual amounts within the cell lysate. However, are the protein levels achieved without added amino acids and tRNA sufficient for biochemical assays that require a certain amount of protein? It is important to note that the focus here is on optimising the simplicity of the buffer rather than the level of protein expression. In fact, the simplicity of the buffer is prioritised over the amount of protein produced. This should be made clear.<br /> - How would the NLuc level compare if all the components were optimised individually and present in an optimised buffer, compared to a buffer optimised for simplicity as described by the authors?

(3) Line 71, Streamlining eCFPS: removal of dispensable components. This title is misleading because it creates the false impression that proteins can be produced in vitro without the addition of certain compounds. While this is true, the level of protein produced may not be sufficient for subsequent biochemical analyses. This should be made clear.

(4) Figure 2: In the legend, change "(A) Protein expression levels of the eCFPS system measured at varying concentrations of KGlu and MgGlu2" to "(A) Protein expression levels of the eCFPS system using an Nanoluciferase (NLuc) reporter DNA measured at varying concentrations of KGlu and MgGlu2".

(5) Lanes 302-303: "The thorough optimization of the seven core components was a critical step in achieving high protein expression levels". What are "high expression levels"? Compared to what?

Comments on revisions:

The authors have adequately addressed the issues I had raised in my initial review.

Reviewer #3 (Public review):

Leshem et al have generated a transcriptional cell atlas of the human outflow tract at two developmental timepoints and its adult valvular derivatives. This carefully performed study provides a useful resource for the study of known genes implicated in outflow tract defects and potentially also to discover new disease genes. The authors reveal neural crest and mesodermal contributions to different outflow tract components and show that GATA6, known to play a role in arterial valve development, controls a set of genes expressed in endocardial derived cells during valve development. Interestingly the results reveal intersection with GLI3 and suggest lineage persistence of gene expression through to the adult timepoint, a main new finding of this study.

Comments on revisions:

The authors have carefully addressed previous comments, including the addition of new analysis pointing to potential cooperation between GATA6 and GLI3.

Reviewer #3 (Public review):

Summary:

The focus of the research is to understand how transcription factors with high expression in neural crest cell derived cancers (e.g., neuroblastoma) and roles in neural crest cell development function to promote malignancy. The focus is on the transcription factor FOXC1 and using murine cell culture, gain- and loss of function approaches and ChIP profiling, among other techniques, to place PKC inhibitor ARHGAP36 mechanistically between FOXC1 and another pathway associated with malignancy, Sonic Hedgehog (SHH).

Strengths:

Major strengths are the mechanistic approaches to identify FOXC1 direct targets, definitively showing that FOXC1 transcriptional regulation of ARHGAP36 leads to dysregulation of SHH signaling downstream of ARHGAP36 inhibition of PKC. Starting from a screen of Foxc1 OE to get to ARHGAP36 and then using genetic and pharmacological manipulation to work through the mechanism is very well done. There is data that will be of use to others studying FOXC1 in mesenchymal cell types, in particular the FOXC1 ChIP-seq.

Weaknesses:

Work is almost all performed in NIH3T3 or similar cells (mouse cells, not patient or mouse-derived cancer cells) so the link to neuroblastoma that forms the major motivation of the work is not clear. The authors look at ARHGAP36 levels in association the neuroblastoma patient survival however the finding, though interesting and quite compelling, is misaligned with what the literature shows about FOXC1 and SHH, their high expression is associated with increased malignancy (also maybe worse outcomes?). Therefore, ARHGAP36 expression may be more complicated in a tumor cell or may be unrelated to FOXC1 or SHH, leaving one to wonder what the work in NIH3T3 cells, though well done, is telling us about the mechanisms of FOXC1 as an oncogene in neuroblastoma cells or in any type of cancer cell. Does it really function as a SHH activator to drive tumor growth? The 'oncogenic relevance' and 'contribution to malignancy' claimed in the last paragraph of the introduction is currently weakly supported with the data as presented. This could be improved with studying some of these mechanisms in patient-derived neuroblastoma cells with high FOXC1 expression. Does inhibiting FOXC1 change SHH and ARHGAP36 and have any effect on cell proliferation or migration? Alternatively, does OE of FOXC1 in NIH3T3 cells increase their migration or stimulate proliferation in some way and is this dependent on ARHGAP36 or SHH? Application of their mechanistic approaches in cancer cells or looking for hallmarks of cancer phenotypes with FOXC1 OE (and dependent on SHH or ARHGAP36) could help to make a link with cellular phenotypes of malignant cells.

In the revised manuscript, the authors did not add studies in any malignant cell type (mouse or human, neuroblastoma or other) with Foxc1 overexpression to test if the mechanisms they identify in the mouse fibroblasts is present in cancer cells nor if this relates to cellular phenotypes of malignancy (migration or proliferation). Therefore strengths and weaknesses identified by this reviewer in the prior version are the same.

Reviewer #3 (Public review):

Summary:

The authors described an exciting 400-drug screening using a MMV pathogen box to select compounds that effectively affects the medically important Toxoplasma parasite bradyzoite stage. This work utilises a bradyzoites culture technique that was published recently by the same group. They focused on compounds that affected directly the mitochondria electron transport chain (mETC) bc1-complex and compared with other bc1 inhibitors described in the literature such as atovaquone and HDQs. They further provide metabolomics analysis of inhibited parasites which serves to provide support for the target and to characterise the outcome of the different inhibitors.

Strengths:

This work is important as, until now, there are no effective drugs that clear cysts during T. gondii infection. So, the discovery of new inhibitors that are effective against this parasite-stage in culture and thus have the potential to battle chronic infection is needed. The further metabolic characterization provides indirect target validation and highlight different metabolic outcome for different inhibitors. The latter forms the basis for new studied in the field to understand the mode of inhibition and mechanism of bc1-complex function in detail.

The authors focused in the function of one compound, MMV1028806, that is demonstrated to have a similar metabolic outcome to burvaquone. Furthermore, the authors evaluated the importance of ATP production in tachyzoite and bradyzoites stages and under atovaquone/HDQs drugs.

Reviewer #3 (Public Review):

Summary:

This study explores the dynamic reprogramming of histone modification H3K4me2 during the early stages of mammalian embryogenesis. Utilizing the advanced CUT&RUN technique coupled with high-throughput sequencing, the authors investigate the erasure and re-establishment of H3K4me2 in mouse germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and early embryos.

Strengths:

The findings provide valuable insights into the temporal and spatial dynamics of H3K4me2 and its potential role in zygotic genome activation (ZGA).

Weaknesses:

The study primarily remains descriptive at this point. It would be advantageous to conduct further comprehensive functional validation and mechanistic exploration.<br /> Key areas for improvement include enhancing the innovation and novelty of the study, providing robust functional validation, establishing a clear model for H3K4me2's role, and addressing technical and presentation issues. The text would benefit from the introduction of a novel conceptual framework or model that provides a clear explanation of the functional consequences and molecular mechanisms underlying H3K4me2 reprogramming in the transition from parental to early embryonic development.

While the findings are significant, the current manuscript falls short in several critical areas. Addressing major and minor issues will significantly strengthen the study's contribution to the field of epigenetic reprogramming and embryonic development.

Reviewer #3 (Public review):

Summary:

This study is significant as it established a protocol for the long-term culture of Schistosoma mansoni newly transformed cercariae, which developed in vitro into sexually dimorphic forms. The impact of two different sera, Fetal Bovine Serum (FBS) and Human Serum (HS), added to the culture medium supplemented with human red blood cells was evaluated. The authors demonstrated that HS-cultured parasites were able to digest red blood cells, a critical step for long-term parasite development. Furthermore, while most FBS-cultured parasites did not progress beyond an early liver stage, sexual dimorphism was clearly evident in the HS-cultured worms, albeit delayed compared to in vivo development.

Strengths:

This study could contribute to further in vitro studies for a better understanding of the unique sexual biology of Schistosoma mansoni and for screening novel schistosomicidal compounds. By increasing parasite development in in vitro studies, this protocol could have a positive impact on the principles of the 3Rs (Replacement, Reduction and Refinement) for animal research.

Weaknesses:

As the authors mentioned, "pairing between male and female parasites was rare. Pairing was observed in approximately ~7% of the experiments, usually after day ~ 80 in culture. Egg production was also not achieved with this protocol.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

I have no further concerns regarding this manuscript.

Reviewer #3 (Public review):

In this work, Tsay et al. examine the challenge of inferring the ordering of amyloid fibrils. There is a clear need for such methodology. In their work, they computationally analyze the case of the expected decay in the DEER signal for spins randomly distributed in one, two, and three dimensions and show that (not unexpectedly) the decay is sensitive to dimensionality for a range of spin label concentrations. More intriguingly, they measure the dimensionality of tau amyloid labeled at several positions. Intriguingly, they show uniform (but unexpected) dimensionality when the label is in the fibril core. Through further simulations, they show that this anomalous dimensionality cannot arise from label attraction or repulsion (which can lead to deviations from random positions). Instead, this dimensionality is interpreted (again using compelling simulations) to arise from mis-registering due to changes in packing. Taken together, this paper convincingly shows that the DEER signal can be used to get site-specific information on amyloid dimensionality and can discriminate between regions of fibril core vs the "fuzz coat". Overall, this paper moves forward the methodology and opens up the technique to attractive applications in the areas of amyloid formation. More substantively, the field of DEER has been fixated on the dipolar modulation, and it is only once in a while now that one comes across a paper with a fresh breath of air - this paper certainly is!

Reviewer #3 (Public review):

Summary:

Various groups over the last several decades have provided many examples of proteins associating with nascent mRNA co-transcriptionally to influence gene expression at subsequent stages, including in the cytoplasm. In this and previously published works, the Choder group has described these events as "mRNA imprinting", which we know as a field that reflects the differential association of proteins with mRNAs in a gene-specific or environmentally induced fashion to regulate gene expression.

In this study, the authors use a proteomics-based approach termed PROFIT to identify factors associated with RNA Pol II in an RNA-dependent manner. The identified interactors have the potential to be part of mRNA-protein complexes (mRNPs) being formed co-transcriptionally with an "mRNA imprinting" function. PROFIT employs a pulldown of RNA Pol II via a tagged Rpb3 subunit, followed by RNase I-mediated elution to isolate proteins associated in an RNA-dependent manner. Proteomics analyses identified known mRNA-associated proteins that have previously been reported as imprinting factors, as well as other proteins involved in gene expression, including factors functioning in the cytoplasm. The authors suggest, based on the RNA-dependence and assumed formation of these interactions with RNA Pol II co-transcriptionally, that these novel hits could be mRNA imprinting factors. Although for most of these factors, it has not been determined whether they associate with RNA-Pol II in the context of transcription with nascent transcripts to contribute to the downstream regulations of these transcripts.

Strengths:

PROFIT successfully identified nuclear factors known to engage mRNA co-transcriptionally. This suggests that the method has the potential to detect imprinting factors. By employing a proximity-labeling technique, termed BioPROFIT, further evidence is provided for some of the novel interactors being in proximity to RNA Pol II. The authors further demonstrate that one of the factors, the eIF3 component Rpg1, exists in two fractions, with a soluble fraction that matures into a ribosome fraction, which is suggestive of Rpg1 traveling along the gene expression pathway with an mRNP to be engaged in translation. In addition, the authors showed that PROFIT detects changes in RNA Pol II associated factors in response to heat shock, consistent with gene expression reprogramming during stress. As such, these methods and proteomics data provide a starting point for a more detailed characterization of mRNP compositions formed in the nucleus and their impact on gene expression at later stages.

Weaknesses:

The authors interpret the interaction data from PROFIT and BioPROFIT under the assumption that this reflects interactions happening co-transcriptionally. There is no discussion of other ways these data may result, or more importantly, controls to prove these assumptions are true. Overall, these assays lack important controls and experimental validations by independent methods to demonstrate that the identified interactions occur co-transcriptionally within the nucleus and do not represent interactions occurring in the cytoplasm or artifacts related to experimental design. For example, the authors focus on Rpg1 as a potential imprinting factor, which would require this protein to shuttle and be localized at transcribing genes. Yet no evidence is presented that Rpg1 enters the nucleus or can be found in association with a transcribed gene, which leaves open the possibility that this interaction is occurring in the cytoplasm or forming post-lysis.

To the possibility of in vitro interactions, in the PROFIT assay, yeast collected from a 3L culture is cryo-ground and resuspended in 7 mL of lysis buffer. This ratio of cell material to buffer will create a highly concentrated cell lysate that is subsequently used over ~6.5 hours, which is the time for centrifugation, DNase I digestion, and immunoprecipitation. These conditions have a very high probability of promoting new interactions between RNA, RNA Poll II, other proteins, and/or RNA Pol II-associated nascent RNA complexes in vitro. Notably, the PROFIT assay detects many highly expressed proteins but does not identify many of the factors known to be loaded into nuclear mRNPs (e.g., Yra1, THO complex, Sub2, or Nab2). The BioPROFIT assay is used to try to address this issue, but biotinylation may occur post-lysis because the desalting process to remove biotin is performed just before the immunoprecipitation, providing ~2 hours for the reaction to happen in vitro. In addition, even if the biotinylation occurs in cells, nothing about this assay indicates this is occurring in the context of transcribing RNA Pol II or nascent transcripts. To address this major issue, the authors should add a mixing control to show that the detected interactions between RNA Pol II and the identified factors are produced in cells, not in the cell lysate. Specifically, mixing cell grindates from two independent yeast strains (e.g., RPB3-FLAG strain mixed with a TIF4631-HA strain) with the lysate used in the PROFIT assay with western blotting. In this case, if the interaction is detected, the interaction is produced in the cell lysate. To verify PROFIT hits associated with transcribing RNA Pol II and nascent transcripts, BIOPROFIT should be performed in cells treated with a transcription inhibitor (e.g., thiolutin) or mutants blocking transcription by Pol II. These types of verifications should be performed for the multiple novel hits reported in the manuscript.

Another in vitro issue must also be addressed. In the PROFIT assay, elution of RNA-associated factors from the immunoprecipitated material is performed by RNase I digestion, but the reaction time is very long (3 hours) at room temperature. During such a long incubation time and at higher temperature (i.e., above 4 Celsius), it is possible that non-RNA-mediated interactors dissociate from the beads and/or protein binding partners. This possibility is made more problematic by the fact that the authors define interactors using fold change over an Rpb3 no tag sample, where the sample does not contain isolated RNA Pol II complexes and their associated protein-binding partners. As such, even a small amount of non-RNA-mediated RNA Poll II interactors that elute would appear significantly enriched. For this point, a comparison of +/- RNase I elution in the Rpb3-FLAG pulldown sample should be performed using PROFIT.

Other points to address:

(1) The cartoon in Figure 1A should be corrected to present the PROFIT experiment as described in the text. Specifically, in the cartoon, UV is shown to be applied to cells, but this is done with cell grindate.

(2) The cartoon in Figure 2A should be corrected. In the cartoon, it shows the biotin ligase biotinylating proximal proteins during DNase digestion as well as on the Sepharose beads, but in theory, the majority of the biotinylation reaction occurs in cells. In addition, the cartoon depicts biotinylation of proximal proteins, but the system described uses wild-type BirA to specifically biotinylate an Avi-tag. To perform non-specific labeling of proximal proteins, BirA* would need to be used. Finally, the cartoon indicates mass spectrometry analysis of labeled proteins, but this is not done in the manuscript.

(3) In the text, the sentence "However, no bio-Spt6-Avi was released from the complexes containing Pol II mutants (Fig. 5C)" appears to have two errors. "Pol II mutants" should likely be "rpb4 mutant" and "Fig. 5C" is probably "Fig. 6C".

(4) In the Figure 6 legend, the sentence "The bulk Spt6 was detected by anti-HIS Abs that bound to (HIS)x6, which was placed upstream of the FLAG" suggests that "FLAG" should be "Avi-tag." Please correct it if necessary and accurately describe it in the strain list.

(5) On page 18, Npl3 is listed and discussed, but never mentioned anywhere prior in the paper. For example, the paragraph states "...our observation that it binds nascent RNA in an Rpb4-dependent manner...", but Npl3 is not listed in the supplemental Table 4, which lists PROFIT hits affected by rpb4∆. If Npl3 is to be discussed, the associated data needs to be properly presented.

Reviewer #3 (Public review):

Summary:

In this study, Zhu et al. use long-read transcriptomes, with correction using short-read RNA-seq, from 12 spider species that span the major evolutionary lineages to investigate the diversification of spider silk proteins (spidroins). Here, they identify 60 spidroin sequences and propose that two highly divergent sequences found in the basal Liphistius sp., where one is an alanine-serine-rich (AS-type), and one is a glycine-serine-rich (GS-type), represent ancestral templates from which all major spidroin families diversified. Using separate phylogenetic analyses for N-terminal domains, C-terminal domains, and repetitive domains, the authors argue that the AS-type lineage remained relatively conserved and gave rise to tubuliform spidroins (TuSp) used in eggcase silk, while the GS-type lineage evolved into minor ampullate spidroins (MiSp) and may have provided the substrate for major ampullate spidroins (MaSp). In addition, they describe a specific flagelliform-like (flag) transcript in a basal clade spider, with MaSp-like terminal domains, and propose that Flag was co-opted into ampullate silk glands before being progressively lost in more derived retrolateral tibial apophysis (RTA) lineages.

Strengths:

The taxon sampling is a strength of this study, covering representative species at key nodes across spider evolution, from the earliest-diverging Mesothelae through Mygalomorphae and into the most derived Araneomorphae lineages, which enables the authors to make comparative inferences about ancestral states. Also, the use of long-read sequencing is well-suited to the problem since spidroin genes contain highly repetitive coding sequences that would be very hard to resolve by short-read assembly alone. Thus, retrieving 30 full-length sequences in this context is notable, and the assembly quality appears reasonable for transcriptomic resources, with BUSCO completeness values reported between 85% and 93% across species.

The decision to analyse N-terminal, C-terminal, and repetitive domains in separate phylogenetic trees is methodologically sound and yields a biologically interesting result: terminal domains show greater diversification in basal lineages than repetitive regions, suggesting that specialisation of silk gland microenvironments preceded compositional innovation in the repetitive sequences.

Weaknesses:

While the paper has strengths in providing a useful comparative resource and generating interesting hypotheses, several of the central evolutionary conclusions are not directly supported by the current data. There are three main elements that require further attention:

(1) The GS-type Liphistius sequence (Liphistius sp._5400) is central to the manuscript's model for the origin of GA-rich ampullate spidroins, but the authors describe it as a spidroin-like transcript whose N- and C-terminal regions lack homology to known spidroins and may not support typical silk-fiber assembly. Since its terminal domains are excluded from the phylogenetic analyses, the proposed scenario, GS-type to MiSp to MaSp, rests largely on repeat-region similarity. Supplementary materials provided in this study further indicate no predicted signal peptide, although this feature alone is not unique among the annotated silk proteins. The manuscript should therefore either provide a stronger justification for treating Liphistius sp._5400 as an ancestral spidroin or more consistently frame it as a spidroin-like, repeat-based intermediate. The distinction between repeat-region clustering and full functional homology should also be made explicit.

(2) The whole-body transcriptome approach is an important sampling limitation that is acknowledged here, where the authors note that they were unable to recover complete spidroin repertoires for each species. Because the newly generated data are not silk-gland-specific, the absence of a transcript in a given species should be interpreted with caution and not equated directly with gene absence. This is particularly relevant to the manuscript's proposed loss of Flag during RTA evolution. In the focal taxa, the inference combines one positive transcript in H. davidbowie with non-detection in H. diardi, while broader support comes from limited synteny-based absence in a small number of external genomes. Therefore, while the Flag-loss scenario could be plausible, it remains suggestive rather than conclusive without more targeted silk-gland sampling or broader genomic validation.

(3) The Flag co-option model is interesting, but as presented now, it is based on limited evidence: a single Flag-like transcript in H. davidbowie, the absence of detection in H. diardi, restricted synteny comparisons, and terminal-domain similarity to ampullate spidroins. The manuscript does not present proteomic evidence that this Flag-like protein is incorporated into ampullate silk fibers, nor does it show a series of pseudogenized or truncated Flag loci across derived RTA lineages. This is a plausible and interesting scenario, but it should be framed more consistently as a testable hypothesis rather than as an established evolutionary pathway.

Reviewer #3 (Public review):

Neural activity in visual cortex has primarily been studied in terms of responses to external visual stimuli. While the variability of neural inputs to a visual area are known to also influence visual responses, the contribution of this stimulus independent component to overall visual responses has not been well characterized.

In this study, the authors analyze datasets from both mice (a previous V1 Ca++ imaging study) and monkeys (data from a previous study and new large-scale electrophysiological recordings from V1-V4). Using regression models, they examine the predictability of neural activity between Layer 4 and Layer 2/3 in mice and between V1 and V4 in monkeys. Their main finding is that significant predictions are possible even in the absence of visual input, highlighting the influence of stimulus independent downstream activity on neural responses. These findings can inform future modeling work of neural responses in visual cortex to account for such non-visual influences.

The authors perform a thorough analysis comparing regression-based predictions for a wide variety of combinations of stimulus conditions and directions of influence. While many of the predictability pattens are largely in line with expectations (eg., downstream layers/areas predicting upstream activity), it is valuable to have these relationships quantified as the authors have done here. Predictability also depended on stimulus type, but these dependencies were not consistent across animals, making it difficult to draw general conclusions. Finally, they show robust predictions even during spontaneous activity which are only partially accounted for by available behavioral metrics. Together, these analyses provide a valuable quantification of stimulus-independent components of visual cortical activity and their potential role in shaping sensory responses.

Reviewer #3 (Public review):

Overview and Strengths:

Accurate evaluation of threat levels allows animals to determine whether to escape. The precise mechanism underlying threat evaluation remains unclear. Smith et al. identified a cluster of neurons in the zebrafish rostrolateral dorsal pallium (Rl) that respond differentially to varying levels of negative-valence stimuli.

This work leverages the small size and optical transparency of the larval zebrafish, using two-photon selective plane illumination microscopy to assay the response of pallial neurons to various negative-valence stimuli. Interestingly, unlike the ventromedial pallium and habenula, which responded to all stimuli tested, neurons in the Rl were activated by a selection of stimuli representing relatively higher levels of threats. By leveraging a zebrafish brain atlas, the authors identified a transgenic line labeling a tiam2a+ cluster of neurons that appears to be the activated population in the Rl. Together, these results demonstrate a subpopulation of pallial neurons that likely categorizes the strength of negative valence in larval zebrafish.

The primary conclusions of this work are well supported by the data. The identification of a neuronal cluster that may underlie the categorization of threat-associated sensory stimuli is significant. Furthermore, this study generates a high-quality functional imaging dataset using cutting-edge microscopy, setting the foundation for understanding the neuronal encoding of emotions in zebrafish.

Results from this work set the stage to answer further exciting questions: How do tiam2a+ Rl neurons modulate the activity of the hindbrain escape circuit? What is the functional role of the Rl population inhibited by threat stimuli? Computationally, how does Rl integrate sensory signals and classify threat levels? How does the activity of Rl change in the context of habituation and conditioning? Future work may use more nuanced stimuli and combine new genetic tools, behavioral recording, and circuit-level analysis to systematically reveal how emotions modulate defensive behaviors.

Weaknesses:

The impact of this work could be further enhanced by incorporating more sophisticated data analysis and by more clearly anchoring the findings within the known framework of zebrafish defensive behavior.

(1) The authors performed statistical analyses across six ROIs per experiment in Figures 1E/J, 3E/J, and 6B/D/F. This increases the probability of Type I errors. Applying multiple comparison corrections would mitigate this concern. Given that most stimuli (except for the "IR heating") are non-directional, the authors may consider first testing for the response symmetry following each stimulus and then combining ROIs from the two hemispheres to calculate a single averaged measurement per region per fish for comparisons of regional dF/F.

(2) I found the topographical mapping of activated and inhibited ROIs very informative. There appear to be two subpopulations of Rl: a posterior-medial population often activated by negative valence stimuli, and an anterior-lateral population that is frequently inhibited. I wonder if it is possible to decode the valence or category of a stimulus based on the topography and response profiles of these neurons? These results would provide additional evidence for the Rl's roles of threat evaluation.

(3) Findings in this paper, especially differential responses of the Rl to full and partial looming, deserve an expanded discussion. The authors should better anchor these findings to established literature to emphasize their significance in the Discussion. For example, how might this potential categorization mechanism contribute to, or differ from, the mechanisms underlying habituation (Fotowat & Engert, 2023, eLife); what are the possible connections between the pallium and the hindbrain escape circuits that could relay these Rl signals (Kunst et al., 2019, Curr Biol)?

(4) The authors make conservative claims associating the tiam2a+ cluster with Rl neurons activated by noxious stimuli, and their data support this conclusion. However, this link could be further strengthened by testing whether the tiam2a+ cluster shows differential responses to full vs partial looming. This could be achieved by performing pERK staining following the stimulus paradigm. While future tools may allow for direct functional imaging of this population, I believe such experiments are beyond the scope of this paper.

(5) Figure 1E/J, Figure 3E/J: Please clarify whether the dashed red vertical lines indicate the onset or the offset of the stimuli. Additionally, different time windows were used for AUC calculations across these experiments; the authors should provide a rationale for these varying windows in the Results or Methods.

Reviewer #3 (Public review):

Summary:

This study provides direct neuroimaging evidence relevant to the integration-segregation theory of exogenous attention-a framework that has shaped behavioral research for more than two decades but has lacked clear neural validation. By combining an inhibition-of-return (IOR) paradigm with a modified Stroop task in an optimized event-related fMRI design, the authors examine how attentional integration and segregation processes are implemented at the neural level and how these processes interact with semantic and response conflicts. The central goal is to map the distinct neural substrates associated with integration and segregation and to clarify how IOR influences conflict processing in the brain.

Strengths:

The study is well-motivated, addressing a theoretically important gap in the attention literature by directly testing a long-standing behavioral framework with neuroimaging methods. The experimental approach is creative: integrating IOR with a Stroop manipulation expands the theoretical relevance of the paradigm, and the use of a genetic-algorithm-optimized fMRI design ensures high efficiency. Methodologically, the study is rigorous, with appropriate preprocessing, modeling, and converging analyses across multiple contrasts. The results are theoretically coherent, demonstrating plausible dissociations between integration-related activity in the fronto-parietal attention network (e.g., FEF, IPS, TPJ, dACC) and segregation-related activity in medial temporal regions (e.g., PHG, STG). Importantly, the findings provide much-needed neural support for the integration-segregation framework and clarify how IOR modulates conflict processing.

Revisions and Evaluation:

The authors have responded thoroughly and convincingly to the concerns raised in the previous round of review. In particular, issues related to the interpretation of dACC activity, the functional characterization of PHG and STG, and reporting clarity have been carefully addressed. The manuscript has been improved in terms of transparency, consistency of reporting, and overall readability.

As a result, I no longer see any major weaknesses. The study is now clearly presented, methodologically sound, and theoretically informative. It makes a valuable contribution to the literature on attention and cognitive control.

Comments on revisions:

I appreciate the authors' efforts in addressing the previous comments. They have responded thoroughly to the concerns raised in the prior round of review. The work is well executed and makes a meaningful contribution to the field.

Reviewer #3 (Public review):

Summary:

Razlan and colleagues provide a detailed anatomical characterization of lamina I projection neurons in the mouse spinal cord that are densely innervated by primary afferents activated by cooling of the skin. The authors validate a Trpm8-Flp mouse line, show synaptic contacts between Trpm8⁺ boutons and projection neurons at the ultrastructural level, and demonstrate at the physiological level that these neurons specifically respond to cooling stimuli. Next, by taking advantage of previous transcriptomic analysis of ALS neurons, the authors identify calbindin as a marker for cold activatetd lamina I projection neurons and map their ascending projections to the rostral lateral parabrachial area, caudal periaqueductal gray, and ventral posterolateral thalamus, well-known thermosensory and thermoregulatory centers. Altogether, these findings provide strong anatomical and functional evidence for a direct line of transmission from Trpm8⁺ sensory afferents through Calb1⁺ lamina I neurons to key supraspinal centers controlling perception of cold and thermoregulatory responses.

Strengths:

The combination of mouse genetics, electron microscopy, ex-vivo physiology, optogenetics and viral tracing provides convincing evidence for a direct cold pathway. The work validates the Trpm8-Flp line by extensive anatomical and molecular characterization. Integration with previous transcriptomic and anatomical data, neatly links the cold-selective lamina I neurons to a molecularly defined cluster of ALS neurons, strengthening the bridge between molecular identity, anatomy, and physiological function.

Weaknesses:

The main limitation remains the relatively small number of neurons that could be recorded electrophysiologically. While understandable given the complexity of the preparation, this necessarily limits generalization.

Reviewer #3 (Public review):

This study aimed to understand the depressing and sensitizing effects of adaptation in mice visual cortex during different behavioral states: locomotion and stationary. There is an impressive characterisation of the responses in different cortical cell types and with different optogenetic manipulations to the inhibitory populations. These form a very interesting dataset to understand the effects of the state on the circuits and gain insight into the mechanisms. This data is then used to constrain a model of the responses. Unfortunately, the model appears to be too flexible, and it was difficult to interpret the insights gained from the different model fits.

Strengths:

The data is impressive. There is a characterisation of responses of PCs and VIP, SST and PV interneurons. Additionally, there is the characterisation of some responses to specific optogenetic manipulations, VIP inactivation, SST or PV activation or inactivation. These data will help develop a good insight into the system. The principle of using the optigenetic manipulations to constrain model parameters is very interesting.

Weaknesses:

Many of the analyses have some concerns in the methodology used, which we list in detail below. Further, the model used to gain insight into the mechanism appears overly complicated and seems hard to gain clear insights from.

Major concerns:

(1) Key concern is the usage of dF/F signals for all analyses, especially when comparing responses.

1a) Figure 1G: Comparison of sensitisers and depressors. It is important to consider what the baseline rates are when making these comparisons, especially when comparing the degree of effects between different cell types. For example, if baseline rates for sensitizers were overall higher, it would mean the difference in gain of response would be lower, and could affect the results in the opposite direction of what is claimed. One option to account for this would be to z-score the overall responses, using the same normalization for locomotion and rest. We also suggest plotting differences in sensitisers, intermediates, and depressors as a function of firing rate. Matching for firing rate across each PC categorization and calculating delta AI for each matched firing rate bin.

1b) Figure 2A-F: The above is an even more significant issue when it comes to estimating spiking rates. The methods do not state how dF/F is calculated. If these are based on using the pre-stim as the reference, the algorithms for spike rate used might not be appropriate if this were used. Using pre-stimulus referencing could result in the estimate going into the wrong range in the calculation of the spike rate.

1c) In both cases above, it could be a problem if baseline firing rates are different between cell types, or states (locomotion/stationary). The latter is established to have effects on many cell types measured, and so needs to be accounted for very carefully.

1d) It would be informative to see per-neuron comparison for adaptive indices during rest and locomotion states. This could be visualized using a scatter plot with AI-rest vs. AI-locomotion for Figures 1D- 1F and 2J- 2L.

1e) Are neurons more strongly modulated between locomotion and rest, also more likely to experience a shift in AI indices (i.e. delta AI). Is there a correlation between the change in firing rate between behavioral states and Delta AI (Loco-Rest)? If so, is this present for all neuron subtypes (e.g. VIP, SST, and PV)?

1f) Optogenetic inhibition of VIP neurons on average abolished the slow depressive effects of adaptation in SST (Figure 3). The strength and prevalence of this effect are unclear. Perhaps one can perform a bootstrap control and opto AI indices and calculate whether AI was significantly reduced following optogenetics inhibition, and if so, on average, how likely was this to occur for the recorded SST neurons? This is important in knowing that the average effects (Figure 3D) aren't driven by a portion of SST neurons, especially as this is later used to confirm the region of parameter space and affects the subsequent results in Figure 4.

(2) Statistics for the effects. There is a mention of Liner mixed models, but no information is given on the actual models being used and tested. This is particularly for the case of Figure 1G, where there is a composition of effect sizes between different populations. What precise significance test is being used? Are the stats on paired cells when considering locomotion and rest?

(3) Model parameters: It is acknowledged that there is a large range of parameters that can model the responses effectively, up to 11% of initial conditions. At 9000 initial conditions, this is around 1000. The parameter estimates are then considered as the mean of each parameter. This seems like a strange choice for a few different reasons:

3a) A mean solution might not be one of the solutions. Let's say the parameters range over a large dimensional space. They could occupy non-overlapping / discontinuous subspaces. In that case, the mean parameters do not necessarily fall within the solution subspaces. Therefore, this reduction to means might not be valid.

3b) Compare distributions rather than means. There are multiple distributions of parameters between conditions. All stats should be on the comparison of distributions rather than just the means.

(4) Visualizing weight matrices: It is very challenging to interpret the weight matrices. Furthermore, it appears that the stationary and locomotion conditions fit independently, and given the large parameter spaces, it is even harder to interpret. Can the fitting instead be done by fitting on one and using those at the initial conditions for the other state? Figure 7 shows an initiative cartoon, but it is not clear how the matrices in Figures 5 and 6 lead to the summary shown in Figure 7. It is also not clear why the connections between inhibitory neurons are not shown in Figure 7. One option is to perhaps run some kind of dimensionality deduction on the parameter space to better interpret the data. When showing deltaWeights, was the model initialised with 'Rest' weights and allowed to change? It is not obvious what the difference is between 'relative change in connection weights' and 'relative change in synaptic weights'.This needs to be clarified.

4a) Model parameters were reduced differently for locomotion and rest (Figure 4). We suggest evaluating the results for locomotion and rest using the same chi-square value of 3 for both behavioral states (at least in controls).

Reviewer #3 (Public review):

Major Comments on first version:

- The manuscript presents a beautiful set of high-quality images showing expression of lens differentiation markers over time in the organoids. The set of experiments is very robust, with high numbers of organoids analysed and reproducible data. The mechanism by which lens specification is promoted in these organoids is, however, poorly analysed, and the reader does not get a clear understanding of what is different in these experiments, as compared to previous attempts, to support lens differentiation. There is a mention to HEPES supplementation, but no further analysis is provided, and the fact that the process is independent of ECM contradicts, as the authors point out, previous reports. The manuscript would benefit from a more detailed analysis of the mechanisms that lead to lens differentiation in this setting.

- The markers analysed to show onset of lens differentiation in the organoids seem to start being expressed, in vivo, when the lens placode starts invaginating. An analysis of earlier stages is not presented. This would be very informative, allowing to determine whether progenitors differentiate as placode and neuroepithelium first, to subsequently continue differentiating into lens and retina, respectively. Could early placodal and anterior neural plate markers be analysed in the organoids? This would provide a more complete sequence of lens vs retina differentiation in this model.

- The analysis of BMP and Fgf requirement for lens formation and differentiation is suggestive, but the source of these signals is not resolved or mentioned in the manuscript. Are BMP4 and Fgf8 expressed by the organoids? Where are they coming from?

- The fact that the lens becomes specified in the centre of the organoid is striking, but it is for me difficult to visualise how it ends up being extruded from the organoid. Did the authors try to follow this process in movies? I understand that this may be technically challenging, but it would certainly help to understand the process that leads to the final organisation of retinal and lens tissues in the organoid. There is no discussion of why the morphogenetic mechanism is so different from the in vivo situation. The manuscript would benefit from explicitly discussing this.

Significance:

This study describes a reproducible approach to differentiate ocular organoids composed of lens and retinal tissues. The characterisation of lens differentiation in this model is very detailed, and despite the morphogenetic differences, the molecular mechanisms show many similarities to the in vivo situation. The manuscript however does not highlight, in my opinion, why this model may be relevant. Clearly articulating this relevance, particularly in the discussion, will enhance the study and provide more clarity to the readers regarding the significance of the study for the field of organoid research, ocular research and regenerative studies.

Comments on revised version:

The authors presented substantial additional experimental evidence that further strengthens their manuscript and addressed with these experiments and their revised results/discussion in the manuscript the comments and suggestions from the reviewers. I think the manuscript has been greatly improved with the additions presented.

Reviewer #3 (Public review):

In this manuscript Moss et al. demonstrate that Hsp70 phosphorylation at a conserved threonine residue integrates DNA damage responses with cell-cycle control. The authors present unbiased biochemical, cell-based, and yeast genetic analyses showing that phosphorylation of human Hsp70 at T495 (and the analogous Ssa1 T492 in yeast) is triggered by base-excision-repair intermediates and downstream DDR kinase activity, leading to delayed G1/S progression after DNA damage. They used orthogonal approaches such as ATPase assays, phospho-specific detection, kinase-inhibition studies, synchronization experiments, and phenotypic analyses of phosphomutants. They presented robust data which collectively supported the conclusion that dynamic Hsp70 phosphorylation functions as a conserved "molecular brake" to prevent inappropriate S-phase entry under genotoxic stress.

Comments on revisions:

The authors have addressed all my questions and concerns.

Reviewer #3 (Public review):

Summary:

The present work was aimed at investigating the specific contributions of thalamic nuclei to associative threat learning and extinction. Using fMRI, it examined activation patterns across pulvinar divisions, the lateral geniculate nucleus (LGN), and the mediodorsal thalamus (MD) during threat acquisition, extinction, and recall. It goals was to uncover whether distinct thalamic systems support different modes of learning-automatic survival mechanisms versus more deliberate processes-and to propose a hierarchical pulvinar model of fear conditioning. The manuscript also tried to refine current neuroanatomical models of threat learning and memory, highlighting the role of thalamic nuclei in it.

Strengths:

(1) Valuable theoretical elaboration and modeling regarding the differential role of pulvinar subdivisions on feedforward (inferior, lateral) and higher-order integration (anterior), and their functional interplay with other relevant subcortical and cortical structures in associative threat and extinction learning.

(2) Large sample sizes and multipronged analytical approaches were used for hypothesis testing.

(3) Exhaustive literature review in the field of associative threat, as well as regarding the role of thalamic nuclei and other brain structures in it.

Weaknesses:

(1) The manuscript has improved methodologically and analytically after the review. Several weaknesses remain, in my opinion, but still findings are valuable and the evidence can be considered as convincing.<br /> a) fMRI data have low resolution (3 cubic mm), which certainly limits the examination of small nuclei such as the ones investigated here, and especially the examination of the LGN and inferior pulvinar.<br /> b) fMRI was normalized to standard space. Analyzing the data in individual-subject space would have given you the options of avoiding altering every participant's brain and of using more precise atlases than the normalized AAL for ROI selection.<br /> c) Motion during scanning was poorly controlled. Including the motion parameters as covariates of no interest in the GLM/analysis does not fully guarantee that motion is not influencing the results, and that motion is not differentially influencing some experimental conditions more than others.

Reviewer #3 (Public review):

Summary:

This is an interesting paper. The process of tooth exfoliation and replacement in vertebrates remains an intriguing and fascinating subject of inquiry. As the scientists noted, there are no mammalian models that can be used to examine signaling pathways in real time.

Strengths:

This work integrates in vivo and high-resolution transcriptomics. The study confirms previous findings and emphasizes the need for additional research into the processes that drive the restoration of missing teeth for future therapeutic uses.

Weaknesses:

I disagree with the use of the phrase "plucking". Instead, the authors use tooth extraction or tooth removal, which is clinically more correct for the procedure they are doing.

The title is rather broad and appears to be more appropriate for a review than an original research work. I would advise specifying the species under research and/or the sort of damage model used in the transcriptome analysis.

It's uncertain whether the findings are exclusively based on regeneration. The presence of tooth remnants, as well as unintended harm to surrounding tissues, may have triggered repair mechanisms, thereby biasing the current data. How did the authors handle this issue? The oral cavity was under severe manipulation, increasing the inflammatory stimuli, a situation that does not take place in physiological exfoliation.

The authors indicated the use of microCT analysis; however, no such information appears in the main text. In fact, this manuscript lacks anatomical information. It is required to conduct histological examinations of the regenerated teeth at various time points.

Although the current findings confirm previously found and verified signaling pathways, the absence of functional data lends uniqueness to this work.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

Specificity of the methods used to target both meningeal macrophages and RAMP1 are limited. A discussion section on potential pitfalls is included to address this.

Reviewer #3 (Public review):

The authors build on the large body of their previous research, which showed that the mouse primary visual cortex is organised into two types of clusters, M2+ and M2-, which exhibit distinct input patterns from thalamus and higher visual cortical areas and distinct visual tuning preferences. The current study reveals that a like-to-like projection from within-cluster neurons to the areas that provide feedback projections and, furthermore, that neurons in the M2- clusters are more strongly affected by non-visual signals about the locomotion of the animal.

The study adds fundamental insights to our understanding of the principles of cortical organisation and computation, specifically how the cortex integrates sensory and action-related signals.

While the tracing data are very convincing, data analysis should be strengthened to support the claims:

(1) The locomotion modulation index (LMI) compares the mean activity during running and not running but does not seem to account for differences between visual stimuli, so that the LMI could be influenced by the neuron's visual tuning rather than its sensitivity to locomotion, e.g. if the mouse was running more when the neuron's preferred stimulus was presented. Trials should first be averaged per stimulus, and then across stimuli. Alternatively, only the preferred stimulus could be considered.

The significance test (unpaired t-test) suffers from the same flaw. Instead an ANOVA (with stimulus parameter as factor) would resolve the problem, or testing whether fitting the data with two tuning curves (one per locomotion state) or a single curve results in a lower error (using cross-validation).

Given that there is evidence that specific visual stimuli can induce more or less running in mice, this issue is very important to account for behavioural differences across stimuli.

(2) All bars in Figure 2b show a lower LMI than the reported mean LMI of 0.19. This should be checked.

(3) Correlation tests: Pearson correlation is only meaningful when applied to continuous data. A more suitable test for discrete data like the M2 patch quantile is a rank test like Kendall's coefficient of rank correlation. This applies to data in Figure 2b,c, 4j,k, Figure 2 - Supplement 2,1a, etc.

(4) How OSI was determined should be clarified. Specifically, were R_pref and R_ortho the mean responses to the two opposite movement directions? Similarly, how was the half-width at half-maximum of orientation determined? From the fits in Figure 2a, it looks like the widths of both Gaussians can be different.

(5) The correlation measures in Figure 3 would greatly benefit from additional analyses to help interpretation of the results.

a) Correlations between neurons typically increase with increasing firing rates (e.g., de la Rocha J, Doiron B, Shea-Brown E, Josić K, Reyes A. 2007. Correlation between neural spike trains increases with firing rate. Nature 448:802-6. doi:10.1038/nature06028). Could the higher correlations in M2+ pairs (Figure 3a) be explained by higher firing rates in M2+ compared to M2- neurons?

b) To determine correlations in Figure 3a, trials during locomotion and stationarity were pooled. As locomotion impacts the firing rate of the neurons, it would be helpful to separate correlations between the two states, locomotion vs stationarity, so the measures reflect something closer to "noise correlations" rather than tuning to locomotion.

c) Similarly, in Figure 3b, I wonder whether the large correlations in M2- pairs are driven by locomotion rather than functional connectivity. As suggested in b, a better test of noise correlations would be to account for locomotion, i.e., separate trials by stimulus identity and locomotion state. To prevent conditions with few trials from having greater weight in the overall noise correlations, I suggest the authors first z-score responses per condition, then determine noise correlations across all trials (as explained in Renart et al., 2010).

d) Correlations in Figure 3a,b should be tested with an ANOVA and a control for multiple tests.

(6) In plots like Figure 4j-l, it would be very informative to show individual measures (per ROI and mouse) in addition to mean +- SEM. As the counts are low (<10) it wouldn't obstruct the plot.

(7) The caption of Figure 4l says that most retrogradely labelled cells are located in L2/3. However, the plot only shows data from L2/3 and a single section of L4, so one cannot compare it to other layers. Can the authors corroborate the claim with data from other layers?

(8) Methods:<br /> The authors should provide more details on the visual stimuli: What was the background on which gratings were presented? How long was the inter-stimulus interval? What was presented during the inter-stimulus interval? How large were gratings used to map tuning to SF, TF, and orientation?

Reviewer #3 (Public review):

Summary:

This manuscript uses a novel paradigm to demonstrate that rotational motion patterns in the retinal image, called curl, directly influence perception of heading direction. This means that it is not necessary to recover the focus of expansion, defined by the point of zero motion when moving along a straight trajectory toward a target, as is commonly thought.

Strengths:

It has long been accepted that the focus of expansion of the optic flow field generated by self-motion is used to guide heading direction. While there have been many challenges to the need to recover the focus of expansion when gaze is not in the direction of travel, it is still not well understood how retinal motion patterns contribute to heading perception. Recent work has demonstrated the complexity of the retinal motion patterns during natural walking, where body motion adds a rotational component. A rotational component also results from curved paths as well as gaze off the direction of travel. This rotational component is called curl. The primary contribution of this manuscript is to demonstrate convincingly that curl influences perception of heading, and that it is not necessary to recover the focus of expansion.

A strength of the manuscript is that realistic retinal motion patterns are generated by recording the image sequences generated by a walker in a virtual environment, and then using those patterns as stimuli in the experiment. This allows the creation of the more complex flow patterns that are a consequence of the bob and sway of natural walking, which are often considered a minor factor. The elegant experimental design allows direct manipulation of the curl signal, and this in turn directly influences measured heading perception. Another strength is that the authors ground their findings in control theory and neural computations, using a model that produces human-like path trajectories.

The study is timely, given the long history of this question, together with the growing understanding of the complexity of naturally generated retinal motion and the absence of direct evidence for the way that these motion patterns are used in heading perception. It adds an important piece of evidence for how retina-centered optic flow may be used by the visual system, which is critical for our understanding of motion processing in the brain.

Weaknesses:

The primary limitation of the paper is that it avoids discussion of some of the inevitable complexities of heading perception. The main issue is what exactly is meant by heading. Different behaviors evolve over different timescales. The geometry of retinal motion defines instantaneous heading, which varies widely through the gait cycle. Time-varying information like this is known to be important in the momentary control of balance. Heading can also be thought of as steering the body toward a distant goal, which evolves over longer timescales. The current manuscript appears to be concerned with heading information integrated over a few seconds and seems to provide evidence that heading is indeed integrated over the gait cycle. The issue of the time scale of the computation is touched on, but it is not related to how it might be used in normal walking or what situations it might apply to. Steering toward a distant goal during walking is not a very difficult problem and may not require evaluation of retinal motion, but control of balance is more challenging and may depend critically on curl. Consequently, the timescale of the computation needs to be considered in order to understand what is meant by heading.

Reviewer #3 (Public review):

Summary:

This study used a pre-registered novel behavioural paradigm and computational modelling to investigate multi-sensory influences on detection and confidence. Participants performed amodal detection of auditory and visual stimuli (indicating that a stimulus was there when either an auditory stimulus or a visual stimulus or both were present), followed by amodal and unimodal confidence ratings. Detection was higher when both stimuli were present, and the presence of one modality increased the confidence in the presence of the other modality. In contrast to previous detection studies, confidence was higher for absent than for present judgements, but metacognitive efficiency was higher for present judgements. Metacognitive sensitivity was higher for bimodal stimuli, but this was not the case for metacognitive efficiency, suggesting that the sensitivity might be driven by first-order performance. The computational model showed that both detection and confidence in absence followed a disjunctive evidence integration rule, while confidence in presence followed a conjunctive integration rule.

Strengths:

The paper has several major strengths. Firstly, it addresses a novel research question using an innovative and well-controlled paradigm. Furthermore, the paradigm and analyses were pre-registered, and all effects that were interpreted were replicated in two independent samples. Finally, the paper uses an advanced computational model to capture counterintuitive patterns in the data.

Weaknesses:

The major weakness of the paper is the narrative structure. It is not always clear how the different analyses relate to the main research question. Many different effects are reported in terms of detection accuracy, bias, confidence and metacognition, as well as cross-modal and unimodal versus bimodal effects. It would help readability if the paper were streamlined in terms of the research question that is being answered, which I believe is specifically about multimodal absence judgements. Relatedly, for a reader not intimately familiar with the metacognition literature, the difference between MRatio, metacognitive sensitivity and metacognitive efficiency is not obvious. It would be good to clarify this more in the manuscript.

In general, the conclusions drawn by the authors seem to be supported by the results. However, I was missing quantitative model comparisons between the conjunctive and the disjunctive models and an explanation of why the models systematically overestimated the confidence ratings. Furthermore, the 'perceptual multisensory interference' section reports on very interesting effects, but these are not supported by statistical tests in the main text. It would help to assess the strength of the claims if the statistical evidence in favour of these claims were presented together in the main text.

One other concern is that in real-world multi-sensory perception, such as the mosquito example in the introduction, the auditory and visual signals have a strong natural association, which means that if you hear the auditory signal, you expect that you will see the visual signal soon and vice versa. As far as I understood, this association was not present in the current paradigm, which might influence the type of effects that one would expect to see.

Reviewer #3 (Public review):

Summary:

Sugarman et al. present a microCT scan of a hatchling octopus from the species Octopus bimaculoides. The scan is publicly available and poses as a valuable tool for the field of cephalopod biology. Using this scan, the authors uncover two undescribed neural pathways: the intermediate longitudinal tract (iLTs) in the axial nerve cord linking the suckers to the brain, and the arm-to-arm u-tracts (AAUTs) interconnecting neighboring arms. How the eight sucker-lined octopus arms are coordinated with one another and with the brain have been long standing questions in the octopus motor control field, and the results presented here have promise for addressing these questions. However, major weaknesses addressed below limit the interpretability of the dataset.

Strengths:

The authors have publicly published a scan of an entire hatchling octopus, with major organs and subdivisions of the nervous system already segmented. Accessing the data is straightforward, and the authors provide adequate instructions on how to navigate the dataset.

The authors provide validation of the AAUTs using lucifer yellow and wheat germ agglutinin. To overcome motion artifact in the hatchling dataset, the connections between the iLTs and the suckers are validated with a microCT scan of a distal section of adult arm.

Weaknesses:

Given the resolution of the dataset, neural connectivity is determined by texture differences alone, which can be misleading. As such, any claims of anatomical connectivity will need further validation, ideally with tracing techniques. While the authors investigated the AAUTs with other techniques, no such validation exists for the iLTs. Furthermore, the authors themselves state that as the iLTs converge with the brachial nerve, they become indistinguishable from other fibers. Any connections of the iLTs to the brain are only hypothesized, despite their claim of demonstrating a clear pathway from the suckers to the brain.

The relevant prior research on octopus neurobiology is not well explained, making it challenging to understand the significance of the results in a broader context.

Reviewer #3 (Public review):

Summary:

The manuscript by Patel et al investigates the hypothesis that CDHR1a on photoreceptor outer segments is the binding partner for PCDH15 on the calyceal processes, and the absence of either adhesion molecule results in separation between the two structures, eventually leading to degeneration. PCDH15 mutations cause Usher syndrome, a disease of combined hearing and vision loss. In the ear, PCDH15 binds CDH23 to form tip links between stereocilia. The vision loss in less understood. Previous work suggested PCDH15 is localized to the calyceal processes, but the expression of CDH23 is inconsistent between species. Patel et al suggest that CDHR1a (formerly PCDH21) fulfills the role of CDH23 in the retina.

The experiments are mainly performed using the zebrafish model system. Expression of Pcdh15b and Cdhr1a protein is shown in the photoreceptor layer through standard confocal and structured illumination microscopy. The two proteins co-IP and can induce aggregation in vitro. Loss of either Cdhr1a or Pcdh15, or both, results in degeneration of photoreceptor outer segments over time, with cones affected primarily.

The idea of the study is logical given the photoreceptor diseases caused by mutations in either gene, the comparisons to stereocilia tip links, and the protein localization near the outer segments. The work here demonstrates that the two proteins interact in vitro and are both required for ongoing outer segment maintenance. The major novelty for this paper would be the demonstration that Pcdh15 localized to calyceal processes interacts with Cdhr1a on the outer segment, thereby connecting the two structures. Unfortunately, the data as presented are inadequate proof of this model.

Strengths:

The in vitro data to support the ability of of Pcdh15b and Cdhr1a to bind is well done. The use of pcdh15b and cdhr1a single and double mutants is also a strength of the study, especially being that this would be the first characterization of a zebrafish cdhr1a mutant.

This is a large body of data.

Weaknesses:

(1) I have serious concerns about the quality of the imaging here. The premise that cdhr1a/pcdh15 juxtaposition is evidence for the two proteins mediating the connection between outer segments and calyceal processes requires very careful microscopy. The SIM images have two major issues - one being that the red and green channels are misaligned and the other being evidence of bleed through between the channels. This is obvious in Fig 2A but likely true across all the panels in Fig 2, and possibly applies to confocal images in Fig 1 as well. The co-labelling with actin shows very uneven, punctate staining for actin bundles.

(2) The newly added TEM and transverse sections include colored regions that obscure the imaging.

(3) The quantification should be done with averages from individual fish. Counting individual measurements as single data points artificially inflates the significance. Also, the cone subtypes are still lumped together for analysis despite their variable sizes.

(4) I highlighted previously that the measurement of calyceal processes was incorrect. The redrawn labels in Fig 7 are now more accurate, although still difficult to interpret. However, the quantification in Fig 7O is exactly the same. How can that be if the measurement region is now different?

(5) Lower magnification views would provide context for the TEM data.

(6) The statement describing the separation between calyceal processes and the outer segment in the mutants is still not backed up by the data.

(7) The authors state "from the fact that rod CPs are inherently much smaller than cone CPs". This is now referenced, but incorrectly. Also, the issue of pigment interference was not addressed.

(8) The images in panels B-F of the Supplemental Figure are uncannily similar, possibly even of the same fish at different focal planes.

Reviewer #3 (Public review):

Summary:

The authors proposed to use 5-layer systems level analysis (genomics, transcriptomics, proteomics / protein degradation, metabolomics, phosphoproteomics) to uncover how post-transcriptional mechanisms regulate stage differentiation in Leishmania donovani.<br /> This enabled the identification of several potential regulatory networks, including the regulation of stage-specific gene clusters by RNA stabilisation or decay, proteasomal degradation and protein phosphorylation.<br /> In the new version of this manuscript, the authors have addressed all questions raised by the reviewers.

Strengths:

Although some observations in this study have already been described in the literature, the integrated analysis applied here provides a novel view on how different levels of post-transcriptional networks regulate Leishmania differentiation. This "5-layer system" represents the first analysis of this depth in kinetoplastid parasites.<br /> The revised version with an increased sample number for the RNA-seq now made the authors assumptions adequate to their obtained data.<br /> The use of a proteasomal inhibitor adds an interesting insight in how protein degradation is involved in the parasite differentiation, confirming previous observations in the literature, and help to explain the discrepancies between mRNA and protein expression in the different stages.

Weaknesses:

While this work provides an impressive and foundational dataset, it opens the door for future research to rigorously validate these initial findings and conclusions.

Significance and Impact in the field.

The different datasets generated in this study will be of great interest to the parasitology community, either to be used for hypothesis generation, to validate data from other sources, etc.

The multi-layered analysis performed here identified a series of potential feedback loops and regulatory networks to be further explored in organisms that lack transcriptional control.

Reviewer #3 (Public review):

Summary:

This study addresses the role of MIRO1 in vascular smooth muscle cell proliferation, proposing a link between MIRO1 loss and altered growth due to disrupted mitochondrial dynamics and function. While the findings are useful for understanding the importance of mitochondrial positioning and function in this specific cell type, the main bioenergetic and mechanistic claims are not strongly supported.

Strengths:

- This study focuses on an important regulatory protein, MIRO1, and its role in vascular smooth muscle cell (VSMC) proliferation, a relatively underexplored context.<br /> - This study explores the link between smooth muscle cell growth, mitochondrial dynamics, and bioenergetics, which is a significant area for both basic and translational biology.<br /> - The use of both in vivo and in vitro systems provides a useful experimental framework to interrogate MIRO1 function in this context.

Weaknesses:

- Some key bioenergetic aspects may require further investigation.

Comments on revisions:

The authors have adequately addressed most of the concerns I raised. I would suggest adding some of the justifications provided to the reviewers to the manuscript to further clarify and aid interpretation of the data, especially for the bioenergetic part (e.g., the proposed interaction with CI components, which might otherwise appear implausible to readers).

Reviewer #3 (Public review):

Summary:

The authors investigate how HIV-1 Env engagement affects the nanoscale organization and dynamics of the CXCR4 coreceptor on target cells. Using single-particle tracking TIRF microscopy, they analyze CXCR4 distribution following exposure to gp120 or HIV virus-like particles, including both wild-type CXCR4 and the WHIM-associated CXCR4.R334X variant. The study further examines the role of CD4-CXCR4 heterodimerization and contrasts Env-induced receptor organization with that elicited by the natural ligand CXCL12.

Evaluation:

A major strength of this work is the integration of high-resolution imaging with functional and comparative analyses that distinguish Env-induced CXCR4 clustering from chemokine-driven effects. The experiments are clearly described, include appropriate controls, and are supported by quantitative analyses that are consistent across experiments. The revised manuscript appears to have addressed many of the technical and interpretive issues raised during initial review, improving clarity around data analysis and strengthening confidence in the conclusions.

I am not an expert in TIRF microscopy or single-molecule tracking and defer to other reviewers regarding limits of imaging and tracking methods. However, I did not identify major inconsistencies between the biological data presented and the conclusions drawn.

The authors data support the conclusion that HIV-1 Env, delivered as gp120 or virus-like particles, promotes CD4-dependent nanoscale clustering of CXCR4, including the CXCR4.R334X variant associated with WHIM syndrome, in a manner distinct from CXCL12-induced receptor organization. The authors are generally careful to frame their conclusions in proportion to the evidence and avoid overinterpretation.

Overall, this study builds on prior work on CXCR4 distribution and HIV entry by providing higher-resolution insight into receptor nanoclustering and its modulation by Env. The findings provide a mechanistic refinement rather than a conceptual paradigm shift but is a valuable dataset useful to researchers studying HIV entry, coreceptor biology, and membrane receptor organization.

Reviewer expertise: HIV-1 Envelope glycoproteins and entry assays, HIV broadly neutralizing antibodies, HIV vaccine design

Comments on revised version:

This reviewer has no further recommendations and thanks the authors for clarifying that the Env content in gp120-VLPs was lower than the NL4-3deltaIN particles but that the percentage of mature particles in the gp120-VLPs was higher.

Reviewer #3 (Public review):

Summary:

Yang et al sought to describe central brain circuits that underlie nociception-induced escape in Drosophila using a combination of neurogenetic tools to silence subsets of neurons and to trace their postsynaptic connections. They present interesting data that identify subsets of DANs and MBONs that are required for a jumping response to an aversive stimulus, but not for baseline locomotion, and present a model for linking peripheral nociception to MB- dependent escape behavior.

Strengths:

They use an innovative avoidance assay to elicit a robust behavioral response and use trans-tango to identify downstream targets of painless and TrpA1-expressing neurons.

Weaknesses:

This reviewer's enthusiasm for the study is lowered due to an incomplete description of methods, methods section, appropriate behavioral controls, immunohistochemistry data, and a complete behavioral screen of DANs and MBONs. Below I list my suggestions, questions, and criticisms.

(1) Behavioral studies are interesting. The assay is simple, yet innovative. However, there is no power analysis or explanation of how sample sizes were selected. I commend the authors for including a positive control; however, although UAS-controls are present, there are no GAL4-controls included in the study. Given that many of the lines used for behavior are split-GAL4's, it's unclear if the additional transgene influenced behavior. This should be addressed.

(2) It is also not clear from the methods how the behavior was run and how it was analyzed. Was baseline locomotion recorded before the laser was introduced? I assume this is the case; however, more importantly, how long after the flies were introduced to the arena were baseline recordings collected? How much data was used to calculate velocity? Were the experimenters blind to the conditions they were assessing? More detail in the methods is essential for understanding the data and providing an opportunity to replicate results.

(3) At times, the authors describe "locomotion velocity" as baseline locomotion, but other times, they describe it as escape velocity (see reference to Figure 1F). The authors should clarify whether escape velocity was calculated.

(4) Immunohistochemistry: There is a lack of detail regarding a description of the flies used for trans-tango experiments. How many brains were evaluated? Was there variability across brains? Were the flies males or females? This is an important detail as sex could impact the level of expression of the ligand and therefore the results. It is also not clear at what age these flies were dissected and at what temperature they were raised. This can also significantly affect the post-synaptic signal that is measured (see Talay et al 2017).

(5) Figure 2 shows the overlap of trans-tango and dopamine signal, but there is no signal for the GAL4-line to evaluate the overlap between presynaptic signal and postsynaptic signal. This expression is an important consideration and should be included.

(6) Expression of the GAL4 lines in the central brain is also important to show because the authors suggest that, because painless and TrpA1 expression does not fully overlap in peripheral tissue, it might converge in the central brain. Does that central brain expression of painless and TrpA1 overlap?

(7) Further, although the authors clearly label the different dopamine subsets (PPL1, PAL, and PAM), some orientation with regard to where these images were taken would be helpful. I recommend a stack showing the location of the cell bodies and then a zoom in to see the overlap.

(8) Behavioral data for DANs and MBONSs: I recommend that the authors discuss the results by the neurons that are targeted and not the driver lines. For instance, the authors suggest they get the largest effects for 433B, 434B, and 298B, but all of these lines target very similar neuronal subsets y4>y1y2. It's also not clear why different split-lines were selected. Several of the lines have overlapping expression, and other compartments were not included at all. In order to determine which MBONs and DANs are required for escape behavior, all MBONs and DANs should be included. See Aso et al for a list of recommended lines for behavior based on specificity and intensity.

(9) Based on trans-tango data, it is not clear why the authors focus exclusively on PPL1 and PAM when PAL, PPM1, 2, 3, and PPL2 also overlap with painless and trpA1. Certainly, PPL1 and PAM DANs innervate the MB, but so do some of the other DANs identified.

(10) For Figure 5, the titles of A and B are DANs and MBONs, but it is really showing the average jumping response when neurons that innervate MB compartments are silenced. Many DANs and MBONs innervate multiple compartments (PPL1-a`2a2, etc.); thus, if the intention is to identify neural circuits that modulate escape response, the analysis should focus on the neurons, not the MB compartments. I recommend reorganizing this data so it highlights the DANs and MBONs instead of the MB compartments. I also recommend showing error bars for averages and/or raw data and organizing the x-axes so DAN and MBON compartments can be easily compared.

(11) Lastly, nuance is lost here in the Behavioral Potency Level, given that some of these compartments are over-represented and not adjusted for the strength of expression in different split-GAL4 lines. Aso et al. (2014) recommended specific split-GAL4 lines based on specificity and intensity. Some of the lines that are included in the average Behavioral Potency are not recommended for behavior based on the intensity of expression, which could significantly influence the potency score.

Reviewer #3 (Public review):

Summary:

This study presents a novel device for wireless control of optogenetic stimulation of the mouse brain, the Blueberry, using Bluetooth Low Energy (BLE) communication for parallel activation of up to 4 devices through an Arduino interface. The authors also present two types of brain implants for light delivery that can be connected to the Blueberry: one using uLEDs for surface cortical stimulation, and another using optical fibers for intra- or sub-cortical implants. The architecture of the system, including electronics, communication, and programming, is thoroughly described. Because the system was especially designed to be integrated with existing software used for neuroscience behavioral experiment for closed-loop experiments, validation of the system is shown on two different scenarios: a learning task in a "infinite" Y-maze, where light delivery at precise locations conditions arm choice for navigation; and a social interaction analysis where 3 animals are simultaneously stimulated in order to alter social dynamics among the group.

Strengths:

(1) The full system can be built by individual labs with simple PCB printing, off-the-shelf components, and readily available hardware (Arduino) for widespread dissemination.

(2) Four headstages can be controlled in parallel for simultaneous experiments with multiple mice.

(3) Validation across different relevant behavioral tests, demonstrating the potential of integrating Bluberry in closed-loop setups.

Weaknesses:

(1) Some details in the manuscript regarding system characterization (latency, battery life, etc) are included only in the supplementary materials.

(2) The practical details of integration with other commercial and open-source software used for the closed-loop experiments, which could help third-party researchers interested in using the system, are lacking sufficient detail.

(3) System range (3 meters reported) is limited for a BLE device.

(4) Light output amplitude is not programmable, limiting the choice of stimulation protocols and LEDs used.

(5) Thermal modeling of the cortical surface stimulator was not performed, and it is unclear if the brain implant for this purpose is within the safety limits.

(6) The paper is missing a comparison with other state-of-the-art devices for wireless control of optogenetic stimulation in mice.

Reviewer #3 (Public review):

Summary:

This modelling study connects synaptic plasticity, connectivity motifs, and representational drift. The authors combine excitatory and inhibitory STDP with weight normalization and intrinsic plasticity in a recurrent spiking network of AdEx neurons. This combination generates heavy-tailed synaptic weight distributions and supports repeating spike sequences under both unstructured and structured inputs. While global network statistics stabilize over time, individual synapses continue to change, creating a form of drift. Structured inputs further stabilize sequences, yet the network retains flexibility to learn new patterns.

Strengths:

(1) Multi-scale turnover analysis:

The authors study the evolution of individual synapses, 3-neuron motifs, follower neurons, and entire neuronal sequences, revealing distinct turnover timescales.

(2) Fan-in/out motif analysis:

A specific connectivity motif (fan-in/out) is shown to be over-represented in the network and preferentially stabilised by the plasticity rules compared to other possible motifs. This generates interesting insights and testable predictions.

(3) Connection to representational drift:

The connection of ongoing synaptic plasticity to drift is timely and interesting, reproducing observations of macro-level stability and synapse-level turnover with a relatively simple mechanism.

(4) Rigour and thoroughness:

The overall quality of the numerical experiments performed in this study is high, with extensive supplementary material performing various controls to solidify the claims.

Weaknesses:

(1) Limited connection to network function:

Sequence detection relies on a rather artificial protocol (forced spiking of a single neuron 1,000 times), which I suspect mostly tests whether the lognormal tail of the weight distribution can propagate activity. This risks being circular. I think performing the same sequence analysis on a random network/a network with the same weight distribution but shuffled would help understand what comes from a generic heavy-tailed weight distribution and the particular weights potentiated by the plasticity rules used here.

The network, which would classically be evaluated as a memory network, is not assessed on this aspect. While the authors do not overclaim, this limits the impact.

Relatedly, the relearning experiment (Figure 5G) shows catastrophic forgetting. This is acknowledged in the discussion, but the suggested solutions (alternating patterns, plastic readout) are speculative without supporting simulations. This limits the applicability of the model as a memory model or, more broadly, as a model of a brain region/function.

Additionally, in the sequence learning experiments with structured input, the ability to learn seems tied to the very specific timescale of pattern presentation (~10 ms per pattern, comparable to the STDP kernel time constants), arguably faster than the timescale of external stimuli. The stability of sequences may also owe more to the normalization scheme than to STDP per se.

(2) Novelty claims and positioning within the literature:

On page 16, the authors write: "Our results demonstrate that spiking sequences can be generated in randomly connected networks trained by synaptic plasticity even under unstructured inputs, which supports STDP being the main actor, while stabilizing mechanisms such as weight normalization and intrinsic plasticity play a complementary role." (c1).

Several aspects of this work are less novel than the presentation suggests:

(a) The fact that STDP can create sequence-like dynamics/asymmetric connectivity matrices in recurrent networks has been studied theoretically [1,2] and in simulations [3,4,5]. While [3] is cited, the manuscript underplays the similarity. [4] (uncited) considers e+iSTDP with a different homeostatic term to represent sequential stimuli in large recurrent spiking networks. [5] (uncited) also considers a recurrent spiking network with several STDP-like rules and shows that many combinations can store and recall sequential inputs.

(b) Lognormal weight distributions emerging from STDP-based plasticity and the autonomous emergence of connectivity structures have extensive literature. While many of these articles are already cited in the manuscript, I fail to see what this work brings to this matter compared to existing work (particularly [6]).

(c) Several published works challenge the manuscript's implicit claim (c1) that sequences require their particular combination of rules. Many other plasticity mechanisms can create sequences [3,4,5,7,8,9]. Some interpretations may also need to be dialed down: [10] (uncited) showed that sequences can be stored and retrieved using EI and IE plasticity alone. iSTDP may be doing more computational work than acknowledged, which complicates the interpretation of which mechanisms are truly driving the phenomena.

Overall, most of the relevant work is already cited in the manuscript, but not necessarily acknowledged adequately.

(3) Justification of plasticity model/robustness analysis:

The parameters in Tables 1 and 2 are quite specific without strong justification (for instance, different sparsity values for each connection type and specific normalization factors). Without parameter sweeps, it is difficult to know whether the key findings are robust or overfit to this particular network configuration. Given the number of parameters, exhaustive sweeps are out of question, and the argument made previously would still prevent the rule combination proposed from being considered as more than one possible mechanism for sequence generation among many others. However, this deserves to be acknowledged, and potentially a few sweeps to be run (e.g., over LTP/LTD ratio, normalization threshold, and network size). I don't think that Figure S12, which shows that removing any component of the model causes it to break down in some way, is enough to cover alternative plasticity rules.

A related concern is that the network is small by current standards (1,200E + 240I neurons), especially with sparse connectivity (6-20%). Small networks with few connections are susceptible to synchronization (other studies typically consider networks of at least 10k neurons). The authors should discuss whether the phenomena they observe would persist at larger scales and under more biologically realistic connectivity. Specifically, are the intrinsic and normalization plasticity terms as crucial in this case?

(4) Fan-in/out motif evidence is correlational:

The evidence linking the fan-in/out motif to sequence stability appears to be correlational. Properly establishing causality would require targeted ablations or rewiring of fan-in/out connections. While designing a clean causal intervention may be difficult, the correlational nature of the evidence should be stated explicitly.

Conclusion:

To summarize, the manuscript would benefit from:

(1) Reframing the contribution:

Multi-scale turnover analysis and the discussion around representational drift as the core novelties. I would reposition sequence emergence and lognormal distributions as reproducing known results under a specific plasticity model and analysis method.

(2) Acknowledging that many rule combinations could produce equivalent outcomes, and not suggesting that the combination chosen here is special.

(3) Adding parameter sensitivity analysis or, at a minimum, discussing robustness.

References:

[1] Kempter, Gerstner and van Hemmen, Hebbian learning and spiking neurons, 1999, PRE

[2] Ocker, Litwin-Kumar and Doiron, Self-organization of microcircuits in networks of spiking neurons with plastic synapses, 2015, plos CB<br /> (Theoretical account of STDP in spiking networks and motifs, though it only looks at 2-synapse motifs (not fan-in/fan-out)).

[3] Fiete et al., Spike-Time-Dependent Plasticity and Heterosynaptic Competition Organize Networks to Produce Long Scale-Free Sequences of Neural Activity, 2010, Neuron

[4] Duarte and Morrison, Dynamic stability of sequential stimulus representations in adapting neuronal networks, 2014, Frontiers in Comp Neuro

[5] Confavreux et al., Memory by a thousand rules: Automated discovery of functional multi-type plasticity rules reveals variety and degeneracy at the heart of learning, 2025, bioRxiv

[6] Zheng, Dimitrakakis and Triesch , Network Self-Organization Explains the Statistics and Dynamics of Synaptic Connection Strengths in Cortex, 2013, plos CB

[7] Zheng and Triesch, Robust development of synfire chains from multiple plasticity mechanisms, 2014, Front Comp Neuro

[8] Ravid Tannenbaum and Burak, Shaping Neural Circuits by High Order Synaptic Interactions, 2016, plos CB

[9] Bell, Duffy, and Fairhall, Discovering plasticity rules that organize and maintain neural circuits, 2024, NeurIPS

[10] Gong and Brunel, Inhibitory Plasticity Enhances Sequence Storage Capacity and Retrieval Robustness, 2024, bioRxiv

Reviewer #3 (Public review):

Summary:

This manuscript investigates whether the theta-gamma phase-amplitude coupling in the human auditory cortex serves as an intrinsically generated neural mechanism for hierarchically parsing speech or not. By analyzing speech corpora across 17 languages alongside human intracranial EEG recordings, the authors demonstrate that these nested oscillatory dynamics are actually inherent, robust acoustic properties embedded within the speech envelope itself. Consequently, they claim that rather than generating parsing windows internally, the early auditory cortex acts as a temporal demultiplexer that segregates syllabic, vocalic, and pitch features into distinct, stimulus-driven neural channels. Furthermore, the study presents evidence for a reversed functional directionality wherein fast-varying gamma activity drives the phase alignment of slower theta rhythms, fundamentally reframing auditory PAC as a stimulus-evoked alignment to a highly structured external signal rather than an endogenous cognitive parsing tool.

Strengths:

(1) The authors demonstrated robust theta-gamma acoustic structure across languages. They analyzed the acoustic speech envelope across 17 typologically distinct languages. This establishes that the nested theta-gamma acoustic structure is a universal feature of human speech, rather than an artifact of one language's specific phonology.

(2) The use of time-resolved, high-SNR intracranial recordings is a critical strength of this study. This approach provides the precise spatiotemporal fidelity required to confidently separate and delineate multiplexed high-frequency dynamics, particularly the low- and high-gamma bands, that are essential for accurate speech decoding but are typically attenuated or lost in non-invasive scalp recordings.

(3) The authors move beyond standard correlational PAC metrics by employing a suite of converging analyses, including the isolation of true oscillations from aperiodic noise and the directional index. Together, these metrics demonstrate that auditory PAC is a stimulus-evoked alignment to a highly structured external speech signal, rather than an intrinsically generated top-down parsing mechanism.

Weaknesses:

(1) A major methodological concern is the use of ICA across SEEG electrode shafts to define distinct neural sources (SEEG-ICs). SEEG electrodes traverse complex macroanatomy, including multiple cortical layers, sulcal banks, and white matter. By constructing components derived from weights across the entire electrode, and subsequently localizing each component solely to the contact with the maximal contribution, the authors risk generating biologically implausible signals. Such an approach potentially mixes true localized cortical gray matter activity with deep structure or white matter signals. Given that a central claim of this manuscript is the spatial and functional segregation of theta and gamma neural populations, the authors could consider further validating these core findings (such as the gamma-to-theta directionality) using single-channel or bipolar-referenced data.

(2) Another methodological concern is the use of GC to evaluate the directional causality between speech and neural signal. As noted in Bastos & Schoffelen (2015) and indeed acknowledged by the authors' own citation of Nolte et al. (2010), Granger Causality is highly sensitive to SNR imbalances and filtering artifacts. Given the inherent SNR disparity between a cleanly extracted acoustic envelope and noisy SEEG data, coupled with the known distortions introduced by distinct filtering pipelines (Barnett & Seth, 2011), the GC results may reflect methodological artifacts rather than true physiological driving.

(3) The third concern is the study's exclusive reliance on linear metrics applied to the envelopes of band-filtered speech and neural signals, e.g., linear Granger Causality and cross-correlations. The human auditory system is an inherently non-linear dynamical system. Complex acoustic features, such as rapid spectrotemporal transitions or dynamic pitch trajectories, often drive non-linear neural responses and complex phase-locking behaviors. While the linear models provide strong interpretable results, by restricting their connectivity and directionality metrics to linear autoregressive models, the authors may be missing substantial non-linear interactions, or conversely, forcing a linear fit onto non-linear data, which can distort estimations of causality and temporal lags. The authors should consider explicitly addressing this limitation in their discussion. Ideally, they should validate their core directional claims on a subset of the data using an information-theoretic, non-linear metric (e.g., Transfer Entropy or Mutual Information), or apply linear methods to nonlinearly abstracted features (e.g., phonemic, syllabic, intonational-level features), to ensure their linear assumptions are not masking or misrepresenting the true underlying dynamics.

Reviewer #3 (Public review):

Summary:

The authors aim to characterize the intrinsic temporal dynamics of the infant visual system by examining how it responds to rhythmic visual stimulation. Using EEG in 8-month-old infants, they present visual stimuli that flicker at different periodic frequencies as well as broadband (aperiodic) luminance sequences to probe resonance properties of the visual system. The central goal is to determine whether the infant brain exhibits a characteristic oscillatory response independent of the external stimulation frequency, analogous to the well-known alpha (~10 Hz) resonance of the adult visual system. The results are then compared with data from a small adult sample to assess whether the dominant processing rhythm of the visual system shifts across development.

Strengths:

This manuscript presents a compelling and carefully executed study with intriguing findings, and I greatly enjoyed reading it. Several strengths deserve particular mention:

(1) Clear and focused research approach. The study addresses a well-defined question regarding the intrinsic rhythmic dynamics of the infant visual system and applies an elegant experimental paradigm to probe these dynamics directly.

(2) Well-designed parametric stimulation paradigm. The use of rhythmic visual stimulation across multiple frequencies (2-30 Hz), combined with broadband stimulation, provides a systematic way to characterize resonance properties of the visual system. This parametric approach allows the authors to clearly visualize the relationship between stimulation frequency and neural response, making the key effects easy to grasp.

(3) Strong statistical power in the infant sample. The relatively large infant sample (N = 42) is a major strength, particularly given the challenges of infant EEG research. This sample size provides sufficient power to support the conclusions about the robustness of the ~4 Hz response in infants.

(4) Converging analytical approaches. The authors combine periodic stimulation analysis with impulse-response-function (IRF) analyses of broadband stimulation, which provides complementary evidence for the presence of a ~4 Hz resonance in the infant visual system. This convergence strengthens the interpretation of the results.

(5) Direct developmental comparison. Although the adult sample is small, including adults in the same paradigm provides a useful benchmark showing the expected alpha-band response (~8-9 Hz), thereby contextualizing the infant findings within a developmental framework.

Weaknesses:

(1) Potential oculomotor contribution to the frontal 4 Hz effect. My main concern relates to the interpretation of the prominent ~4 Hz response in infants, particularly at frontal electrodes. The frequency range is close to what might be expected for oculomotor activity such as microsaccades, and the scalp distribution appears suggestive of such a contribution. Notably, the topography of the 4 Hz response differs substantially from the topography of the harmonic responses (Figure 2B), which show the expected occipital dominance. The latter is more clearly visual, whereas the former is more complex, definitely going beyond visual responses. This should be considered more in the discussion.

(2) Differences in topography between periodic and IRF effects. The spatial distribution of the 4 Hz response during periodic stimulation also appears to differ from the topography of the 4 Hz impulse response function (IRF; Figure 2B vs 3D). The IRF response appears not really "visual" in its spatial distribution, as compared to, e.g. the harmonic responses in 2B. This difference could indicate distinct underlying generators, but the implications of this discrepancy are not discussed in detail.

(3) Strength of the interpretation of neural resonance. Taken together, these observations make it difficult to determine conclusively whether the observed 4 Hz activity reflects genuine neural resonance of the visual system or potentially other processes (e.g., oculomotor dynamics). While the current findings remain interesting under either interpretation, the manuscript tends to favor the neural resonance account quite strongly without fully addressing alternative explanations.

(4) Relation to known developmental shifts in resting-state oscillations. The dominance of lower-frequency rhythms (theta range) in infancy is well documented in the resting-state EEG literature. Although this point is briefly mentioned in the discussion, it would be interesting to relate the current findings more directly to this literature. For example, it would be informative to know whether peak frequencies observed here align with resting-state theta peaks in infants and whether similar spatial distributions are observed.

(5) Limited follow-up of the proposed theoretical accounts. The discussion introduces both mnemonic and inhibition accounts for infant theta activity. However, these frameworks are not fully developed in relation to the present data. In particular, the mnemonic account might generate testable predictions within the current dataset, for example, whether theta responses change over time with repeated stimulus exposure or learning.

(6) Characterization of the adult alpha response. A minor point concerns the characterization of the adult resonance frequency. The manuscript often refers to a 10 Hz alpha resonance, whereas the data presented here show a peak around ~8 Hz (Figure 5A). In that frequency range, that is a lot. Also, there seems to be some variability, such that for the topography, the authors use the "individual alpha frequency". It would be interesting to see the distribution of peak frequencies across participants to appreciate the actual range. Interestingly, the spatial distribution of the alpha response also appears quite similar to the infant 4 Hz effect (Figure 5B) and differs from the harmonic responses, which may deserve further discussion. A comparison with resting-state alpha characteristics could also be informative here (e.g., does the peak IAF during visual stimulation relate to IAF recorded at "rest").

Reviewer #3 (Public review):

Summary:

Shin et al. examine hippocampal-prefrontal interactions during sleep using simultaneous CA1 and prefrontal cortex recordings in rats performing a spatial memory task. They identify high-frequency oscillation (HFO) events in PFC during REM sleep that occur in theta-modulated chains and are associated with increased CA1-PFC coherence and sequential, sparse reactivation of cortical ensembles. This pattern contrasts with the synchronous reactivation observed during NREM cortical ripples. Together with a simple cholinergic network model, the authors propose that REM HFO chains represent a distinct mechanism for hippocampal-cortical coordination that complements NREM ripple-mediated processing during sleep.

Strengths:

A major strength of the work is the extensive electrophysiological dataset, which includes simultaneous recordings of large neuronal populations in both hippocampus and prefrontal cortex across behaviour and subsequent sleep. The analyses linking high-frequency events to population dynamics, interregional coherence, and ensemble reactivation are technically sophisticated and provide an incredibly detailed description of REM-associated cortical activity patterns. In particular, the demonstration that REM HFOs occur in chains aligned to theta phase and organise sequential activation of cortical assemblies represents a potentially important advance in understanding the neural structure of REM sleep activity. The integration of experimental data with a computational model further provides a useful framework for interpreting the observed differences between REM and NREM network states in terms of neuromodulatory influences.

Weaknesses:

While overall this study provides a highly valuable body of work, there are two primary limitations, which, if overcome, would provide substantially more significance to the overall characterisation of REM HFOs. Specifically:

(1) Distinction from wake HFOs

The results largely support the authors' claim that REM HFO chains represent a distinct pattern of neural coordination compared to NREM cortical ripples. The analyses consistently show differences between REM and NREM events in terms of neuronal modulation, ensemble structure, and interregional coupling. However, similar high-frequency events during wake are not examined. Since REM sleep shares several network features with wakefulness, including strong theta oscillations, evaluating whether comparable PFC HFOs occur during wake would provide clarity on whether these events are specific to REM sleep (and its associated functions) or represent a more general theta-associated phenomenon.

(2) Link to memory consolidation

The manuscript proposes throughout that REM HFO chains may contribute to memory consolidation by coordinating hippocampal-cortical reactivation, but the evidence for this functional role remains indirect. The authors do highlight this as a limitation of the study - the inability to link their findings to learning - but it is not clear why. Further details of the behaviour results should be included. If no learning occurred across the eight behavioural sessions, this should be reported. If learning did occur, but could not be linked to HFO events, this should also be reported.

Reviewer #3 (Public review):

Summary:

This important work provides a web-based tool to contextualize effect sizes in psychiatry with respect to reliability and base rates (collectively referred to as predictive utility analysis). The methods for the tool incorporate established psychometric principles that I think are of use for multiple fields in this seemingly easy-to-use tool. I agree with the critical importance of this tool and the methodological points made in this manuscript. Enthusiasm for the manuscript is weakened by a lack of clarity on the formulation of the paper and stated goals of the examples used, with the inferences and impact on clinical decision making from various parameterizations via this tool left open-ended.

Strengths:

This paper presents a well-considered and, what I think will be highly useful, web-based tool to contextualize effect sizes with respect to reliability and base rates. As the authors rightly point out, such a tool could be used in conjunction with widespread analytic power analysis tools in study planning. The paper also well contexualizes the need for such a tool in the relatively recent history of concerns of power, reliability, and inference in psychiatry specifically, and more general meta-scientific debates in psychology and neuroscience.

Weaknesses:

My primary feedback on this manuscript is the lack of clarity in what the paper itself, specifically, separate from the tool, is hoping to achieve. There is a central, but unresolved, tension in whether the reader is supposed to:

(1) focus on the specifics of the examples used and whether to reevaluate the substantive claims from the studies, (2) buy in to how various reliability and base rate parameters impact modeling outcomes, (3) receive an introduction to the tool itself.

In my estimation, the largest contribution to the field here is in (2) and (3), but currently much of the real estate of the paper is dedicated to several examples of (1). While these specific examples may be illustrative to some degree, I think given the number and brevity of such, they are unlikely to incidentally achieve points (2) and (3) above. Specific examples include the assertion of kappas for DSM diagnoses, without much nuance (e.g., see https://psycnet.apa.org/buy/2015-27500-001). Given the relatively limited space given to this example, however, it's hard to be entirely certain what the reviewer should take away.

A second point of concern is where this tool would be situated in the research pipeline. I agree with the authors that this tool could be used in ways that parallel power analysis. With that in mind, it seems the most common use of this tool for an individual investigator is likely to be in a priori study planning. In contrast, and with my point above in mind, the use of the tool for existing results is likely best done with multiple estimates of effect sizes, reliability, and base rates, as is common in meta-analysis or consensus reviews. Nevertheless, there is no real example or guidance around how this influences new study planning.

A third point is that more nuance would be useful in the introduction about the current state of psychiatry research. For example, I share many of the authors' concerns about reliability, power, reproducibility, and barriers to translation. That said, it is the case that while effect sizes should be considered considerably more, they are widely considered in psychiatry research via the common place of meta-analysis and other data pooling approaches. Another such example that the authors state in the context of reliability: "However, this [reliability] attenuation is rarely accounted for in routine analyses in psychiatry". This is true in practice, but somewhat misleading insofar as the method by which to do this remains unclear. For example, should we all report disattenuated associations, assuming there is no error and everything is perfectly reliable? This, of course, would be unrealistic to expect zero error. That we can achieve this with the new tool is clear, but the nuance of how and under what circumstances it should be done is not clear, and such nuance should be better reflected in the framing of the problem. That is, there is also a lack of clarity on what ought to be best practices and field-wide goals, rather than simply the lack of an ability to model these factors.

Minor point

For conceptual clarity, it would benefit the manuscript to at least briefly mention the role of validity in translational importance. Of course, the current psychometric issues of reliability, base rate, power, etc are critical, but it should at least be mentioned, given the potential wide audience of this manuscript, validity is important as well. For example, highly reliable measures may not be valid indicators of underlying disease etiology (e.g., fMRI head motion is a highly reliable trait-level feature, but typically not considered an important predictor or consequence of mental health worth investing translational resources in). Relatedly, confounding as a general topic would be useful to mention just briefly, to help with the spirit of considering underlying issues in translation.

Reviewer #3 (Public review):

Summary:

The study aims to elucidate the dual molecular mechanisms of the RNA-binding protein MATR3 in oocyte growth and maturation. The authors propose that MATR3, highly expressed in growing oocytes (GOs), regulates oocyte quality through two pathways: epigenetically, by recruiting KDM3B to remove the repressive H3K9me2 mark at the Gdf9 locus to activate transcription; and post-transcriptionally, by binding Rdx mRNA to maintain microvillus structure for GDF9 secretion. This mechanism ensures oocyte-granulosa cell communication and female fertility. The study also explores the link between MATR3 and human oocyte maturation arrest (OMA).

Strengths:

The study proposes an innovative dual-mechanism model encompassing "epigenetic transcriptional activation and cytoskeletal regulation," which not only expands the functional understanding of RNA-binding proteins in chromatin regulation but also reveals the coordination between nuclear transcription and organelle structure. By integrating scRNA-seq and LACE-seq, the authors constructed a comprehensive regulatory network for MATR3, identifying both key targets and numerous potential molecules, thereby providing rich resources for future mechanistic studies. Furthermore, the inclusion of oocyte samples from human OMA patients directly links the basic findings to clinical reproductive disorders. Despite the limited sample size, this approach demonstrates strong translational potential.

Weaknesses:

The partial phenotypic improvement achieved by exogenous GDF9 supplementation suggests that the downstream effector pathways may involve a more complex network regulation, implying that the current interpretation of GDF9's central role could be further explored. Regarding the developmental abnormalities of granulosa cells in the conditional knockout model, their pathological origins require in-depth analysis to determine whether they represent primary alterations or secondary adaptive responses resulting from the loss of oocyte signaling.

Reviewer #3 (Public review):

Summary:

PLCβ3 is activated by both Gαq and Gβγ subunits. This paper follows previous solutions and cryoEM studies of PLCβ3 / Gβγ, trying to understand the molecular details of activation using cellular BRET assays and cryoEM.

Strengths:

The authors find evidence for multiple binding sites on PLCβ3 for Gβγ and suggest that Gβγ is not bone fide activator per se but enhances Gαq activation by positioning the catalytic site towards substrate, although this is not completely convincing. Although these sites may not naturally be operative, the authors might want to develop the potential role of these sites.

The authors also find that this activation is not through recruitment of the enzyme to the membrane by Gβγ released upon G protein activation, in accord with other PLCβ enzymes, but not for PLCβ3, and again, the authors might want to develop this point further.

Weaknesses:

(1) I'm confused as to why the authors feel that their mechanism is distinct from the two-state enzyme, the synergistic activation proposed by Ross in 2011, using a primarily thermodynamic argument. As written, the authors appear to be very reliant on structural and BRET studies that do not give the details that would disprove this interpretation. The main issue is that the author's mechanism does not fully explain how Gβγ activation occurs for PLCβ2 in reconstituted systems in the absence of Gαq subunits.

(2) In a recent study, McKinnon presents a model showing that Gαq and Gβγ activate PLCβ3 by two distinct pathways and that activation by Gβγ occurs through membrane recruitment. It is not surprising that the authors find that this is not true since the pelleting method used by McKinnon is subject to error. The authors should directly address the limitations of this previous work and the changes in proteoliposomes with sedimentation that alter partition coefficients. Although the inability of Gβγ to drive membrane binding is in accord with the quantitative studies of Scarlata, showing that the affinity of PLCβ3 to Gβγ is fairly weak as compared to the intrinsic membrane partition coefficient.

(3) It was proposed many years ago that in signaling complexes Gαq - Gβγ may not have to fully dissociate when binding PLCβ, but rather shift their relative orientation when binding to PLCβ to allow activation. Is their model consistent with this? Is it possible that PLCβ3 keeps Gβγ from diffusing to enhance the rate of Gq / Gβγ re-association?

(4) The authors find that Gβγ binds multiple sites, and it is clear that the PH domain site is the primary one in accord with previous work. Could these weaker sites be an artifact of the elevated concentrations used in cryoEM and BRET assays?

(5) Although their assays infer differences in binding affinities, it would strengthen the paper if the authors could estimate the association energies of these different binding sites. This estimation would also address the concern stated above.

Reviewer #3 (Public review):

At the core of the bacterial type III secretion system (T3SS), a nanomachine used to inject effector proteins into eukaryotic cells, five highly conserved proteins, SctRSTUV, form the export apparatus, which is the actual gate for effector proteins. Not only are these proteins the most strongly conserved parts of the system, but also their gene order is conserved, which is not the case for most other components of the T3SS. Interestingly, this order does not completely recapitulate the assembly order, which is SctR5-T4-S-U-V. Looking into the reasons for the conserved synteny, the authors noted a stem-loop in the mRNA of the Salmonella SPI-1 sctS gene, which is present in many other T3SS as well (and in fact had been found in Yersinia before). They then use an array of clever gene permutations and modifications to discern the benefit of this order for the bacteria. The combination of thorough sequence analysis with different, partly quantitative, protein expression and secretion assays and growth curves, both in the native Salmonella background and in heterologous systems, provides strong evidence for the interpretation of the authors: The stem-loop in sctS prevents the premature expression of SctT, which can otherwise assemble into "futile multimers" that can lead to ion loss. The presence of stem-loops in many other sctS/T genes gives weight to this finding.

This is a very nice and thorough study addressing an important point in the assembly of type III secretion systems. I only have a few suggestions.

(1) Conserved gene orders have been shown for many complexes, and the findings presented in this manuscript might be applicable to other membrane complexes.

The conservation of gene order and the presence of the stem loop give weight to the authors' findings. However, it is only mentioned quite late in the discussion that a similar stem loop was found in Yersinia upstream sctT earlier, and was interpreted differently. The authors' current discussion is somewhat evasive on this point. Why would these similar structures be used differently? Why would temperature not play a role in Salmonella SPI-1? And wouldn't the stem-loop also couple sctS and sctT expression in Yersinia? This should be addressed, if possible, by experiments (at least, the influence of temperature on the SPI-1 mRNA structure should be testable for the authors) and by a more detailed discussion (given the redundancy of RNA thermometers in the Yersinia T3SS, the interpretation in the current paper might well be the more compelling one).

(2) A point that deserves more attention is that a similar finding in Yersinia has been interpreted differently before (as a temperature sensor rather than translational coupling) - are these systems really different? Testing the different interpretations in the respective other system (at least the influence of temperature in the Salmonella SPI-1 system used in this manuscript) would have made the interpretation even more compelling.

(3) Another point that should be discussed in more detail is why this mechanism is present when replacement of the sctT ATG by weaker start codons and the simple omission of a separate SD sequence upstream sctT would achieve the same outcome. This could be tested in one of the nice heterologous systems, as used in Figure 4.

Reviewer #3 (Public review):

Summary:

In this study, Tang, Yu & colleagues investigate the impact of continuous flash suppression (CFS) on the responses of V1 neurons using 2-photon calcium imaging. The report that CFS substantially suppressed V1 orientation responses. This suppression happens in a graded fashion depending on the binocular preference of the neuron: neurons preferring the eye that was presented with the marker stimuli were most suppressed, while the neurons preferring the eye to which the grating stimuli were presented were least suppressed. Binocular neuron exhibited an intermediate level of suppression.

Strengths:

The imaging techniques are cutting-edge.

Weaknesses:

The strength of CFS suppression varies across animals, but the authors attribute this to comparable heterogeneity in the human psychophysics literature.

Comments on previous revisions:

The authors have addressed my comments from the previous round of review, and I have no further comments.

Reviewer #3 (Public review):

The manuscript by Amara et al. provides novel mechanistic insight into how SafA, a spore coat morphogenetic protein, self-assembles and is later crosslinked by the Tgl transglutaminase during spore coat assembly. Through rigorous, carefully executed biochemical analyses of SafA's oligomerization and crosslinking states, the authors demonstrate that SafA forms dimers that promote disulfide bond formation between two cysteine pairs found in its C30 region; this disulfide bond-mediated crosslinking promotes, but is not essential for, Tgl-mediated crosslinking of lysine residues within SafA. Specifically, one pair in its N-terminal C30 region promotes the formation of higher-order oligomers, while the second pair in its C-terminus C30 region promotes its ability to form a tetramer. Mutation of both cysteine pairs prevents higher-order SafA structures and reduces the efficiency of Tgl-mediated crosslinking via lysines in close proximity to the cysteines. They further show that disulfide bond formation promotes, but is not essential for, SafA to self-assemble into structures ~1200 kDa via SAXS analyses and kinetic analyses of Tgl-mediated crosslinking of purified SafA in vitro.

Major Comments:

(1) While the authors' detailed and thorough biochemical analyses advance our understanding of how SafA forms higher-order structures in the presence and absence of Tgl, they could broaden the significance of their findings with additional functional analyses of their mutants in B. subtilis. Figure 8 shows that loss of Tgl and SafA disulfide bond formation renders SafA more extractable (presumably leading to a less resilient spore coat), and FRAP analyses indicate that SafA in ∆tgl sporulating cells is more mobile than in its lysine crosslinked form. Some ideas that the authors could test to try and identify additional functions for the Cys and Lys residues in SafA:<br /> - Analyze the Cys mutants in the FRAP assay?<br /> - Does loss of SafA-mediated crosslinking via the Cys and/or Lys mutations affect its localization to the forespore or the recruitment of its client proteins like GerQ?<br /> - Have the authors tested higher concentrations of lysozyme? Or chloroform?

(2) While the authors show in supplementary data that the safA point mutants they generated do not affect spore germination in the single condition tested, the Rudner group previously showed that SafA plays a role in spore germination by affecting CwlJ localization to the forespore. Perhaps the authors might see a more significant phenotype on spore germination with their Cys and Lys mutants if they tried to complement a ∆safA∆sleB double mutant with mutant safA constructs? For the germination assays, it was unclear to me whether the authors used heat activation prior to inducing spore germination.

(3) Have the authors looked at whether the Cys or Lys mutations affect the sensitivity of spores to oxidative insults, especially since the Cys residues might temper the effects of oxidizing agents?

(4) Did the authors test the effect of single Cys mutations on disulfide bond formation, since intermolecular disulfide bond formation might still be possible even if one of the Cys residues has been changed?

(5) Finally, I was unsure how many times each experiment was replicated and how many experiments had been conducted in total.

Reviewer #3 (Public review):

Summary:

This research shows that a-mangostin, a proposed nutraceutical, with cardiovascular protecting properties, could act through the activation of large conductance potassium permeable channels (BK). The authors provide convincing electrophysiological evidence that the compound binds to BK channels and induces a potent activation, increasing the magnitude of potassium currents. Since these channels are important modulators of the membrane potential of smooth muscle in vascular tissue, this activation leads to muscle relaxation, possibly explaining cardiovascular protecting effects.

Strengths:

The authors have satisfactorily answered my previous comments and present evidence based on several lines of experiments that a-mangostin is a potent activator of BK channels. The quality of the experiments and the analysis is high and represents an appropriate level of analysis. This research is timely and provides a basis to understand the physiological effects of natural compounds with proposed cardio protective effects.

Weaknesses:

The identification of the binding site continues to be the least developed point of the manuscript. The authors show that the binding site is probably located in the hydrophobic cavity of the pore and show that point mutations reduce the magnitude of the negative voltage shift of activation produces by a-mangostin. This binding site should be demonstrated in the future using structural techniques such as cryo-EM.

Reviewer #3 (Public review):

Summary:

Prudhomme et al report a detailed analysis of the role of vinculin in maintaining neuroepithelial integrity during cranial neurulation.

Strengths:

The authors use complementary experiments involving super-resolution microscopy, laser ablation, and live imaging of conditional knockout and ESC-derived embryos to demonstrate that loss of vinculin produces wide gaps between the adherens junctions of neuroepithelial cells at later stages of cranial neural fold elevation. The data presented are of extremely high quality, logically presented in a compelling story, and represent a very substantial contribution.

Weaknesses:

The authors are invited to consider the largely minor questions recommended below.

(1) The laser ablations reported are a correlate of cell border, or 'junctional' tension. Please avoid broad statements such as 'mechanical forces are upregulated' (abstract), which invoke gene-like regulation of tissue-level forces (in Newtons). Changes in junctional tension are likely to relate to changes in force generated, but their relationship is not simple: higher tensile stress withstood by the shorter length of junctions in cells with smaller apical surfaces does not necessarily translate into greater force being produced by that cell. The junctional tension readout measured is perfectly relevant to the paper, more so than tissue-level forces would have been.

(2) What is the mechanical mechanism by which loss of vinculin prevents neural fold elevation? The authors present exciting findings about the cellular consequences of losing Vcl at the late elevation stages when the tissue is quantifiably dysmorphic. A clear argument of how Vcl loss could lead to this dysmorphology would strengthen the paper, particularly given that junctional tension defects are excluded and apical non-constriction at the late stage is only mild.

(3) Can the authors comment on the likely impacts of Vcl deletion on the basal domain of the cell? For example, they could cite live-imaging of distinct behaviours in Williams et al Dev Cell 2014, and the NTD phenotypes of some integrin/focal adhesion mutant mice.

(4) The apparent uncoupling of apical area (larger in Vcl KO) from junctional tension (equivalent) in this model is noteworthy. Can the authors speculate on its potential basis?

(5) Live imaging in Figure 7C appears to show a marked reduction in apical area before cleavage furrow formation (T0-18min), suggesting a large apical constriction event (post-mitotic?), as previously reported (e.g., Ampartzidis et al Dev Biol 2023). Do junctional gaps appear during these constrictions?

(6) The live imaging setup used is clearly sufficient to identify differences between genotypes, so this is only a minor point. The gassing conditions listed in the methods specify 5% CO2, but E8.5 embryos also need low O2 to complete cranial closure. Was the O2 level controlled? Was tissue-level shape change observed to be consistent with ongoing neurulation during live-imaging?

(7) Neither the multi-cell laser ablations in the pre-print by De La O cited here, nor the narrower junctional ablations in Bocanegra-Moreno et al., Nat Phys, (2023), identified differences in recoil between developmental stages. Why might those results be different from the findings reported here (e.g., analysis region - not specified in the latter paper)? Limitations to interpreting junctional ablations between cells with different junction lengths include more of the recoil being dissipated by retraction of the longer ablated border.

(8) Is a truncated Vcl expressed in the ESC model, which could bind catenin without an F-actin anchor? The very high-contrast western shown is cropped so it is not clear whether the catenin-binding N-terminus is present. Does the antibody used recognise the head domain (this reviewer could not readily find the information)?

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors challenge a dogma in cell biology, namely that cells are at any time point engulfed by a continuous plasma membrane. Liang et al. find that during C elegans embryogenesis, a high number of cells are not entirely surrounded by a plasma membrane but show membrane openings (MOs). These openings are enriched at the embryo's periphery, towards the eggshell. The authors propose that plasma membrane discontinuities emerge during metaphase of mitosis and that independent extension of "sister membranes" engulfs the daughter cells.

Strengths:

On the positive side, the authors find plasma membrane discontinuities not only by electron microscopy but also by fluorescence microscopy and provide information about the dynamics of membrane openings and their emergence. While this is assuring, the authors conclude that MOs emerge during metaphase. From what the authors show, this particular information cannot be deduced, as there is no dynamic capture of a membrane scission event together with a chromatin marker that would indicate mitosis. The authors could, however, attempt to find such events in live movies, given the high incidence of MOs reported from their EM data.

Weaknesses:

In order to convincingly demonstrate the absence of any plasma membrane in the respective regions of the embryonic periphery or between cells of the embryo, the authors would have to show consecutive serial TEM sections where MOs are detected over more z-planes, beyond the mere 3D reconstructions. Although the authors state in the methods section that continuous ultrathin sections were cut for the metaphase sample (page 21, line 472), consecutive sections are never shown in TEM. While we do see the 3D reconstructions, better documentation of the underlying TEM data is missing. It would be necessary to show a membrane opening in consecutive z sections. Alternatively, the authors could seek the possibility to convincingly back up their claims with volume imaging by focused ion beam scanning EM (FIBSEM), where cellular volumes can be sectioned in almost isotropic resolution.

Another critical issue concerns the detection of the membrane discontinuities in electron micrographs, which, in my opinion, is ambiguous. How do the authors reliably discriminate in their TEM images whether there is a plasma membrane or not? The absence - or weak appearance - of the stain of the electron dense material at membranes, which seems to be their criterion for MOs, is also apparent at other, intracellular membranes, like at the NE or at the ER (for example, see Figure 1C). Also, the plasma membrane itself appears unevenly stained in regions that the authors delineate as intact (for example, Figure 1C, 2B/1).

Reviewer #3 (Public review):

This manuscript investigates computational mechanisms underlying increased risk-taking behavior in adolescent patients with suicidal thoughts and behaviors. Using a well-established gambling task that incorporates momentary mood ratings and previously established computational modeling approaches, the authors identify particular aspects of choice behavior (which they term approach bias) and mood responsivity (to certain rewards) that differ as a function of suicidality. The authors replicate their findings on both clinical and large-scale non-clinical samples.

The main problem, however, is that the results do not seem to support a specific conclusion with regard to suicidality. The S+ and S- groups differ substantially in the severity of symptoms, as can be seen by all symptom questionnaires and the baseline and mean mood, where S- is closer to HC than it is to S+. The main analyses control for illness duration and medication but not for symptom severity. The supplementary analysis in Figure S11 is insufficient as it mistakes the absence of evidence (i.e., p > 0.05) for evidence of absence. Therefore, the results do not adequately deconfound suicidality from general symptom severity.

The second main issue is that the relationship between an increased approach bias and decreased mood response to CR is conceptually unclear. In this respect, it would be natural to test whether mood responses influence subsequent gambling choices. This could be done either within the model by having mood moderate the approach bias or outside the model using model-agnostic analyses.

Additionally, there is a conceptual inconsistency between the choice and mood findings that partly results from the analytic strategy. The approach bias is implemented in choice as a categorical value-independent effect, whereas the mood responses always scale linearly with the magnitude of outcomes. One way to make the models more conceptually related would be to include a categorical value-independent mood response to choosing to gamble/not to gamble.

The manuscript requires editing to improve clarity and precision. The use of terms such as "mood" and "approach motivation" is often inaccurate or not sufficiently specific. There are also many grammatical errors throughout the text.

Claims of clinical relevance should be toned down, given that the findings are based on noisy parameter estimates whose clinical utility for the treatment of an individual patient is doubtful at best.

Comments on revisions:'

The authors adequately addressed my comments and I find the manuscript substantially strengthened.

Reviewer #3 (Public review):

Summary:

This multi-omics study by Zhou et al elucidates the context-dependent roles of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway (JSP) across different cellular compartments in the breast cancer tumor microenvironment. While bulk JSP activity is associated with a favorable prognosis, single-cell analysis reveals a paradoxical landscape: high JSP in T cells drives anti-tumor cytotoxicity and reduces exhaustion, whereas high activity in tumor epithelial cells promotes malignancy and immunosuppression via the MIF-CD74 signaling axis. The JSP score (immune-related) serves as a robust predictive biomarker for response to anti-PD-1 immunotherapy, particularly in triple-negative breast cancer (TNBC). Furthermore, the study identifies the STAT4/SLC47A1 axis as a critical mechanism through which tumor cells resist ferroptosis, facilitating disease progression. These findings suggest that broad JAK-STAT inhibition may be counterproductive in cancer therapeutics; instead, therapeutic success depends on precise modulation and carefully timed interventions to preserve its T-cell-associated functions. This study may inspire future studies to explore specific factors that selectively modulate JAK-STAT activity in immune cells to achieve favorable therapeutic outcomes.

Strengths:

Significant therapeutic implications.

Weaknesses:

Limited molecular mechanisms.

Reviewer #3 (Public review):

Summary:

Razlan and colleagues provide a detailed anatomical characterization of lamina I projection neurons in the mouse spinal cord that are densely innervated by primary afferents activated by cooling of the skin. The authors validate a Trpm8-Flp mouse line, show synaptic contacts between Trpm8⁺ boutons and projection neurons at the ultrastructural level, and demonstrate at the physiological level that these neurons specifically respond to cooling stimuli. Next, by taking advantage of previous transcriptomic analysis of ALS neurons, the authors identify calbindin as a marker for cold activatetd lamina I projection neurons and map their ascending projections to the rostral lateral parabrachial area, caudal periaqueductal gray, and ventral posterolateral thalamus, well-known thermosensory and thermoregulatory centers. Altogether, these findings provide strong anatomical and functional evidence for a direct line of transmission from Trpm8⁺ sensory afferents through Calb1⁺ lamina I neurons to key supraspinal centers controlling perception of cold and thermoregulatory responses.

Strengths:

The combination of mouse genetics, electron microscopy, ex-vivo physiology, optogenetics and viral tracing provides convincing evidence for a direct cold pathway. The work validates the Trpm8-Flp line by extensive anatomical and molecular characterization. Integration with previous transcriptomic and anatomical data, neatly links the cold-selective lamina I neurons to a molecularly defined cluster of ALS neurons, strengthening the bridge between molecular identity, anatomy, and physiological function.

Weaknesses:

The main limitation remains the relatively small number of neurons that could be recorded electrophysiologically. While understandable given the complexity of the preparation, this necessarily limits generalization.

Reviewer #3 (Public review):

Summary:

Razlan and colleagues provide a detailed anatomical characterization of lamina I projection neurons in the mouse spinal cord that are densely innervated by primary afferents activated by cooling of the skin. The authors, building on their previous anatomical work, validate a Trpm8-Flp mouse line, show synaptic contacts between Trpm8⁺ boutons and projection neurons at the ultrastructural level, and demonstrate at the physiological level that these neurons specifically respond to cooling stimuli. Next, by taking advantage of their previous transcriptomic analysis of ALS neurons, they identify calbindin as a marker for cold-activated lamina I projection neurons and map their ascending projections to the rostral lateral parabrachial area, caudal periaqueductal gray, and ventral posterolateral thalamus, well-known thermosensory and thermoregulatory centers. Altogether, these findings provide strong anatomical and functional evidence for a direct line of transmission from Trpm8⁺ sensory afferents through Calb1⁺ lamina I neurons to key supraspinal centers controlling perception of cold and thermoregulatory responses.

Strengths:

The combination of mouse genetics, electron microscopy, ex vivo physiology, and viral tracing provides convincing evidence for a direct cold pathway. The work validates the Trpm8-Flp line by extensive anatomical and molecular characterization. Integration with previous transcriptomic and anatomical data neatly links the cold-selective lamina I neurons to a molecularly defined cluster of ALS neurons, strengthening the bridge between molecular identity, anatomy, and physiological function.

Weaknesses:

While anatomical evidence for direct synaptic connectivity between Trpm8+ afferents and lamina I projection neurons is compelling, a physiological demonstration of strict monosynaptic transmission is not shown. The conclusion that these inputs are exclusively monosynaptic should be toned down. Similarly, the statement that "Lamina I ALS neurons that are surrounded by Trpm8 afferents are cold-selective" should also be toned down as only a few neurons have been tested and it cannot be excluded that other neurons with similar characteristics may be polymodal.

Reviewer #3 (Public review):

Summary:

Fengwen Huang et al. used multiple neuroscience techniques (transgenetic mouse, immunochemistry, bulk calcium recording, neural sensor, hippocampal-dependent task, optogenetics, chemogenetics, and interfer RNA technique) to elucidate the role of the excitatory cholecystokinin-positive pyramidal neurons in the hippocampus in regulating the hippocampal functions, including navigation and neuroplasticity.

Strengths:

(i) The authors provided the distribution profiles of excitatory cholecystokinin in the dorsal hippocampus via the transgenetic mice (Ai14::CCK Cre mice), immunochemistry, and retrograde AAV.

(ii) The authors used the neural sensor and light stimulation to monitor the CCK release from the CA3 area, indicating that CCK can be secreted by activation of the excitatory CCK neurons.

(iii) The authors showed that the activity of the excitatory CCK neurons in CA3 is necessary for navigation learning

(iv) The authors demonstrated that inhibition of the excitatory CCK neurons and knockdown of the CCK gene expression in CA3 impaired the navigation learning and the neuroplasticity of CA3-CA1 projections.

Weaknesses:

(i) The causal relationship between navigation learning and CCK secretion remains nebulous; answering this question will require a more sensitive CCK-BR sensor in future work.

Reviewer #3 (Public review):

Summary:

This study uses isolated frog brainstem preparations to test whether inspiratory rhythm generation is confined to a narrowly defined neural center or instead reflects the activity of a distributed and adaptable network. Building on prior rodent work, the authors examine structural and functional parallels between the frog Buccal Area and the mammalian preBötzinger complex. By increasing excitatory drive, they assess whether a localized rhythmogenic region can expand into a broader network that participates in buccal rhythm generation, providing insight into how respiratory circuits are dynamically reconfigured across physiological states.

Strengths:

The work presents compelling evidence that ventilatory rhythm generation is supported by a flexible, state-dependent network rather than a fixed anatomical locus. The experimental preparation is well-suited to address these questions, and the data are generally of high quality. The demonstration that increased excitation recruits a more distributed network parallels observations in mammalian systems and strengthens the translational relevance of the findings. Overall, the analyses are thoughtful, and the interpretations are largely well supported by the results.

Weaknesses:

Some issues limit the strength of the conclusions. First, the study does not address the transition from eupnea to gasping in mammals, which could provide important physiological context for the observed AMPA-induced network reorganization. Second, the reported transformation of lung-active neurons into buccal-active neurons would benefit from additional analyses to clarify whether neurons switch identities or acquire dual activity. Finally, the necessity and sufficiency experiments in Figure 9 require further support, particularly through AMPA dose-response analyses and more comprehensive GABA manipulations, to confirm that network expansion does not obscure the continued functional importance of the core buccal region.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

This study examines the role of dentate gyrus neuronal populations, reflecting neurogenesis and anatomical location (suprapyramidal vs infrapyramidal blade), in a mnemonic discrimination task that taxes the pattern separation functions of the dentate. The authors measure dentate gyrus activity resulting from cognitive training and test whether adult neurogenesis is required for both the anatomical patterns of activity and performance in the cognitive task. The authors find that more cognitively challenging variants of the task evoked more dentate activity, but also distinct patterns of activity (more activity in the suprapyramidal blade, less in the infdrapyramidal blade). Using chemogenetic approaches they silence mature vs immature dentate gyrus neurons and find that only mature neurons (either the general population or specifically mature adult-born neurons), and not immature adult-born neurons, are required for the difficult version of the task. Inhibition of mature adult-born neurons furthermore increased overall activity in the dentate and reduced the biased pattern of activity across the blades, consistent with evidence that adult-born neurons broadly regulate dentate gyrus activity.

Comments on revisions:

I appreciate the efforts the authors have taken to revise this manuscript. I have only minor concerns with this revised version of the manuscript:

Methods state that significance is defined as P<0.05 but some results are interpreted as significant when P=0.05. Either the alpha value needs to change or the interpretation needs to change.

I believe the statistical results for group and blade effects for the ANOVAs, in Figs 2,3 & 4, appear to be switched (blade should be significant, not group).

I appreciate that sometimes there is not a perfect overlap between immunohistochemical signals, but I continue to believe that the spatially-non-overlapping TRAP and EDU signals in Fig 3 is caused by these 2 markers being in different cells. A Z-stack or orthogonal projection could verify/disprove this concern.

Reviewer #3 (Public review):

Summary

Kong and coauthors describe and implement a method to correct local deformations due to beam induced motion in cryo-EM movie frames. This is done by fitting a 3D spline model to a stack of micrograph frames using cross-correlation-based local patch alignment to describe the deformations across the micrograph in each frame, and then computing the value of the deformed micrograph at each pixel by interpolating the undeformed micrograph at the displacement positions given by the spline model. A graphical interface in cisTEM allows the user to visualise the deformations in the sample, and the method is proved to be successful by showing improvements in 2D template matching (2DTM) results on the corrected micrographs using five in situ samples.

Impact

This method has great potential to further streamline the cryo-EM single particle analysis pipeline by shortening the required processing time as a result of obtaining higher quality particles early in the pipeline, and is applicable to both old and new datasets, therefore being relevant to all cryo-EM users.

Strengths

(1) The key idea of the paper is that local beam induced motion affects frames continuously in space (in the image plane) as well as in time (along the frame stack), so one can obtain improvements in the image quality by correcting such deformations in a continuous way (deformations vary continuously from pixel to pixel and from frame to frame) rather than based on local discrete patches only. 3D splines are used to model the deformations: they are initialised using local patch alignments and further refined using cross-correlation between individual patch frames and the average of the other frames in the same patch stack.

(2) Another strength of the paper is using 2DTM to show that correcting such deformations continuously using the proposed method does indeed lead to improvements, as evidenced by the number of particles found and the quality of the detections (measured using 2DTM SNR). This is shown using five in situ datasets, where local motion is quantified using statistics based on the estimated motions of ribosomes. The same analysis is performed using other deformation correction tools, with Unbend showing superior performance in terms of particle detected or 2DTM SNR of the detections.

Reviewer #3 (Public review):

Summary:

The authors tested the hypothesis that interactions among size- and age-matched rivals will lead to the emergence of social roles, accompanied by divergence in four aspects of individual phenotypes: growth, feeding behavior, fighting behaviors, and gene expression in clownfish.

Strengths:

The data on growth, feeding rate, and fighting behaviors support the authors' claims.

Weaknesses:

Gene analysis conducted in this study is not sufficient to clarify how the relevant genes actually regulate growth and behavior.

The information obtained from whole-body gene expression analysis is very limited. Various gene expression is associated with the regulation of fighting behaviors, food intake, growth, and metabolism, and these genes are regulated differently across tissues, even within a single individual. Gene expression analysis should be performed separately for each tissue.

Clownfish undergo sex change depending on social status and body size, as the authors mention in the manuscript. Numerous gene expressions are affected by sex change. It is unclear how this issue was addressed.

Reviewer #3 (Public review):

Summary:

The manuscript builds on the observation that, at some synapses, low-frequency stimulation causes synaptic depression, which can be reversed by subsequent high-frequency stimulation. Such low-frequency depression (LFD) cannot be easily explained by the depletion of a single vesicle pool. Here, Silva and colleagues propose a model of activity-dependent vesicle trafficking to explain LFD at synapses between cerebellar granule cells and molecular layer interneurons.

Strengths:

Overall, LFD is interesting and worthy of examination, and the authors provide new experimental results that are of the high quality expected from this group.

Weaknesses:

The study proposes a novel model of vesicle trafficking that is not explained by known biological mechanisms, and the manuscript does not adequately compare or discuss alternative models.

I have several concerns about how the authors interpret the data. First, the manuscript's primary conceptual advance is the idea that LFD involves vesicle undocking, rather than depletion. However, most experiments were performed under conditions that promote vesicle depletion (3 mM extracellular Ca2+). When experiments were repeated in physiological Ca2+, there appeared to be little or no LFD (stats are not provided). Second, the RS/DS/DU/undocking model, though not outside the realm of possibility, is not readily explained by known mechanisms and is only loosely supported by experimental findings. Third, when simulating LFD, the authors do not compare alternative models and use inappropriate language to imply that a model fit represents the truth (e.g., "the finding of identical experimental and simulated values confirms that the undocking mechanism accounts for LFD"). Finally, the model is presented in an overly complicated manner. The sheer amount of terms and nomenclature makes the manuscript confusing and difficult to read. Overall, the manuscript would benefit from added experiments and more statistics, a better justification and evaluation of the model, and more nuanced language.

Major concerns:

(1) Most experiments were performed under conditions that exacerbate depletion

In order to attribute LFD to vesicle undocking rather than depletion, it is important to show LFD under conditions where depletion is minimal. As mentioned above, the authors only report significant LFD in elevated extracellular Ca2+. In a small number of experiments performed in more physiological Ca2+ (1.5 mM), there is no depression after a single stimulus, and it is not clear that there was statistically significant depression during a low-frequency train. Several studies cited in support of LFD share this problem:

• Abrahamsson et al., (2007) recorded from Schaffer collaterals in 4 mM Ca, 3-4X physiological Ca2+.

• Doussau et al., (2010) recorded from aplysia synapses in 3X Ca compared to seawater.

• Rudolph et al., (2011) is cited as an example of LFD. However, this study performed experiments at high release probability cerebellar climbing fibers, and reported depression that increased monotonically with

stimulation frequency, so it does not resemble the phenomenon studied in this paper. Lin et al., (2022) also largely describe monotonic depression at the calyx.

The authors note that their results differ from those of Atluri and Regehr, but do not mention that a possible reason for the difference is the increased release probability in their experiments.

The authors should provide statistics for the data obtained in 1.5 mM Ca, and discuss why LFD is increased in conditions that also elevate vesicle release probability.

(2) Lack of biological mechanisms supporting the model

The model is presented without compelling biological support. The evidence in support of vesicle undocking comes from experiments by the Watanabe lab, which showed fewer-than-expected docked vesicles under EM when cultured synapses were stimulated immediately prior to high-pressure freezing. Kusick et al were careful to note that these vesicles may have been lost to fusion.

The putative undocking Kusick describes is immediate (< 5 ms after stimulation), and was not shown to be Ca2+ sensitive. This manuscript describes "calcium-dependent undocking" that proceeds from 10 ms - 200 ms. Multiple studies from the Watanabe lab show that a single stimulus lowers the number of docked vesicles, and subsequently, there is a transient redocking of vesicles that can be blocked by EGTA or Syt7 knockout.

I also question the rationale for the authors' model that 2 vesicles are coupled in series to a single release site. Previous papers from this lab cited EM studies from frog and neuromuscular that showed filamentous connections between vesicles (do these synapses show LFD?). Here, the authors primarily cite their previous models to support their arguments. I encourage them to continue searching for ultrastructural evidence for 2-vesicle-docking-units and to cite such studies.

(3) Comparison to other vesicle models

The authors use overly assertive language to suggest that the model proves a mechanism. "Altogether, these results indicate that the slow phase of LFD ... reflects a δ decrease without significant changes in pr, in ρ or in IP size". Simulating data does not conclusively "indicate" the underlying mechanism, but the authors could state their data can be "explained by a model where..".

However, LFD does not require activity-dependent undocking. Instead, the phenomenon has been explained by high-release probability, paired with an activity-dependent increase in either docking or release probability (Chiu and Carter, 2024; Doussau et al., 2017). Does the new model do a better job of replicating some facet of the data? If multiple models can explain the same data, how can we determine which model is correct? The "Alternative Presynaptic Depression Mechanisms" should be expanded to discuss these issues.

Reviewer #3 (Public review):

Summary:

This study examines how training regimes influence the formation of cognitive maps. Participants learned two relational maps over three days through pairwise transitions: one map was trained with grouped sequences that followed rows or columns, while the other was trained with disjoint transitions sampled randomly across the map. In addition, the study manipulated the temporal spacing of training blocks (blocked vs. semi-blocked) and tested whether the results generalized across two map geometries (a 5×5 grid and a 4×4 torus).

Furthermore, they run a follow-up experiment (or condition) testing rows and columns shuffled in the grouped condition.

While grouped training produced better performance during learning, the authors report that disjoint training led to superior performance at test on tasks probing the global map knowledge.

Summarising experimental design:

(1) Map geometry (between-subjects): 5×5 grid vs 4×4 torus

(2) Training block schedule (between-subjects): Blocked vs Semi-blocked

(3) Training regime/transition sampling (within-subject): Grouped or Disjoint (Day 1 and Day 2)

Strengths:

The study addresses a clear and timely theoretical question about how the training regime affects the formation of cognitive maps. A further strength is the well-controlled experimental design, allowing the authors to test their hypotheses in a systematic and informative way.

Weaknesses:

(1) If I understood correctly, participants learned one map on the first day and the other on the second day, with the training regime (grouped vs. disjoint) counterbalanced across maps. This raises the possibility that experience with one training regime on day one could influence performance on the second day. For example, it would be interesting to examine whether participants who experienced the disjoint regime first showed any differences when learning the grouped regime on the following day. While it may be difficult to fully disentangle such transfer effects from the main training regime effects, it would be informative to test whether performance on the second day depends on the regime experienced on the first day (e.g., whether prior exposure to the disjoint regime predicts performance on the subsequent grouped training, but not vice versa).

(2) The author mentions a control experiment. Did the participants in the control experiment complete only the training phase or also the testing tasks used in the main experiment? If testing was included, it would be informative to report whether performance at test was comparable to that observed in the main experiment. Given that this condition appears to involve blocked transitions while moving across both rows and columns, I would expect performance to fall somewhere between the grouped and disjoint conditions.

(3) Participants' performance did not differ between conditions in the map reconstruction task, suggesting that participants in both the grouped and disjoint regimes were ultimately able to form a cognitive map. Was this task always administered last during the testing session? I wonder whether the explicit request of the reconstruction task could have influenced participants' awareness of the map structure.

(4) The manuscript describes the study as consisting of four experiments (two groups per map shape, differing in the blocked versus semi-blocked schedule). However, based on the design described in the Methods, this appears more accurately characterized as a single experiment with two between factors: map geometry (grid vs. torus) and blocking schedule (blocked vs. semi-blocked) manipulated between participants, and training regime (grouped vs. disjoint) manipulated within participants.

(5) It is not entirely clear to me from the Results section whether performance at test differed between the two map geometries (grid and torus), or whether the reported effects of training regime were consistent across them.

Reviewer #3 (Public review):

Summary:

The authors aimed to test whether hypoxia disrupts the migration of human cortical interneurons, a process long suspected to underlie brain injury in preterm infants but previously inaccessible for direct study. Using human forebrain assembloids and ex vivo developing brain tissue, they visualized and quantified interneuron migration under hypoxic conditions, identified molecular components of the response, and explored the effect of pharmacological intervention (specifically ADM) on restoring the migration deficits.

Strengths:

The major strength of this study lies in its use of human forebrain assembloids and ex vivo prenatal brain tissue, which provide a direct system to study interneuron migration under hypoxic conditions. The authors combine multiple approaches: long-term live imaging to directly visualize interneuron migration, bulk and single-cell transcriptomics to identify hypoxia-induced molecular responses, pharmacological rescue experiments with ADM to establish therapeutic potential, and mechanistic assays implicating the cAMP/PKA/pCREB pathway and GABA receptor expression in mediating the effect. Together, this rigorous and multifaceted strategy convincingly demonstrates that hypoxia disrupts interneuron migration and that ADM can restore this defect through defined molecular mechanisms.

Overall, the authors achieve their stated aims, and the results strongly support their conclusions. The work has significant impact by providing the first direct evidence of hypoxia-induced interneuron migration deficits in the human context, while also nominating a candidate therapeutic avenue. Beyond the specific findings, the methodological platform-particularly the combination of assembloids and live imaging-will be broadly useful to the community for probing neurodevelopmental processes in health and disease.

Comments on revisions:

The authors have fully addressed my concerns by incorporating the relevant discussion into the manuscript, especially regarding how well the migration observed in hSO-hCO assembloids reflects in vivo condition. I have no further comments.

Reviewer #3 (Public review):

Summary:

Franchet et al. sought to characterize the impact of Nora virus on host lifespan and sensitivity to a variety of infectious or stressful treatments. Through careful and rigorous analyses, they provide evidence that the Nora virus greatly impacts fly survival to infection, overall lifespan, and intestinal integrity. The authors have been thorough and rigorous, and the experimental evidence including proper isolation of the virus and Koch's Postulate reinoculation of the organism is excellent. The additional work is valuable and to the gold standard of the field, characterizing the pathology of the gut, including data showing gut leakage, the presence of the virus in the intestinal stem cells, and the importance of stem cell proliferation for virus replication and spread using elegant genetic tools to block stem cell proliferation or enterocyte death.

Strengths:

The authors have been rigorous and careful. The initial finding is presented through the lens of two related strains differing in virus infection. From there, the authors characterized the virus and isolated a purified culture, which they used to reinoculate a cleared strain to demonstrate proper Koch's Postulate satisfaction. The authors have also probed various parameters in terms of dietary importance in relevant conditions for many experiments. The additional work to characterize the pathology of the gut is compelling, using genetic tools to block or allow intestinal stem cell proliferation and enterocyte death through JAK-STAT and JNK signalling alongside the tracing of virus presence using a Nora virus antibody. JAK-STAT and JNK are previously described as regulators of these processes, making these tools appropriate and convincing. It is also interesting to see good evidence that the virus itself is damaging, rather than simply permitting coinfection by gut microbes (which does happen).

Reviewer #3 (Public review):

Summary:

In this study, the authors seek to explain what influences the temporal resolution of visual perception and its associated metacognitive monitoring, interindividual differences in such processes, and the neural mechanisms associated with these interindividual differences. More specifically, they investigated the factors influencing the perception of a rapid alternating stream of visual patterns as a single fused percept versus two segregated stimuli, and how these factors relate to stable features of ongoing brain activity. They introduce a novel sustained-stream temporal integration paradigm designed to address limitations of traditional two-flash tasks, and combine this with resting-state electroencephalography (EEG) to examine how individual alpha peak frequency and the aperiodic component of the power spectrum relate to temporal integration thresholds, perceptual history effects, and subjective confidence. Their overarching aim is to move beyond a purely oscillatory account of temporal sampling and to test whether periodic (alpha) and non-periodic (aperiodic) neural dynamics jointly shape perceptual decisions.

Strengths:

The study has several notable strengths. First, the experimental paradigm represents a thoughtful and innovative refinement of earlier approaches. By presenting alternating gratings within a continuous stream and varying the duration of each element rather than introducing discrete blank intervals, the authors mitigate well-known confounds of classical two-flash paradigms, particularly the possibility that "fusion" reports reflect missed detections rather than genuine temporal integration. The psychometric functions are well characterized, and the sample size is large for an individual-differences EEG study, with an a priori power analysis supporting the adequacy of the sample. Second, the use of spectral parameterization to separate oscillatory alpha peak frequency from the aperiodic component of the spectrum is methodologically rigorous and timely, as this distinction is increasingly recognized as important to avoid confounds in oscillatory activity estimation and the measurement of neural noise/excitatory-inhibitory balance (i.e., the aperiodic component of the power spectrum). The present work contributes to this emerging direction by relating both to behavioral indices within the same dataset. Third, the integration of perceptual thresholds, serial dependence, and subjective confidence within a unified framework provides a richer account of temporal perception than studies focusing on a single measure. In particular, the demonstration that resting alpha frequency predicts integration thresholds and that the aperiodic exponent relates to variability of the psychometric function is broadly consistent with the authors' central claims.

Weaknesses:

(1) At the same time, several aspects of the interpretation require caution. One conceptual issue concerns the interpretation of the psychometric slope parameter as an index of "temporal precision." The manuscript consistently equates steeper slopes with higher perceptual precision or lower internal noise. However, the slope of a binary psychometric function does not uniquely index sensory temporal resolution. It reflects the steepness of the transition between response categories and can arise from multiple sources, including variability in sensory encoding, instability of decision criteria, lapse rates, or other decisional processes. Even in the literature cited by the authors, slope is often described more generally as reflecting perceptual variability or sensory and/or decision noise rather than a pure measure of perceptual precision. An abrupt transition from "fused" to "segregated" responses, therefore, does not necessarily imply finer temporal resolution at the sensory level; it may instead reflect more consistent categorization or reduced decisional variability. The present data convincingly demonstrate relationships between spectral measures and the steepness of behavioral transitions, but they do not by themselves establish that this steepness reflects perceptual temporal precision rather than broader sources of behavioral variability.

(2) A related concern involves the causal language used to describe the relationship between neural measures and behavior. The EEG metrics are derived from resting-state recordings and therefore reflect stable, trait-like individual differences. Nonetheless, the Discussion sometimes adopts mechanistic phrasing suggesting that slower alpha rhythms or flatter spectra lead the brain to compensate by weighting prior information more heavily, or that neural noise is being "regulated." Such formulations imply within-task adaptive processes that are not directly measured. The results demonstrate robust between-participant associations, but further research is needed to establish whether individuals regulate neural noise or adjust prior weighting dynamically.

(3) Another point that merits clarification concerns the control analyses. The authors appropriately use spectral parameterization to dissociate oscillatory alpha peak frequency from the aperiodic component in the main analyses; however, their subsequent control analyses examining other frequency bands appear to rely on conventional band-power measures. Because band power can be influenced by the aperiodic background, null effects in other bands are difficult to interpret without similarly accounting for aperiodic structure.

(4) In addition, the temporal structure of the stimulus stream introduces an interpretational nuance. Varying the duration of each Gabor in a continuous alternation produces quasi-periodic stimulation rates, and several of these ISIs fall within the alpha frequency range. Rhythmic visual stimulation at alpha-range frequencies is known to produce strong stimulus-locked responses and can interact with intrinsic alpha rhythms in a frequency-dependent manner (Keitel et al., 2019; Gulbinaite et al., 2017). Although the present study does not record EEG during task performance and therefore cannot directly assess stimulus-driven steady-state responses, this aspect of the design complicates a purely intrinsic sampling interpretation. The observed relationship between resting alpha frequency and integration thresholds may reflect intrinsic sampling speed, but it could also be influenced by how closely an individual's alpha rhythm aligns with alpha-range temporal structure in the stimulus.

Conclusion:

Despite these limitations, the study achieves many of its primary aims. The sustained-stream paradigm reliably elicits graded temporal integration behavior and robust serial dependence effects. Individual alpha frequency is convincingly associated with integration thresholds, and the aperiodic exponent relates to behavioral variability measures. These findings support the broader conclusion that temporal perception reflects an interaction between rhythmic neural dynamics and the background spectral structure of ongoing activity. The work is likely to have a meaningful impact for researchers studying perceptual timing, perceptual history, individual differences in brain rhythms, and the functional role of aperiodic neural activity.

References:

Keitel, C., Keitel, A., Benwell, C. S., Daube, C., Thut, G., & Gross, J. (2019). Stimulus-driven brain rhythms within the alpha band: The attentional-modulation conundrum. Journal of Neuroscience, 39(16), 3119-3129.

Gulbinaite, R., Van Viegen, T., Wieling, M., Cohen, M. X., & VanRullen, R. (2017). Individual alpha peak frequency predicts 10 Hz flicker effects on selective attention. Journal of Neuroscience, 37(42), 10173-10184.

Reviewer #3 (Public review):

Summary:

This paper addresses an important question (the relationship between DN and dATN, and the role of retinotopic coding) and uses a set of novel analyses.

Strengths:

Important question, novel analytical approaches (pRF-informed functional connectivity analysis).

Weaknesses:

Some of the key claims are not fully supported by the data presented. There is also a concern about over-interpretation of the results. Key issues:

(1) The authors claim that retinotopic coding scaffolds the interaction between DMN and dATN. However, retinotopically tuned voxels account for a mere 9% of DMN voxels. So this appears to be a major overstatement. For instance, the statement that "these findings would position retinotopy as a unifying framework for brain-wide information processing" is not justified given the presented data.

(2) Given that positive pRF voxels in DMN positively correlate with dATN voxels and negative pRF voxels in DMN negatively correlate with dATN voxels, there is a concern that these results could be contributed to by imprecise brain network parcellations. E.g., could some of the positive pRF voxels in DMN be erroneously assigned to DMN and actually belong to one of the other task-positive networks? There is insufficient validation of network parcellation to put this worry to rest, especially since it depends on ICA, which has a degree of arbitrariness built in.

(3) The claim that retinotopic coding is intrinsic to the DN network is not supported by rigorous analysis and results. The analysis here has many arbitrary factors, including: the threshold of the 99th percentile of resting-state distribution; the designation of DN as "top-down" and dATN as "bottom-up"; the definition of "anti-matched" voxels instead of using randomly selected voxels; and the statistics being paired between matched and anti-matched voxels instead of using comparisons to baseline. Overall, I do not think that the result supports the conclusion that retinotopic coding in DN is intrinsic instead of being bottom-up-driven, given the very high threshold (99%) used and the fact that many other networks could also send bottom-up input to DN. Furthermore, the idea that bottom-up inputs only occur when the dATN (or any other RSN)'s spontaneous BOLD activity is above a certain threshold is a huge and unvalidated assumption.

Reviewer #3 (Public review):

Summary:

Environments change over time; therefore, optimal decision-making ought to discount older observations of the environment in favor of newer ones in a manner consistent with the amount of temporal instability. Computational models of perceptual decision-making model this temporal discounting with a 'leak' parameter that determines the rate at which older information is discarded. In this study, McGaughey and Gold examine the neurophysiological mechanisms that could underlie adaptation to different degrees of temporal instability. They developed a novel variant of the well-established perceptual decision-making random-dot-motion paradigm, in which the stimulus being evaluated was preceded by an 'adapting' stimulus with either high or low temporal stability. When the test stimulus was preceded by the adapting stimulus with lower temporal stability, NHPs showed reduced psychometric slopes, indicative of increased temporal discounting ('leak'). While the NHPs performed this task, single-unit neural activity was recorded in area MT, along with pupillometric data. The authors use these neural and pupil datasets to investigate two potential sources of adaptive discounting under varying amounts of temporal instability: sensory adaptation (changes in instantaneous evidence encoding), and arousal-related changes in evidence accumulation. MT neurons respond differently to the test stimulus under conditions of high vs low temporal stability of the adapting stimulus - when the adapting stimulus is more stable, MT neurons have larger and more selective responses to the test stimulus. In addition, evoked pupil responses to the test stimulus were modulated by the adapting stimulus. Both the strength of the difference in MT responses across contexts and the difference in pupil diameter across contexts were correlated with context-dependent modulation of the monkeys' behavior over sessions. The paper concludes that both sources appear to independently contribute to adaptive evidence accumulation, likely operating at different processing stages in the brain.

Strengths:

(1) While computational models of perceptual decision-making have been very useful for explaining behavior and neural responses in decision-making areas, we are still in search of some of the neural mechanisms that could implement such models. Studies such as this one, which aim to identify neural correlates of simplified model parameters, are quite crucial.

(2) Analysis is generally careful and well-executed.

(3) Prompts some interesting follow-up questions that could be answered with simultaneous recordings and causal manipulations, as the authors state in the Discussion - e.g., which areas are affected by arousal-related neuromodulation correlated with evoked pupil size and how.

Weaknesses:

(1) The task design may not be optimal. While the amount of time the monkey is exposed to each motion direction during the adapting stimulus is matched, it's hard to know if the reduced MT responses to the test stimulus are truly due to the greater frequency of switches during the HSF adapting stimulus or because the monkeys have been exposed to more repetitions of the stimulus. It's increased sensory adaptation in either case, but it makes it problematic to interpret this as temporal context-dependent adaptation specifically. I think this could potentially be partially addressed by an analysis that is in the paper, but could potentially be emphasized/fleshed out more, specifically the results shown in Figure 4D that seem to show that most of the reduction in neural response for adapting units occurs between the first and second stimuli.

(2) The pupillometric analysis seems to be an indirect way of assessing whether the accumulator itself might be modulated by temporal context, but the link could be made clearer. The authors show that context-dependent behavior is related to pupil size, which is related to arousal/neuromodulation, but it would be helpful to have some idea of what neural mechanisms underlying adaptive decision-making are actually impacted by this neuromodulation. Lacking neural data to address this question (e.g., from a brain region proposed to be involved in the accumulation process), at least more discussion of this would be helpful. Essentially, I'm unsure of how to interpret the pupil results: the argument that temporal context affects instantaneous evidence encoding in MT that then drives the accumulator is very clear, but I am a bit confused about what, mechanistically, I should think about the effect of neuromodulation doing.

Reviewer #3 (Public review):

In this manuscript, the authors investigate the role of attention in foveal processing during a naturalistic task. They record neural activity from extrastriate visual areas V4 and inferotemporal cortex, as well as from the lateral prefrontal cortex, in macaques performing a free-gaze visual search task. In this task, animals searched for a face or house target among multiple complex stimuli, with no constraints on eye movements. Unlike classic studies of visual attention, which often rely on controlled fixation, this work examines neural activity in both foveal and peripheral receptive fields during naturalistic eye movements.

The main question addressed by the authors is how feature-based attention is distributed and coordinated across foveal and peripheral visual fields during active search, and how this attentional processing influences saccade behavior. The authors show that foveal units in visual areas exhibit feature-based attentional enhancement, with stronger responses when a fixated stimulus is a target compared to when the same stimulus serves as a distractor. Peripheral units in visual and prefrontal areas show both feature-based and spatial attentional modulation, consistent with prior work. Finally, the authors show that attentional modulation depends primarily on stimulus category rather than response magnitude, with neurons showing similar enhancement for all images within the target category regardless of how strongly individual images drive the cell.

There are several notable strengths of this paper, including:

(1) Disentangling feature-based and spatial attention during naturalistic vision remains a central challenge. This paper tackles both simultaneously, parsing neural populations by object selectivity (face-selective, house-selective, non-selective) and RF position (foveal vs. peripheral).

(2) The unconstrained search task (Figure 1A) moves beyond the dominant fixed-gaze, cued-attention designs (Zhou & Desimone, 2011) to study attention as it operates during natural behavior, with sequential fixations and voluntary saccades.

(3) The scale of the multi-area recordings is a major strength and is well aligned with current trends in primate and human neuroscience toward large-scale, multi-area recordings. Simultaneous recordings from visual and prefrontal areas, comprising over 4,900 foveal units and more than 1,500 peripheral units, enable meaningful cross-area latency comparisons and area-specific analyses of attentional modulation. This study builds on the authors' previous analyses of this dataset by expanding the scope to show that feature-based attention generalizes across neuronal classes and operates on categorical identity rather than response magnitude.

(4) The combination of simultaneous multi-area recordings and a rich behavioral paradigm provides a dataset that is well-suited for population decoding, cross-area interaction analyses, and trial-by-trial prediction of saccade choices, which could substantially deepen mechanistic understanding beyond the largely univariate comparisons presented here.

While the data broadly support the paper's main conclusions, several issues limit the strength of the mechanistic interpretation and should be taken into consideration:

(1) Receptive field size is not explicitly quantified and may confound foveal-peripheral comparisons. Units are classified as foveal or peripheral based on responsiveness to the cue versus the search array (Methods, p. 17), but the manuscript lacks essential information about receptive field sizes, eccentricities, and the number of search stimuli falling within each receptive field and related proper controls. This is critical because receptive fields in visual area V4 at foveal eccentricities are relatively small (Gattass et al., 1988; Desimone & Schein, 1987), whereas receptive fields in inferotemporal cortex can span several degrees to tens of degrees and often include the fovea (Op de Beeck & Vogels, 2000; DiCarlo & Maunsell, 2003; Zoccolan et al., 2007). Given the 2{degree sign} × 2{degree sign} stimulus size, multiple search items could potentially fall simultaneously within peripheral receptive fields. This introduces a potential confound, as attentional modulation is known to be strongest when multiple stimuli appear within a single receptive field (Reynolds et al., 1999). Although the authors acknowledge this issue for visual area V4 (p. 17), it is neither quantified nor controlled for. Without explicit receptive field mapping relative to the search array, comparisons between foveal and peripheral units, as well as between visual areas, are difficult to interpret cleanly.

(2) Attentional modulation is difficult to dissociate from saccade planning and decision-related signals. The free-gaze paradigm enhances ecological validity but introduces a temporal confound: mean distractor fixation durations are approximately 156 ms (p. 9), while attentional effects emerge between 137 and 170 ms after fixation onset (Figure 2). As a result, the reported attentional modulation coincides with the preparation of the subsequent saccade. Neural activity measured in the primary analysis window (150-225 ms; p. 19), therefore, likely reflects a mixture of visual, attentional, motor planning, target recognition, and behavioral relevance signals, all of which are known to modulate responses in visual areas at similar latencies (e.g., Chelazzi et al., 1998). Moreover, target fixations (~257 ms) and distractor fixations (~156 ms) occur on fundamentally different behavioral timescales, which may inflate apparent foveal attentional effects. While the authors suggest that these timing differences support the idea that foveal feature-based attention facilitates prolonged fixation on target stimuli, this interpretation is not fully supported by the current analyses. That said, the saccade-aligned analyses of peripheral units (Figure S3) partially mitigate this concern by demonstrating that feature-based modulation persists through saccade execution.

(3) The "attention-out" condition for spatial attention lacks directional control. In the spatial attention analyses (Figures 4D-F), the "attention-out" condition appears to include all fixations followed by saccades directed away from the receptive field, regardless of saccade direction. This differs from classic spatial attention designs, which typically use controlled anti-saccades or saccades to fixed locations opposite the receptive field (e.g., Moore & Armstrong, 2003; Gregoriou et al., 2009). Saccades directed toward locations adjacent to, but outside, the receptive field may still partially engage spatial attention mechanisms near the receptive field via broad attentional fields or motor preparation gradients (Bisley & Goldberg, 2010). In addition, the "attention-out" condition likely contains a heterogeneous mixture of trials in which the stimulus in the receptive field is either a target or a distractor, since feature-based attention effects are derived from this same pool of trials. As a result, spatial and feature attention effects are not fully orthogonal, and variance related to feature attention may already be embedded in the spatial attention baseline.

Reviewer #3 (Public review):

Overview:

In this manuscript, the authors describe two additions to an existing toolbox (SpikeInterface, Buccino et al., 2020, eLife). The first addition is an empirical simulator for extracellular recordings, in which spikes from predefined templates are added up with Gaussian noise. The second addition involves granting user-level access to intermediate processing steps along spike sorting algorithms. The authors demonstrate the toolbox by evaluating functions (e.g., event detection) or sets of functions (e.g., feature extraction + clustering) on their simulated data, and suggest that a specific combination of function implementations provides performance improvement relative to kilosort4 (Pachitariu et al., 2024, Nature Methods).

If the authors are interested in making this manuscript a suitable scientific contribution, the entire work has to be revised extensively. In particular, the simulator has to be extended and improved; the implementation of existing spike sorters has to be improved; the feedforward architecture of the modules has to be extended; the reporting of results has to follow standard reporting standards; new algorithms have to be explained in sufficient detail; and the manuscript has to undergo extensive proofreading.

Notably, even assuming perfect implementation and descriptions, it is unclear to me whether the scope of the present work warrants a publication in a scientific journal, or is more suitable for an internal technical report or an e.g., a GitHub version release. To go beyond a scientifically-sound technical report, the authors may choose to demonstrate the utility of their new proposed sorter ("Lupin") and compare it to existing tools on multiple datasets.

General comments:

(1) The simulator itself has to be improved and extended. Right now, it simply generates, for every unit, a mother waveform from a sum of exponentials, scales that over channels, and then adds up multiple instantiations of every unit on every channel, along with noise. This is not a biophysical simulator: it is an ad hoc procedure, and the sentence "we firmly believe that.." (lines 482-483) does not make the procedure convincing. To make the simulator credible, the authors should: (1) use a set of biophysical equations, with multi-compartmental modeling of currents and return currents; (2) use noised data from extracellular recordings; or (3) some combination thereof.

(2) The simulated dataset has to be extended in time. Maybe I missed something, but 500 units over 10 minutes, with some units having firing rates as low as 0.1 spikes/s, corresponds to some of the units firing an expected 60 spikes. This is clearly too short, and does not replicate the standard situation in extracellular experiments.

(3) The simulated dataset has to be extended in space. The choice of using NeuroPixels 1.0 geometry is a poor one. Many labs use other monolithic electrode arrays (MEAs, silicon probes, other rigid arrays); tetrodes remain a major tool, and flexible probes (polyimide, mesh) are evolving. Assessing algorithms over a single spatial architecture is likely to lead to local maxima in performance and potentially erroneous conclusions.

(4) The existing spike sorters evaluated are not completely described. Some sorters (e.g., SpyKING Circus and KS4) were described in previous publications, but it is unclear whether the implementation that was used for the present tests is exactly the same as those previously published. More importantly, some of the sorters evaluated (e.g., TDC, TDC2, SpyKING Circus 2) were never described in a peer-reviewed paper. This does not mean that they cannot be evaluated - but if they are, they must be described in full. Relying on the fact that the code is open source cannot replace a complete and accurate scientific description.

(5) Related to the above, all relevant code should be made available online in permanent repositories, not only in author-controlled ones.

(6) It is unclear why SpyKING Circus 2 and TDC2 are evaluated - these could potentially be described as straw men. I recommend reorganizing the manuscript so that after every module is evaluated separately based on a limited ground truth dataset, a single "best" sorter would be constructed, and then tested extensively (and compared to the de facto state of the art). Such reorganization would both demonstrate the utility of a modular approach and clarify the general usefulness of the outcome.

(7) The new algorithms developed, for example, clustering and template matching, have to be described in more detail, and demonstrated graphically on simple datasets. This can be done in supplementary material if the authors prefer not to extend the manuscript too much.

(8) This reviewer finds the description and interpretation of the results to be inadequate. As an example, focusing on Figure 5: The results in Figure 5A have to be supplemented and summarized as a scalar point estimate (e.g., median accuracy), an estimate of dispersion (e.g., using MAD, IQR, or SD), evaluated over multiple runs, and compared using statistical tests between tools and conditions (e.g., using a multi-dimensional analysis of variance, a mixed effect model, etc.). The results in Figure 5D must have an indication of dispersion. Any conclusions based on the numerical experiments must be based on these metrics and statistical evaluations.

(9) The entire MS would benefit from expert proofreading; there are many language errors, mostly in indefinite articles and grammatical numbers.

Reviewer #3 (Public review):

Summary:

In this manuscript, Yang et al introduce a new method for automatically identifying marmosets in their home cage using a supervised deep learning method that recognizes the face and colored beads on marmoset collars. The authors show a high precision rate of identifying marmosets to levels comparable to a human experimenter. The method overall seems robust at identifying marmosets at different life stages and different settings; however, given the current form, I'm struggling to see the generalizability and experimental utility of this method.

Strengths:

(1) The authors provide a near-perfect automatic identification of marmosets in their home cage.

(2) This method is robust across lightning, camera angles, etc., making it potentially useful for marmoset (and other NHP) identification outside the housing cage as well

Weaknesses:

(1) Despite the almost perfect precision, in its current form, I'm failing to see how this method can be useful to other labs.

(2) This is a nice methods manuscript, but the authors do not present results to show how their method can be used outside of identifying marmosets inside their home cages in a small field of view.

(3) Reading the manuscript is strenuous, given its repetitive nature. Consolidating and shortening the results, as well as adding some definitions to the results section, would be helpful.

Reviewer #3 (Public review):

Summary:

Malhotra and colleagues present evidence that the integrin LFA-1 on NK cells is a ligand for the Plasmodium falciparum protein GBP130 on the infected erythrocyte surface and that this interaction plays a role in the clearance of infected erythrocytes by NK cells.

The authors first select a subdomain contained within the CD11a subunit of LFA-1 as a probe to discover possible binding proteins on the infected erythrocyte surface. Parasite-infected erythrocytes stained positively with this probe; the level of staining increased as the parasites progressed through the life cycle. Using the LFA-1-based probe in cross-linking pull-down experiments, GBP130 was identified by mass spectrometry as a co-purifying parasite protein. The N-terminal portion of GBP130 was recombinantly expressed and shown to interact with LFA-1 alpha-I by biolayer interferometry experiments. The full-length extracellular domain of GBP130 was then recombinantly expressed and used to stain primary human NK cells and THP-1 cells. Knocking down LFA-1 by siRNA reduced staining by GBP130. To assess the contribution of GBP130 to the activation of NK cells, CHO cells exogenously expressing GBP130 were incubated with primary NK cells. Transfecting CHO cells with GBP130 led to increased activation of co-incubated NK cells compared to mock-transfected and compared to GBP130 transfected cells, with the inclusion of anti-CD11a to block NK cell adhesion. Finally, CHO cells expressing GBP130 led to increased activation of NK cells compared to mock-transfected CHO cells.

Overall, although the authors present data from NK cell killing assays that include appropriate controls, the data suggesting a direct interaction between PfGBP-130 and LFA-1 does not include the same necessary controls, for example, the use of blocking antibodies. Most critically, the biolayer interferometry experiments use a recombinant fragment of PfGBP-130, which does not include the residues predicted to be important for mediating specific interaction with LFA1. The biolayer interferometry data instead suggest non-specific interactions between PfGBP-130 and LFA1, as binding does not reach saturation.

Reviewer #3 (Public review):

Summary:

Rosero and Bai report an unconventional role of AFD neurons in mediating tactile-dependent locomotion modulation, independent of their well-established thermosensory function. They partially elucidate the signaling mechanisms underlying this AFD-dependent behavioral modulation. The regulation does not require the sensory dendritic endings of AFD but rather the AFD neurons themselves. This process involves a distinct set of cGMP signaling proteins and CNG channel subunits separate from those involved in thermosensation or thermotaxis. Furthermore, the authors demonstrate that AIB interneurons connect AFD to mechanosensory circuits through electrical synapses. They conclude that, beyond its primary function in thermosensation, AFD contributes to context-dependent neuroplasticity and behavioral modulation via broader circuit connectivity.

While the discovery of multifunctionality in AFD is not entirely unexpected, given the limited number of neurons in C. elegans (302 in total), the molecular and cellular mechanisms underlying this AFD-dependent behavioral modulation, as revealed in this study, provide valuable insights into the field.

Strengths:

(1) The authors uncover a novel role of AFD neurons in mediating tactile-dependent locomotion modulation, distinct from their well-established thermosensory function, providing an important conceptual contribution to our understanding of how individual neurons can support multiple, mechanistically separable behavioral functions.

(2) They provide meaningful mechanistic insight into how AFD, GCY-18-dependent cGMP signaling, and AFD-AIB electrical coupling contribute to this AFD-dependent behavioral modulation.

(3) The neural behavior assays utilizing two types of microfluidic chambers (uniform and binary chambers) are innovative and well-designed. In the revised manuscript the authors introduce a removable-barrier assay that physically separates exploration and assay phases. This independent behavioral approach addresses prior concerns about ongoing sensory input and confirms that tactile experience alone is sufficient to modulate locomotion.

(4) By comparing AFD's role in locomotion modulation to its thermosensory function throughout the study, the authors present strong evidence supporting these as two independent functions of AFD.

(5) The finding that AFD contributes to context-dependent behavioral modulation is significant, further reinforcing the growing evidence that individual neurons can serve multiple functions through broader circuit connectivity.

Weaknesses:

While the requirement for AFD, GCY-18, and AFD-AIB electrical coupling is well supported, the directionality of information flow and the precise mode of interaction between mechanosensory neurons, AIB, and AFD remain unclear and an area of future studies.

Overall, the authors successfully achieve their primary aim of identifying and characterizing a novel role for AFD in tactile experience-dependent locomotion modulation. This work contributes meaningfully to the growing body of literature demonstrating multifunctionality and context-dependent reconfiguration of individual neurons within compact nervous systems.

Reviewer #3 (Public review):

Summary:

The authors studied the organisation of orientation and direction-selective retinal ganglion cells' boutons in the mouse superior colliculus. They confirmed the results already published (Molotkov, 2023; de Malmazet, 2024; Vita, 2024; Laniado, 2025), that retinal ganglion cells' boutons in the superior colliculus conserve the retinal organisation. Thereby, orientation and direction preferences of retinal boutons at each collicular location reflect the tuning of retinal ganglion cells found at the corresponding retinal location, that is covering a matching receptive field location.

The authors also studied the organization of orientation and direction-selective neurons in the superior colliculus. They report a lack of functional organisation in the superior colliculus for neurons preferring the same stimulus orientation or direction of movement. This goes against several published reports (Ahmadlou and Heimel, 2015; Liang et al., 2023; De Malmazet et al., 2018; Feinberg and Meister, 2014; Kasai and Isa, 2021; Li et al., 2020) but echoes a study from Chen et al. (Chen, 2021). The latter authors contested the strength of the anatomical clustering of tuned alike direction-selective neurons. They found, however, that in about a quarter of their recordings, direction-selective cells with similar preferred directions did cluster anatomically in the superior colliculus.

Here, the authors of the current manuscript under review report that local clustering of tuning was weak in all neural populations and confined to very small spatial scales (10-20 μm). This is one order of magnitude smaller than previously reported clusters of around 100-300μm wide. Therefore, the authors conclude that orientation and direction tuning in the mouse superior colliculus follows a salt and pepper organisation.

Strengths & Weaknesses:

Although the authors performed a solid analysis contesting the functional clustering of direction and orientation selective neurons, there seemed to be some elements in their data in favour of a functional clustering of neurons.

As an illustration, the authors plotted in Figure 1Q the distribution of preferred orientations from all their recorded orientation-selective cells. The curve shows a clear bias, indicating that neurons preferring horizontal orientations were found two times more often than neurons encoding any other orientations. Moreover, the authors recorded all their neurons from a defined anatomical location of the colliculus, marked by the dotted rectangle in Figure 3A-C. Therefore, this suggests that orientation-selective cells in this particular collicular location are biased towards preferring horizontal orientations. This supports an anatomical clustering of tuned alike orientation-selective cells in the superior colliculus.

Similarly, Figure 1P shows a bias in the preferred directions of direction-selective neurons in the same recording area. Cells tended to respond more to upward and forward-moving stimuli. The bias is more modest than the one described above for preferred orientations. However, it still seems significant. For example, cells preferring upwards movements appeared to be four times more abundant than cells preferring downward movements. As a consequence, it indicates that preferred directions might not be uniformly distributed and equally represented across the superior colliculus.

These anatomical biases are also visible in the receptive field analysis of the paper. In Figure 3G, the authors plotted the distribution of preferred orientations for every 10-degree bins within the recorded field of view. Out of 26 bins containing more than one neuron, only 6 seemed to include cells not overwhelmingly preferring a single orientation. These were located towards the top right of the figure. Therefore, over almost 80% of the recorded superior colliculus, the data seem in agreement with the view that orientation-selective cells tend to prefer the same orientation at a given receptive location.

The patch analysis in Figures 4G and H also seems to show some degree of coherence in the preferred orientation and direction of neighbouring tuned collicular cells. In both Figures 4 G and H, clear patches of similar preferred orientation and direction appeared to emerge. For example, in Figure 4H, there is a predominance of horizontally tuned patches. This was expected given the recording bias consisting of a majority of horizontally tuned cells. In addition, vertical and 45-degree patches are also visible, in blue and red, respectively. These patches overlap with the corresponding retinotopic locations in Figure 3G, where the histograms show that cells tend to prefer the same orientations, horizontal, vertical or 45 degrees.

It is important to note that in the previous studies on functional clustering of orientation and direction, variability in the tuning of cells within clusters was always reported (Ahmadlou and Heimel, 2015; Chen et al., 2021; De Malmazet et al., 2018; Feinberg and Meister, 2014; Kasai and Isa, 2021; Li et al., 2020). This was more marked for direction-selective cells than for orientation-selective cells. In general, cells preferring all four cardinal directions were often recorded at any given collicular location. Similarly, orientation-selective cells could be found to prefer deviant orientations compared to adjacent cells. Therefore, it is not surprising to see locally mixed tuning in collicular neurons. However, what appeared significant in these studies was the overall proportion of cells with similar tuning in patches of the superior colliculus. As described above, this also seems to be the case in the data of this manuscript.

To conclude, it seems that authors tend to overlook the sources of agreement between their data and previous reports showing functional clustering of cells in the superior colliculus. Instead, the authors tend to emphasise the dissimilarities and variability to put forward a contentious view on the organisation of orientation and direction selectivity in neurons of the superior colliculus. This, I fear, is detrimental to the field because it creates a sort of manufactured chaos that produces unnecessary confusion for readers who do not attentively read the manuscript. It would be valuable for the authors to consider rewriting the manuscript, acknowledging where their data, in fact, support some level of functional clustering.

Reviewer #3 (Public review):

Summary:

Exposure to heat shock results in major changes to gene expression programs within the cell, and over the past decades, there has been extensive characterization of the mechanisms through which heat shock activates transcription. However, heat shock also leads to widespread repression of many genes, and the transcriptional mechanisms that mediate this repression have not been well understood. Here, the authors show that the transcription factor CLAMP mediates this heat shock-dependent repression via changes in local 3D chromatin looping. Intriguingly, CLAMP is already bound to chromatin prior to heat shock, but is necessary for the loss of local chromatin loops at its bound sites and repression of genes located within the loops. This study is significant because it defines chromatin looping, depending on a key transcription factor CLAMP, as the major mechanism through which genome-wide changes in gene repression occur in response to an inducible stimulus, heat shock.

Strengths:

The use of the SLAM-seq and Micro-C techniques to measure the necessity of CLAMP for heat shock-dependent transcription repression and local chromatin looping is excellent, and these approaches provide valuable insight into the role of CLAMP in heat shock-dependent repression that was not apparent with older approaches. The HiChIP approach provides an excellent method to test whether CLAMP is bound at sites where there are changes in looping upon heat shock, providing good support for their conclusions that CLAMP induces heat shock repression by decreasing loops. Appropriate controls are present, and there is robust statistical analysis of the bioinformatics data.

Weaknesses:

The study does not provide insight into how CLAMP mechanistically affects loops upon heat shock, although the discussion raises the possibility that this could result from biophysical changes since CLAMP is an intrinsically disordered protein.

Reviewer #3 (Public review):

Summary:

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

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

Comments on revisions:

The authors answered all my questions. I have no further requests.

Reviewer #3 (Public review):

Summary:

In this paper it is established that high fever-like 39oC temperatures cause parasite infected red blood cells become stickier. It is thought that high temperatures might help the spleen to destroy parasite infected cells, so they become stickier to remain trapping in blood vessels, so they stop passing through the spleen.

Strengths:

The strength of this research is that it shows that fever-like temperatures can cause parasite infected red blood cells to stick to surfaces designed to mimic the walls of small blood vessels. In a natural infection this would cause parasite infected red blood cells to stop circulating through the spleen where the parasites would be destroyed by the immune system. It is thought that fevers could lead to infected red blood cells becoming stiffer and therefore more easily destroyed in the spleen. Parasites respond to fevers by making their red blood cells stickier, so they stop flowing around the body and into the spleen. The experiments here prove fever temperatures increase the export of Velcro-like sticky proteins onto the surface of the infected red blood cells and are very thorough and convincing.

Weaknesses:

Minor weaknesses in the original version have now been satisfactorily addressed with additional work which is very convincing.

Reviewer #3 (Public review):

Summary:

This paper focused on how to navigate the complex decision-making process of whether to go into human trials. This is a critical topic considering the well-documented challenges in replicating and translating findings. While these are two distinct topics (i.e., replication and translation), they are related, and the authors simulated many conditions to assess the utility of replication assessment metrics.

Strengths:

A major strength of the study is the detailed approach to identifying relevant conditions and metrics, and to providing rich results that outline the strengths and weaknesses of each metric. Any simulation study is challenged by trying to identify the most relevant variables of interest, and this study provided sound justification for its chosen variables of interest. While this study does not make a strong recommendation (which I see as a strength), it does provide a comprehensive overview of the various metrics and conditions that were investigated.

Weaknesses:

The weaknesses of the study are the limited focus on specific metrics, the assumptions, particularly in the limited number of human study variables, and the less-than-ideal approachable summary of findings for a non-technical audience.

Conclusion:

This paper provides a much-needed investigation and discussion of how decisions are made when assessing whether to go into human trials. This is an important topic that productively challenges the status quo, considering documented challenges in replication and translation in biomedical research.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al., investigates how herbivorous insects overcome plant receptor-mediated immunity by targeting plant receptor-like proteins. The authors identify two independently evolved salivary effectors, BtRDP in whiteflies and NlSP694 in brown planthoppers, that promote the degradation of plant RLP4 through the ubiquitin-dependent proteasome pathway.

Strengths:

This work highlights a convergent evolutionary strategy in distinct insect lineages and advances our understanding of insect-plant coevolution at the molecular level.

Comments on revisions:

The authors have satisfactorily addressed all the issues I raised.

Reviewer #3 (Public review):

Summary:

This paper uses a novel method to look at how stable brain states and the transitions between them promote memory formation during encoding and post-encoding rest in children. I think the paper has some weaknesses (detailed below) that mean that the authors fall short of achieving their aims. Although the paper has an interesting methodological approach, the authors need better logic, and are potentially "double dipping" in their results - meaning their logic is circular. I think the method that they are using could be useful to the broader neuroimaging community, although they need to make this argument clearer in the paper.

Strengths:

The paper is interesting in that they use a novel method to look at brain state dynamics and how they might support memory.

Weaknesses:

The paper has several weaknesses:

(1) The authors use children as their study subjects but fail to reconcile why children are used, if the same phenomena are expected to be seen in adults (or only children), and if and how their findings change with age across an age range that ranges from middle childhood into early adolescence. They need to include more consideration for the development of their subject population. The authors should make it clear why and how memory was tested in children and not adults. Are adults and children expected to encode and consolidate in a similar manner to children? Do the findings here also apply to adults? Do the findings here also apply to adults? How was the age range of 8-13-year-old children selected? Why didn't the authors look at change with age? Does memory performance change with age? Do the BSDS dynamics change with age in the authors' sample?

(2) The authors look for brain state dynamics within a preselected set of ROIs that are selected because they display a subsequent memory effect. This is problematic because the state that is most associated with subsequent memory (S3, or State 3) is also the one that shows most activity in these regions (that have already been a priori selected due to displaying a subsequent memory effect). This logic is circular. It would be helpful if they could look at brain state dynamics in a more ROI agnostic whole brain approach so that we can learn something beyond what a subsequent memory analysis tells us. I think the authors are "double dipping" in that they selected regions for further analysis based on a subsequent memory association (remembered > forgotten contrast) and then found states within those regions showing a subsequent memory effect to further analyze for being associated with subsequent memory. Would it be possible instead to do a whole-brain analysis (something a bit more agnostic to findings) using the BSDS framework, and then, from a whole-brain perspective, look for particular brain states associated with subsequent memory? As it stands, it looks like S3 (state 3) has greater overall activation in all brain regions associated with subsequent memory, so it makes sense that this brain state is also most associated with subsequent memory. The BSDS analysis is therefore not adding anything new beyond what the authors find with the simple subsequent memory contrast that they show in Figure 1C. This particularly effects the following findings: (a) active-encoding state occupancy rate correlated positively with memory accuracy, (b) transitions to the active-encoding state were beneficial / Conversely, transitions toward the inactive state (S4) were detrimental, with incoming transitions showing negative correlations with memory accuracy / The active-encoding state serves as a "hub" configuration that facilitates memory formation, while pathways leading to this state enhance performance and transitions away from it impair encoding.

(3) The task used to test memory in children seems strange. Why should children remember arbitrary scenes? How this was chosen for encoding needs to be made clear. There needs to be more description of the memory task and why it was chosen. Why was scene encoding chosen? What does scene encoding have to do with the stated goal of (a) "Understanding how children's brains form lasting memories", (b) "optimizing education" and (c) "identifying learning disabilities"? What was the design of the recognition memory test? How many novel scenes were included in the test, and how were they chosen? How close were the "new" images to previously seen "old" images? Was this varied parametrically (i.e., was the similarity between new and old images assessed and quantified?)

(4) They ultimately found four brain states during encoding. It would be helpful if they could make the logic and foundation for arriving at this number clear.

(5) There is already extant work on whether brain states during post-encoding rest predict memory outcomes. This work needs to be cited and referred to. The present manuscript needs to be better situated within prior work. The authors should look at the work by Alexa Tompary and Lila Davachi. They have already addressed many of the questions that the authors seek to answer. The authors should read their papers (and the papers they cite and that cite them) and then situate their work within the prior literature.

More minor weaknesses:

(1) The authors should back up the claim that "successful episodic memory formation critically depends on the temporal coordination between these systems. Brain regions must coordinate their activity through dynamic functional interactions, rapidly reconfiguring their activity and connectivity patterns in response to changing cognitive demands and stimulus characteristics." Do they have any specific evidence supporting this claim?

(2) These claims seem overstated: "this work has broad implications for understanding memory function in children, for developing educational interventions that enhance memory formation, and enabling early identification of children at risk for learning disabilities." Can the authors add citations that would support these claims, or if not, remove them?

Reviewer #3 (Public review):

The manuscript by Sadeqi et al. studies the interactions between the mitochondrial protein Ups1 and reconstituted membranes. The authors apply synthetic liposomal vesicles to investigate the role of pH, curvature, and charge on the binding of Ups1 to membranes and its ability to extract PA from them. The manuscript is well written and structured. The authors provide all relevant information and reference the appropriate literature in their introduction. The underlying question of how the energy barrier for lipid extraction from membranes is overcome by Ups1 is interesting, and the data presented by the authors offer a valuable new perspective on this process. It is also certainly a challenging in vitro reconstitution experiment, as the authors aim to disentangle individual membrane properties (e.g., curvature, charge, and packing density) to study protein adsorption and lipid transfer.

Reviewer #3 (Public review):

Summary:

This manuscript by Kim and Parsons presents an overview of the nitroreductase/metronidazole (NTR/MTZ) cell ablation system.

Strengths:

This manuscript nicely places the NTR/MTZ system in the context of other cell ablation methods, with a discussion of their respective advantages and disadvantages. This review is particularly useful for highlighting the many ways the NTR/MTZ system has been applied to study the regeneration of multiple cell types and to model different degenerative human diseases. The review concludes with a discussion on recent improvements made to the system and practical considerations and "best practices" for NTR-based experiments. This review could be a helpful resource, especially for researchers new to regeneration or cell ablation studies.

Weaknesses:

Although the NTR/MTZ system has been used in other model organisms, this review is primarily focused on its uses in zebrafish. While this is understandable given the wide adoption of NTR/MTZ in the zebrafish field, discussion of the unique considerations and/or challenges for non-zebrafish systems would be an interesting addition and could broaden the potential audience for this review. Additional minor revisions, as suggested below, could also improve readability.

Reviewer #3 (Public review):

Summary:

The article by Chen et al. describes the comprehensive metabolic profiling of DP16 mice, a Down syndrome model that carries a duplicated segment of the mouse chromosome syntenic to human chromosome 21. The authors note that this model is superior to previously used models, based on genetics, as ~65% of the chromosome 21 orthologues. The metabolic phenotypes also appear to be more consistent with those observed in humans with Down Syndrome. The study lays the groundwork for a more detailed genetic dissection of dosage-sensitive genes that contribute to the metabolic deficits observed in Down Syndrome.

Strengths:

There is an enormous amount of data in this manuscript, and the methods are described with adequate attention to detail. A strength of the manuscript is that both male and female mice were analyzed, so that concordant and discordant phenotypes were identified. Both males and females had evidence of insulin resistance. Transcriptomic and metabolomic data revealed impaired pathways for lipid metabolism, a pro-inflammatory state, reduced mitochondrial health and oxidative stress. Although the effects of a high-fat diet on weight gain were divergent, this diet caused worsened insulin resistance in both males and females.

The discussion is excellent. Limitations of the study are well described. This reviewer does not identify any critical missing data.

Weaknesses:

It might have been helpful to have included blood pressure measurements, given the differences in 19-Nor-deoxycorticosterone. The discussion references several articles that describe sex-dependent differences in metabolic phenotypes in humans with Down syndrome, and it might have been helpful to state more explicitly whether these differences correlate with those observed here in mice.

Reviewer #3 (Public review):

Summary:

Several recent findings indicate that forces perpendicular to the microtubule accelerate kinesin unbinding, where perpendicular and axial forces were analyzed using the geometry in a single-bead optical trapping assay (Khataee and Howard, 2019), comparison between single-bead and dumbbell assay measurements (Pyrpassopoulos et al., 2020), and comparison of single-bead optical trap measurements with and without a DNA tether (Hensley and Yildiz, 2025).

Here, the authors devise an assay to exert forces along the microtubule axis by tethering kinesin to the microtubule via a dsDNA tether. They compared the behavior of kinesin-1, -2, and -3 when pulling against the DNA tether. In line with previous optical trapping measurements, kinesin unbinding is less sensitive to forces when the forces are aligned with the microtubule axis. Surprisingly, the authors find that both kinesin-1 and -2 detach from the microtubule more slowly when stalled against the DNA tether than in unloaded conditions, indicating that these motors act as catch bonds in response to axial loads. Axial loads accelerate kinesin-3 detachment. However, kinesin-3 reattaches quickly to maintain forces. For all three kinesins, the authors observe weakly attached states where the motor briefly slips along the microtubule before continuing a processive run.

Strengths:

These observations suggest that the conventional view that kinesins act as slip bonds under load, as concluded from single-bead optical trapping measurements where perpendicular loads are present due to the force being exerted on the centroid of a large (relative to the kinesin) bead, needs to be reconsidered. Understanding the effect of force on the association kinetics of kinesin has important implications for intracellular transport, where the force-dependent detachment governs how kinesins interact with other kinesins and opposing dynein motors (Muller et al., 2008; Kunwar et al., 2011; Ohashi et al., 2018; Gicking et al., 2022) on vesicular cargoes.

Weaknesses:

The authors attribute the differences in the behaviour of kinesins when pulling against a DNA tether compared to an optical trap to the differences in the perpendicular forces. However, the compliance is also much different in these two experiments. The optical trap acts like a ~ linear spring with stiffness ~ 0.05 pN/nm. The dsDNA tether is an entropic spring, with negligible stiffness at low extensions and very high compliance once the tether is extended to its contour length (Fig. 1B). The effect of the compliance on the results should be addressed in the manuscript.

Compared to an optical trapping assay, the motors are also tethered closer to the microtubule in this geometry. In an optical trap assay, the bead could rotate when the kinesin is not bound. The authors should discuss how this tethering is expected to affect the kinesin reattachment and slipping. While likely outside the scope of this study, it would be interesting to compare the static tether used here with a dynamic tether like MAP7 or the CAP-GLY domain of p150glued.

In the single-molecule extension traces (Figure 1F-H; S3), the kinesin-2 traces often show jumps in position at the beginning of runs (e.g., the four runs from ~4-13 s in Fig. 1G). These jumps are not apparent in the kinesin-1 and -3 traces. What is the explanation? Is kinesin-2 binding accelerated by resisting loads more strongly than kinesin-1 and -3?

When comparing the durations of unloaded and stall events (Fig. 2), there is a potential for bias in the measurement, where very long unloaded runs cannot be observed due to the limited length of the microtubule (Thompson, Hoeprich, and Berger, 2013), while the duration of tethered runs is only limited by photobleaching. Was the possible censoring of the results addressed in the analysis?

The mathematical model is helpful in interpreting the data. To assess how the "slip" state contributes to the association kinetics, it would be helpful to compare the proposed model with a similar model with no slip state. Could the slips be explained by fast reattachments from the detached state?

Reviewer #3 (Public review):

Summary:

The authors argue that establishing the expression pattern and subcellular localisation of an animal's proteome will highlight many hypotheses for further study. To make this point and show feasibility, they developed a pipeline to knock in DNA encoding fluorescent tags into C. elegans genes.

Strengths:

The authors effectively make the points above. For example, they provide evidence of two populations of mitochondria in the C. elegans germline that differ qualitatively in the proteins they express. They also provide convincing evidence that labelling the whole proteome is an achievable goal with relatively limited resources and time.

Weaknesses:

Cell biology in C. elegans is challenging because of the small size of many of its cells, notably neurons. This can make establishing the sub-cellular localisation of a fluorescently tagged protein, or co-localizing it with another protein, tricky. The authors point out in their introduction that advances in light microscopy, such as diSPIM, STED, and ISM (a close relative of SIM), have increased the resolution of light microscopy. They also point out that recent advances in expansion microscopy can similarly help overcome the resolution limit.

(1) Have the authors investigated if the three fluorescent tags they use are appropriate for super-resolution microscopy of C. elegans, e.g., STED or SIM? Would Elektra be better than mTAGBFP2? How does mScarlet3-S2 compare to mScarlet 3?

(2) Have the authors investigated what tags could be used in expansion microscopy - that is, which retain antigenicity or even fluorescence after the protocol is applied? It may be useful to add different epitope tags to the knock-in cassettes for this purpose.

The paper is fine as it stands. The experiments above could add value to it and future-proof it, but are not essential. If the experiments are not attempted, the authors could refer to the points above in the discussion.

Reviewer #3 (Public review):

Summary:

In the present work, the authors offer evidence for the therapeutic potential of trained immunity in the context of inflammatory bowel disease (IBD). Prior research has demonstrated that innate cells pre-treated (trained) with β-glucan show an enhanced pro-inflammatory response upon a second challenge with the same or different stimulus. While an increased immune response can be beneficial and protect against bacterial infections, there is also the risk that it will worsen symptoms in various inflammatory disorders.

Remarkably, the authors show that β-glucan training of bone marrow hematopoietic progenitors and peripheral monocytes mitigates the pro-inflammatory effects of colitis, with protection extending to naïve recipients of the trained cells. Additionally, the authors demonstrate that mice preconditioned with β-glucan have enhanced resistance to Staphylococcus aureus and Salmonella typhimurium infections, indicating heightened immune responses.<br /> Using a dextran sulfate sodium (DSS)-induced model of colitis, β-glucan pre-treatment significantly dampens disease severity. Importantly, the use of Rag1^-/- mice, which lack adaptive immune cells, confirms that the protective effects of β-glucan are mediated by innate immune mechanisms. Further, experiments using Ccr2^-/- mice underline the necessity of monocyte recruitment in mediating this protection, highlighting CCR2 as a key factor in the mobilization of β-glucan trained monocytes to inflamed tissues. In addition, β-glucan training highlights a distinct monocyte subpopulation with enhanced activation and phagocytic capacity. These monocytes, marked by increased expression of Cx3cr1, are suggestive of an increased ability to infiltrate inflamed colonic tissue and differentiate into macrophages.

Transcriptomic profiling reveals that β-glucan training upregulates genes associated with pattern recognition, antimicrobial defense, immunomodulation, and interferon signaling pathways, suggesting broad functional reprogramming of the innate immune compartment. Moreover, among the trained monocyte and macrophage subsets, gene expression signatures are associated with tissue and mucosal repair, suggesting a role in promoting resolution and regeneration following inflammatory insult. Furthermore, this was coupled with analysis of chromatin accessibility in publicly available data.

Strengths:

By employing a range of well-characterized murine models, the authors investigate specific mechanisms involved in the effects of β-glucan training. Furthermore, the study provides functional evidence that the protection conferred by the trained cells persists within the hematopoietic progenitors and can be transferred to naïve recipients. The integration of transcriptomic profiling allows the identification of changes in key genes and molecular pathways underlying the trained immune phenotype.

Weaknesses:

Further studies would benefit from investigating the cytokine responses of intestinal macrophages, particularly CX3CR1⁺ macrophages, following ex vivo stimulation of previously BCG-trained cells. Moreover, assessing the metabolic state of these macrophages would provide valuable insight into the mechanisms underlying trained immunity in this context.

Impact:

Overall, the authors present a mechanistically insightful investigation that advances our understanding of trained immunity in IBD. This is an important study that demonstrates that β-glucan-trained innate cells can confer protection against colitis and promote mucosal repair through trained-immunity related mechanisms. These findings underscore the potential of harnessing innate immune memory as a therapeutic approach for chronic inflammatory diseases.

Reviewer #3 (Public review):

Summary:

Several recent findings indicate that forces perpendicular to the microtubule accelerate kinesin unbinding, where perpendicular and axial forces were analyzed using the geometry in a single-bead optical trapping assay (Khataee and Howard, 2019), comparison between single-bead and dumbbell assay measurements (Pyrpassopoulos et al., 2020), and comparison of single-bead optical trap measurements with and without a DNA tether (Hensley and Yildiz, 2025).

Here, the authors devise an assay to exert forces along the microtubule axis by tethering kinesin to the microtubule via a dsDNA tether. They compared the behavior of kinesin-1, -2, and -3 when pulling against the DNA tether. In line with previous optical trapping measurements, kinesin unbinding is less sensitive forces when the forces are aligned with the microtubule axis. Surprisingly, the authors find that both kinesin-1 and -2 detach from the microtubule more slowly when stalled against the DNA tether than in unloaded conditions, indicating that these motors act as catch bonds in response to axial loads. Axial loads accelerate kinesin-3 detachment. However, kinesin-3 reattaches quickly to maintain forces. For all three kinesins, the authors observe weakly-attached states where the motor briefly slips along the microtubule before continuing a processive run.

Strengths:

These observations suggest that the conventional view that kinesins act as slip bonds under load, as concluded from single-bead optical trapping measurements where perpendicular loads are present due to the force being exerted on the centroid of a large (relative to the kinesin) bead, need to be reconsidered. Understanding the effect of force on the association kinetics of kinesin has important implications for intracellular transport, where the force-dependent detachment governs how kinesins interact with other kinesins and opposing dynein motors (Muller et al., 2008; Kunwar et al., 2011; Ohashi et al., 2018; Gicking et al., 2022) on vesicular cargoes.

Weaknesses:

The authors attribute the differences in the behaviour of kinesins when pulling against a DNA tether compared to an optical trap to the differences in the perpendicular forces. However, the compliance is also much different in these two experiments. The optical trap acts like a ~ linear spring with stiffness ~ 0.05 pN/nm. The dsDNA tether is an entropic spring, with negligible stiffness at low extensions and very high compliance once the tether is extended to its contour length (Fig. 1B). The effect of the compliance on the results is not fully considered in the manuscript.

Compared to an optical trapping assay, the motors are also tethered closer to the microtubule in this geometry. In an optical trap assay, the bead could rotate when the kinesin is not bound. The authors should discuss how this tethering is expected to affect the kinesin reattachment and slipping. While likely outside the scope of this study, it would be interesting to compare the static tether used here with a dynamic tether like MAP7 or the CAP-GLY domain of p150glued.

In the single-molecule extension traces (Fig. 1F-H; S3), the kinesin-2 traces often show jumps in position at the beginning of runs (e.g. the four runs from ~4-13 s in Fig. 1G). These jumps are not apparent in the kinesin-1 and -3 traces. What is the explanation? Is kinesin-2 binding accelerated by resisting loads more strongly than kinesin-1 and -3? In their response, the authors provide an explanation of the appearance of jumps due to limited imaging speeds. The authors state that the qualitative difference in the kinesin-2 traces compared to the kinesin-1 an -3 traces may be due to the specific rebinding kinetics of kinesin-2.

When comparing the durations of unloaded and stall events (Fig. 2), there is a potential for bias in the measurement, where very long unloaded runs cannot be observed due to the limited length of the microtubule (Thompson, Hoeprich, and Berger, 2013), while the duration of tethered runs is only limited by photobleaching. Was the possible censoring of the results addressed in the analysis? The authors addressed this concern by applying a Markov model to estimate the duration parameter.

The mathematical model is helpful in interpreting the data. To assess how the "slip" state contributes to the association kinetics, it would be helpful to compare the proposed model with a similar model with no slip state. Could the slips be explained by fast reattachments from the detached state? In their response, the authors addressed this question by explaining that a three-state model is required to model the recovery time distributions.

Reviewer #3 (Public review):

Summary:

This study reports KRH, a SaCas9 variant computationally engineered via UniDesign to recognize an expanded NNNRRT PAM with substantially enhanced editing efficiency at non-canonical sites. KRH achieves genome- and base-editing efficiencies comparable to or exceeding the evolution-derived KKH variant across multiple human cell types, demonstrating that computational design can effectively remodel PAM specificity while preserving nuclease activity.

Strengths:

The research follows a clear line of reasoning, and the results appear sound. The computational design strategy presented offers a valuable alternative to directed evolution, with potential applicability beyond Cas9 engineering.

Weaknesses:

The benchmarking of the UniDesign method is insufficient. How its performance compares to other protein design algorithms, whether the energy function parameters were systematically optimized, and if the design strategy can be generalized to other Cas9 orthologs or genome engineering tasks.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate whether disruption of intracrine steroid hormone metabolism contributes to meibomian gland dysfunction and proposed a "vicious cycle" of gland dysfunction and inflammation, using a global Had3b6 knockout mouse model. The work addresses an important aspect of MGD, but its impact may be limited unless the intracrine mechanism can be more clearly distinguished from systemic hormonal effects.

Strengths:

This study addressed an important question. The hormonal regulation of the meibomian gland has long been recognized. If clarified, the concept of local steroid metabolism influencing gland homeostasis could have implications for understanding disease mechanisms and identifying therapeutic targets.

Weaknesses:

The use of a global knockout makes it difficult to separate local intracrine effects from systemic hormonal changes, and key controls and hormone measurements are lacking.<br /> LPS-induced inflammation may not reflect the chronic nature of MGD.

Reviewer #3 (Public review):

Summary:

The manuscript by Kim et al. describes a MuSC subpopulation that loses VCam expression in geriatric muscle and shows reduced ability to contribute to muscle regeneration. They propose that this population underlies the reported decline of MuSCs in aged mice, suggesting that these cells remain present in geriatric muscle but are overlooked due to low or absent VCam expression. The identification of a subpopulation that changes with aging would be compelling and of interest to the field.

Strengths:

The authors employ a wide range of assays, from in vitro to in vivo systems, to characterize Vcam-low/negative cells from geriatric muscle. The loss of Vcam appears strong in geriatric mice. They further identify CD63 and CD200 as potential surface markers that remain stable with age, thereby enabling the isolation of MuSCs across different age groups.

Weaknesses:

Some issues remain before establishing whether this population represents a true functional subset or explains the reported decline in MuSC numbers in aged mice. Stronger fate assessment of Vcam-low/negative cells is needed to assess their propensity for cell death and whether this contributes to the conclusions. Comparisons include young, middle-aged, and geriatric mice, but not aged (~24 months) mice, which would help comparisons to previous reports of age-related MuSC decline. The suggestion that the Vcam-low/negative population reflects a senescence-like state remains unclear, as these cells display limited canonical senescence markers, exhibit reversible cell-cycle exit, and yet are reported to be sensitive to senolytic treatment. Validation of CD63 and CD200 as reliable age-independent MuSC markers requires further testing, specifically using the Pax7-YFP tracing model and co-labeling in geriatric mice. Finally, the grouping patterns in some analyses suggest that the Vcam-low/negative fraction may be present in only a subset of geriatric mice, raising the possibility that it reflects health status or pathology rather than a consistent aging-associated phenotype.

Reviewer #3 (Public review):

Summary:

This study aimed to investigate pseudouridylation across various RNA species in multiple bacterial strains using an optimized BID-seq approach. It examined both conserved and divergent modification patterns, the potential functional roles of pseudouridylation, and its dynamic regulation across different growth conditions.

Strengths:

The authors optimized the BID-seq method and applied this important technique to bacterial systems, identifying multiple pseudouridylation sites across different species. They investigated the distribution of these modifications, associated sequence motifs, their dynamics across growth phases, and potential functional roles. These data are of great interest to researchers focused on understanding the significance of RNA modifications, particularly mRNA modifications, in bacteria.

Reviewer #3 (Public review):

Zhang et al. investigated how germline tumors influence the development of neighboring wild-type (WT) germline stem cells (GSC) in the Drosophila ovary. They report that germline tumors generated by differentiation-arrested mutations (bam and bgcn) inhibit the differentiation of neighboring WT GSCs by arresting them in an undifferentiated state, resulting from reduced expression of the differentiation-promoting factor Bam. They find that these tumor cells produce low levels of the niche-associated signaling molecules Dpp and Gbb, which suppress bam expression and consequently inhibit the differentiation of neighboring WT GSCs non-cell-autonomously. Based on these findings, the authors propose that germline tumors mimic the niche to suppress the differentiation of the neighboring wild-type germline stem cells.

Strengths:

The study uses a well-established in vivo model to address an important biological question concerning the interaction between germline tumor cells and wild-type (WT) germline stem cells in the Drosophila ovary. If the findings are substantiated, this study could provide valuable insights that are applicable to other stem cell systems.

Weaknesses:

The authors have addressed some of my concerns in the revised submission. However, the data presented do not allow the authors to distinguish whether the failed differentiation of WT stem cells/germline cells results from "arrested differentiation due to the loss of the differentiation niche" or from "direct inhibition by tumor-derived expression of niche-associated molecules Dpp and Gbb". The critical supporting data, HCR in situ results, are not sufficiently convincing.

Reviewer #3 (Public review):

Summary:

This work uses a convolutional neural network to optimize a voltage clamp protocol to identify features and parameters from human pluripotent stem cell-derived cardiomyocytes.

Yang et al. introduce an innovative experimental framework that integrates computational modeling and deep learning to generate a digital twin of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs).

Strengths:

The major strength is the methodology used to bridge in silico prediction of cell behavior and mechanistic insights from the experimental dataset.

The approach used in this study represents a significant step toward precision medicine by enabling in silico prediction of cellular behavior and mechanistic insight from experimental datasets. The study addresses an important and timely challenge in stem cell-based and personalized medicine, and the authors compellingly leverage state-of-the-art methods alongside strong expertise in computational modeling and cardiac electrophysiology

Weaknesses:

While the overall approach is highly compelling and the potential impact is substantial, there are two areas where clarification and refinement, particularly in the phrasing and framing used throughout the manuscript, would further strengthen the work.

(1) While the overall goal of the study is compelling, the manuscript would benefit from clearer articulation of how the proposed framework is intended to be used in practice. In particular, it is not entirely clear whether the authors envision this approach as:

a) a method to extract population-level trends that, when paired with biological data, enhance statistical power and interpretability, or

b) a strategy capable of constructing a population-based model from limited single-cell recordings. If the latter is intended, additional guidance on the number of action potentials required per cell and the assumptions underlying this extrapolation would greatly clarify the scope and applicability of the method.

(2) The manuscript would also benefit from a clearer explanation of how electrophysiological heterogeneity observed in hPSC-CMs is linked to inter-patient variability. Although the authors state that this framework can be generalized to compare patient-specific hiPSC-CM lines, it remains unclear how this generalization is achieved, given the substantial sources of variability intrinsic to hiPSC-CMs (e.g., batch effects, reprogramming strategy, differentiation protocol, and maturation state). As acknowledged by the authors, addressing this level of variability likely requires large datasets; further clarification of how the proposed approach mitigates or accommodates these challenges would strengthen the translational claims.

Below are my suggestions that could help strengthen the claims in the manuscript:

(1) Adding a dedicated section describing the electrophysiological phenotype of the hPSC-CMs used in this study would help justify the choice of the underlying ionic model and the selection of the six ion currents analyzed. These currents are not only developmentally regulated but may also vary substantially across different hPSC-CM lines, which has implications for generalizability.

(2) If feasible, inclusion of patch-clamp data from an additional hPSC-CM line would significantly strengthen the claim that this framework can harmonize and generalize across datasets and cell sources.

(3) The authors note that the experimental cells exhibited high variability in action potential morphology. This is an important observation that directly supports the motivation for the study and should be explicitly presented, even if only in the supplementary materials.

(4) In the hERG-blocker experiments, further clarification is needed regarding the biological relevance of the reported 3% incidence of early afterdepolarizations (EADs). Additionally, an interrupted sentence in this section makes it unclear whether the goal is to demonstrate that the digital twin can capture rare arrhythmic risk events or whether the digital twin is necessary to determine whether this level of risk is clinically meaningful.

(5) The manuscript states that some action potentials were excluded from the experimental dataset. A brief explanation of the exclusion criteria, along with guidance on how to distinguish high-quality from low-quality recordings, would improve transparency and reproducibility.

Reviewer #3 (Public review):

Summary:

The paper describes the structure of gp5.4, the spike tip of phage T4. This structure was released in the PBD in 2013. The paper further investigates the role of this protein in virion assembly, stability, and infection by comparing the behaviour of the WT phage and a phage without the protein, resulting from an amber mutation in the phage genome. A competition assay between the WT and mutant phage shows a clear increase in the fitness of the WT. A further screening of a transposon bank allowed for the identification of a host strain that is resistant to the mutant phage while still sensitive to the WT phage.

Strengths:

(1) Beautiful structure, at very high resolution (1.15 Å).

(2) Very sophisticated microbiology experiments to allow mutant phage characterisation and dissect the role of the spike tip in phage fitness.

Weaknesses:

(1) The paper is very descriptive, and the lack of a general conclusion, not to say discussion, is frustrating. What do the findings of the paper bring to the knowledge of infection? What would be the fate of the spike and tip? A discussion in the context of the data available in the literature would greatly increase the interest of the paper.

(2) Why didn't the authors include the description of the structure of the homologous Pvc10 and PhiKV gp5.4 in complex with gp5ß, which they also solved a while ago?

(3) Because microbiology is sophisticated, special care should be taken to introduce the strains used (both E. coli and T4). E.g. it is still not clear to me what the difference is between the supF and the supD coli strains in terms of mutant phage produced (both should produce T4(5.4am)-gp5.4?).

(4) For the same reason, strains should always be called by the same name.

(5) In some sections, the conclusion seems lost in the description of controls (e.g. in the "The spike is translocated into the periplasmic space during infection" paragraph).

Appraisal:

The authors show that the sharp tip of the membrane-perforating tube of T4 contractile tail contributes to perforating the outer membrane. In particular, this protein is necessary in a host bearing mutated LPS.

Reviewer #3 (Public review):

Summary:

Gosselin et al. use the sybody technology to study effects of in vivo inhibition of the Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the "transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc "neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA.<br /> The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

My main criticism is that the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only identify sybodies that bind to a rather small part of the large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potentially binding to different parts of Smc.

Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially available. However, this test is not important for the overall study, it would just clarify a minor point.

Fig. 2B: is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the "counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

Testing binding sites of sybodies to the SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

Significance:

The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

Reviewer #3 (Public review):

Summary:

Tkacik et al. systematically characterized all three RAF kinase isoforms in vitro with all three types of RAF inhibitors (Type I, I1/2, and II) to investigate the mechanism underlying paradoxical activation.

In this study, the authors reconstituted heterodimers of A-, B-, and C-RAF kinase domains bound to non-phosphorylable MEK1 (SASA), mimicking the monomeric auto-inhibited state of RAF. These "RAF monomers" were tested for MEK phosphorylation with an increasing concentration of all three types of RAF inhibitors (Type I, I1/2, and II). This study is reminiscent of a previous study of the same team measuring RAF kinase activity in the presence of all three types of inhibitors in the context of dimeric RAF isoforms stabilized by 14-3-3 proteins (Tkacik et al 2025 JBC). RAF monomers had little to no activity at low concentrations of inhibitors (consistent with their "monomeric state"). Addition of type I1/2 inhibitor did not induce paradoxical activation as, in this context, they do not induce RAF dimerization required for activation, as observed by MP. Addition of type I and type II inhibitors led to paradoxical activation consistent with the RAF dimerization induced by these inhibitors, as observed by MP. Interestingly, type II inhibitors induced activation only for B- and C-RAF and not A-RAF.

At high concentrations of type II inhibitors, kinase activity is inhibited with a strong or weak positive cooperativity for BRAF and CRAF, respectively. This observation is very similar to what the authors previously observed with their dimeric RAF system. Interestingly, when the NtA motif is modified by phosphomimetic mutations in A- and C-Raf, basal kinase activity is stronger, but most importantly, inhibitor-induced paradoxical activation is much stronger with both type I and II inhibitors. This demonstrates that mutation of the NtA motif of ARAF and CRAF sensitized them to paradoxical activation by type II inhibitors.

The authors also tested the effect of ATP in the paradoxical activation observed in their RAF "monomer" system. As previously published in their assay with 14-3-3 stabilized dimeric RAF, the authors observed an expected shift of the IC50 with Type I inhibitors, while Type II inhibitors seem to behave as a non-competitive inhibitor. The authors next reconstituted the MAP kinase pathway (with RAF monomers at the top of the phosphorylation cascade) to test paradoxical activation amplification. Again, Type I1/2 inhibitors did not induce paradoxical activation, while Type I and II inhibitors did. The authors tested the inhibitors with FL auto-inhibited RAF/MEK/14-3-3 complexes, where, contrary to the "RAF monomers" experiments, FL B- and C-RAF were not paradoxically activated but were inhibited by all three types of inhibitors.

Overall, Tkacik et al. tackle an important question in the field for which definitive experiments and thorough biochemical investigation to understand the molecular mechanisms for the inhibitor-induced paradoxical activation are still missing, and of high importance for future drug development.

Strengths:

The biochemical experiments here are rigorously executed, and the results obtained are highly informative in the field to decipher the intricate mechanisms of RAF activation and inhibitor-induced paradoxical activation.

Weaknesses:

The interpretation of the results in the context of the current state of the art is ambiguous and raises questions about the relevance of introducing a new model for inhibitor-induced paradoxical activation, particularly since the findings presented here do not clearly contradict established paradigms. I believe some clarification and precision are required.

Main comments:

(1) Figure 2:

The authors comment on the expected greater increase (for a cascade assay) in the magnitude of ERK phosphorylation compared to what was observed for MEK phosphorylation. However, this observation might be reflective of the stoichiometries used in the assay, with 40 times more MEK compared to RAF concentration (250nm vs 6nM), which might favour pERK vs pMEK.

- The authors should clarify their rationale for the protein concentration used in this assay and explain how protein stoichiometry was taken into account for the interpretation of their results.

- In addition, the authors should justify comparing pMEK and pERK TR-FRET values when different anti-phospho antibodies were used. Antibodies may have distinct binding affinities for their epitopes. Could this not lead to differences in FRET signal amplitudes that complicate direct comparison?

(2) Supplementary Figure 2:

The author mentioned that the inhibitors did not activate the FL auto-inhibited RAF complexes; however, they did inhibit the TR-FRET signal.

- Can the authors comment on the origin of the observed basal activity? Would the authors expect self-release of the RAF kinase protein from the auto-inhibited state in the absence of RAS, leading to dimerization and activation? Alternatively, do the inhibitors at low-concentration relieve the auto-inhibited state, thereby driving dimerization and activation?

- Did the author test the addition of RAS protein in their in vitro system to determine whether "soluble" RAS is sufficient to release the protective interactions with RBD/CRD/14-3-3 and lead to inhibitor-induced paradoxical activation of FL RAF?

(3) Figure 5B:

The authors said that the Kd values obtained from their MP assay are consistent with prior studies of RAF homodimerization and RAF:MEK heterodimerization. While this is true from the previous studies of RAF:MEK interaction by BLI (performed from the same team), the Kd of isolated RAF kinase homodimerization has been measured around ~30µM by AUC in the cited ref (24,27 & 37).

- The authors should discuss the discrepancy between their Kd of homodimerization and the reported Kd values in the literature. At the concentration used for MP, it is surprising to observe RAF dimerization while the Kd of homodimerization has been measured at ~30µM (in the absence of MEK).

- Would the authors expect the presence of MEK to influence the homodimerization affinity for the isolated KD?

(4) Conclusions:

Several times in the introduction and the conclusion, the authors suggest that the negative allostery model (where "inhibitor binding to one protomer of the dimer promotes an active but inhibitor-resistant conformation in the other") is a model that applies to all types of RAF inhibitors (I, I1/2, and II).

However, from my understanding and all the references cited by the authors, this model only applies to type I1/2 inhibitors, where indeed the aC IN conformation in the second (inhibitor-free) protomer of the RAF dimer might be incompatible with the type I1/2 inhibitors inducing aC OUT conformation. The type I and type II inhibitors are aC IN inhibitors and are expected to bind both protomers from RAF dimers with similar affinities. Therefore, the negative allostery model does not apply to the type I and type II inhibitors. The difference in the mechanism of action of inhibitors is even used to explain the difference in the concentration range in which inhibitor-induced activation is observed in cells. The description of the state of the art in this study is confusing and does not help to properly understand their argumentation to revise the established model for paradoxical RAF activation.

- Can the authors clarify their analysis of the state of the art on the different mechanisms of action for the paradoxical activation of RAF by the different types of RAF inhibitors?

5) Conclusions:

"Our results suggest that negative allostery (or negative cooperativity) is not a requisite feature of paradoxical activation. The type I and type II inhibitors studied here induce RAF dimers and exhibit paradoxical activation but do so without evidence of negative cooperativity, nor do they appear to inhibit intentionally engineered RAF dimers with negative cooperativity (25). Indeed, type II inhibitors exhibit apparent positive cooperativity while type I inhibitors are non-cooperative inhibitors of RAF dimers (25)."

- Can the authors explain how results on the paradoxical activation induced by type I and type II inhibitors inform or challenge a model that specifically applies to type I1/2 inhibitors?

The authors often refer to their previous study (reference 25), where they tested the inhibition of all three types of inhibitors with engineered RAF dimers. While I agree with the authors that in reference 25 the Type I and type II inhibitors inhibit RAF dimers without exhibiting negative cooperativity (as expected from the literature and the current model), the authors did observe some negative cooperativity for Type I1/2 inhibitors in their study most particularly for the type I1/2 PB (with hill slope ranging from -0.4 to -0.9, indicative of negative cooperativity).<br /> While the observations that type II inhibitors display positive cooperativity is both novel and very interesting, from what I understand the results from thakick et al 2025 and the current study appear more in line with the current paradigm in the field (which describe paradoxical activation with negative cooperativity for type I1/2 inhibitors and no negative cooperativity for the Type I and II inhibitors) rather than disapproving of the current model and supporting for a new model.

- In this context, can the authors clarify how their results challenge the current model for paradoxical activation?

(6) Conclusions:

The authors describe the JAB34 experiment from Poulikakos et al. 2010 to conclude that "While this experiment cleanly demonstrates inhibitor-induced transactivation of RAF dimers, it is important to recognize that the differential inhibitor sensitivity of the two subunits in this experiment is artificial - it is engineered rather than induced by inhibitor binding as the negative allostery model proposes."

Indeed, the JAB34 experiment demonstrated the inhibitor-induced transactivation, but the Poulikakos et al. 2010 study does not discuss differential inhibitor sensitivity. The negative allostery model was proposed later by poulikakos team in other papers (Yao et al 2015 and Karoulia et al, 2016), in which JAB34 was not used.

- Can the authors clarify how the JAB34 experiments question differential inhibitor sensitivity?

(7) Conclusions:

"Considering that the conformation required for binding of type I.5 inhibitors destabilizes RAF dimers, it is unclear how an inhibitor binding to one protomer would be able to transmit an allosteric change to the opposite protomer, if that inhibitor's binding causes the existing dimer to dissociate."

- The authors should comment on whether 14-3-3 proteins might overcome negative regulation by type I1/2 inhibitors, similar to what has been shown for ATP, which acts as a dimer breaker like type I1/2 inhibitors.

(8) Conclusions:

"Furthermore, the complex effects of type I.5 inhibitors on dimer stability and the clear resistance of active RAF dimers to these inhibitors complicates interpretation of inhibition data - weak or incomplete inhibition of an enzyme can be difficult to discern from true negative cooperativity (43). As we discuss below, the clear resistance of RAF dimers to type I.5 inhibitors is alone sufficient to explain their ineffective inhibition during paradoxical activation, without invoking negative allostery."

- The authors should explain how they reconcile this statement and their proposal of a new model that does not rely on negative allostery with their previous findings showing negative cooperativity for RAF dimer inhibition with type I1/2 inhibitors.

(9) Conclusions:

Here, the authors propose a new universal model to explain paradoxical activation of RAF by all types of RAF inhibitors:<br /> " Our findings here, in light of structural studies of RAF complexes and prior cellular investigations of paradoxical activation, lead us to a model for paradoxical activation that does not rely on negative allostery and is consistent with activation by diverse inhibitor classes. In this model, the open monomer complex is the target of inhibitor-induced paradoxical activation (Figure 6). Binding of ATP to the RAF active site stabilizes the inactive conformation of the open monomer, which disfavors dimerization. Displacement of ATP by an ATP-competitive inhibitor, irrespective of class, alters the relative N- and C-lobe orientations of the kinase to promote dimerization (30, 35). Once dimerized, inhibitor dissociation from one or both sides of the dimer would allow phosphorylation and activation of MEK."

From my understanding, the novelty of this new model is twofold: a) the open monomer is the target of the inhibitor-induced paradoxical activation and b) once dimerized, inhibitor dissociation from one or both sides of the dimer would allow phosphorylation and activation of MEK.

Novelty a) implies, as the authors stated, that "Inhibitor-induced activation and inhibition act on distinct species - activation on the open monomer and inhibition on the 14-3-3-stabilized dimer". The authors should explain what they mean by "activation of the open monomer", while only RAF dimers are catalytically active (except for BRAF V600E mutant)?

For novelty b), the authors should explain more clearly what experimental results support this new model.

Reviewer #4 (Public review):

This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications to understand how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms.

The paper has significantly improved after addressing all the Reviewers' comments and I did not observe significant weaknesses in the study.

Reviewer #3 (Public review):

Significance of the Findings:

The study by Liu et al. presents a novel method, DNA-O-MAP, which combines locus-specific hybridisation with proximity biotinylation to isolate specific genomic regions and their associated proteins. The potential significance of this approach lies in its purported ability to target genomic loci with heightened specificity by enabling extensive washing prior to the biotinylation reaction, theoretically improving the signal-to-noise ratio when compared with other methods such as dCas9-based techniques. Should the method prove successful, it could represent a notable advancement in the field of chromatin biology, particularly in establishing the proteomes of individual chromatin regions-an extremely challenging objective that has not yet been comprehensively addressed by existing methodologies.

Strength of the Evidence:

The evidence presented by the authors is somewhat mixed, and the robustness of the findings appears to be preliminary at this stage. While certain data indicate that DNA-O-MAP may function effectively for repetitive DNA regions, a number of the claims made in the manuscript are either unsupported or require further substantiation. There are significant concerns about the resolution of the method, with substantial biotinylation signals extending well beyond the intended target regions (megabases around the target), suggesting a lack of specificity and poor resolution, particularly for smaller loci. Furthermore, comparisons with previous techniques are unfounded since the authors have not provided direct comparisons with the same mass spectrometry (MS) equipment and protocols. Additionally, although the authors assert an advantage in multiplexing, this claim appears overstated, as previous methods could achieve similar outcomes through TMT multiplexing. Therefore, while the method has potential, the evidence requires more rigorous support, comprehensive benchmarking, and further experimental validation to demonstrate the claimed improvements in specificity and practical applicability.

Reviewer #3 (Public review):

Summary:

In their manuscript, Koch et al. describe a novel strategy to synchronize cells of the budding yeast Saccharomyces cerevisiae in metaphase I and metaphase II, thereby facilitating comparative analyses between these meiotic stages. This approach, termed SynSAC, adapts a method previously developed in fission yeast and human cells that enables the ectopic induction of a synthetic spindle assembly checkpoint (SAC) arrest by conditionally forcing the heterodimerization of two SAC components upon addition of the plant hormone abscisic acid (ABA). This is a valuable tool, which has the advantage that induces SAC-dependent inhibition of the anaphase promoting complex without perturbing kinetochores. Furthermore, since the same strategy and yeast strain can be also used to induce a metaphase arrest during mitosis, the methodology developed by Koch et al. enables comparative analyses between mitotic and meiotic cell divisions. To validate their strategy, the authors purified kinetochores from meiotic metaphase I and metaphase II, as well as from mitotic metaphase, and compared their protein composition and phosphorylation profiles. The results are presented clearly and in an organized manner.

Significance:

Koch et al. describe a novel methodology, SynSAC, to synchronize budding yeast cells in metaphase I or metaphase II during meiosis, as well and in mitotic metaphase, thereby enabling differential analyses among these cell division stages. Their approach builds on prior strategies originally developed in fission yeast and human cells models to induce a synthetic spindle assembly checkpoint (SAC) arrest by conditionally forcing the heterodimerization of two SAC proteins upon addition of abscisic acid (ABA). The results from this manuscript are of special relevance for researchers studying meiosis and using Saccharomyces cerevisiae as a model. Moreover, the differential analysis of the composition and phosphorylation of kinetochores from meiotic metaphase I and metaphase II adds interest for the broader meiosis research community. Finally, regarding my expertise, I am a researcher specialized in the regulation of cell division.

Reviewer #3 (Public review):

Summary

This study provides a detailed investigation of neural auditory responses and spontaneous movements in infants listening to music. Analyses of EEG data (event-related potentials and steady-state responses) first highlighted that infants at 3, 6 and 12 months of age and adults showed enhanced auditory responses to music than shuffled music. 6-month-olds also exhibited enhanced P1 response to high-pitch vs low-pitch stimuli, but not the other groups. Besides, whole body spontaneous movements of infants were decomposed into 10 principal components. Kinematic analyses revealed that the quantity of movement was higher in response to music than shuffled music only at 12 months of age. Although Granger causality analysis suggested that infants' movement was related to the music intensity changes, particularly in the high-pitch condition, infants did not exhibit phase-locked movement responses to musical events, and the low movement periodicity was not coordinated with music.

Strengths

This study investigates an important topic on the development of music perception and translation to action and danse. It targets a crucial developmental period that is difficult to explore. It evaluates two modalities by measuring neural auditory responses and kinematics, while cross-modal development is rarely evaluated. Overall, the study fills a clear gap in the literature.

Besides, the study uses state-of-the-art analyses. Detailed investigations were performed, as well as exploratory analyses in supplementary information. The discussion is rich in neurodevelopmental interpretations and comparisons with the literature. All steps are clearly detailed. The manuscript is very clear, well-written and pleasant to read. Figures are well-designed and informative. The authors' responses to previous reviews are also detailed and informative.

Comments on revisions:

The authors answered all my questions.

Reviewer #3 (Public review):

Summary:

In this manuscript, Guin et al. use a CRISPR KO screen of ~1000 candidates in two human cell lines along with high-throughput image analysis to demonstrate that orderly progression through mitosis shapes centromere organization. They identify ~50 genes that perturb centromere clustering when depleted in both RPE1 and HCT116 cells and validate many of these hits using RNAi. They then use auxin-mediated acute depletion of four factors (NCAPH2, KI67, SPC24 and NUF2) to demonstrate that their effects on centromere clustering require passage through mitosis. They further suggest that lack of these factors during mitosis leads to disorganization of centromeres on the mitotic spindle and these effects persist in the subsequent interphase. Overall, the manuscript is clear, well-written, the experiments performed are appropriate and the data is interpreted accurately. In my opinion, the main strength of this manuscript is the discovery of several hits associated with altered centromere clustering. These hits will serve as a solid foundation for future work investigating the functional significance of centromere clustering in human cells. On the other hand, how the changes in centromere clustering relate to other aspects of interphase genome architecture (A/B compartments, chromosome territories etc) remains unclear and represents the main limitation of this manuscript.

Reviewer #3 (Public review):

Summary:

The authors aim to investigate the mechanisms of anxiety. The paper focuses on supramammillary nucleus (SuM) based on a fos screen and recordings showing that footshock and social defeat stress increases activity in this region. Using activity-dependent tagging, they show that reactivation of stress-activated neurons in SuM has an anxiety-like effect, reducing open-arm exploration in the elevated zero task. They then investigate the ventral subiculum as a potential source of anxiety-related information for SuM. They show that ventral subiculum (vSub) inputs to SuM are more strongly activated than dSub when mice explore open arms of the elevated zero. Finally, they show that DREADD-mediated inhibition of vSub-SuM projections alleviates stress-enhanced anxiety. Overall the results provide good evidence that SuM contains a stress-activated neuronal population whose later activity increases anxiety-like behavior. It further provides evidence that vSub projects to SuM are activated by stress and their inhibition alleviates some effects of stress.

Strengths:

Strengths of this paper include the use of convergent methods (e.g., fos plus electrode recordings, footshock and social defeat) to demonstrate that the SuM is activated by different forms of stress. The activity-dependent tagging experiment shows that footshock-activated SuM neurons are reactivated by social defeat but not sucrose is also compelling because it provides evidence that SuM neurons are driven by some integrative aspect of stress rather than by a simple sensory stimulus.

Weaknesses:

The strength of some evidence is judged to be incomplete. The paper provides good evidence that SuM contains stress-responsive neurons, and the activity of these neurons increases some measure of anxiety-like behavior. However, the evidence that the vSub-SuM projection "encodes anxiety" and that the SuM is a key regulator of anxiety is judged to be incomplete. I am not convinced that the identified SuM cells have a specific anxiety function. As the authors mention in the introduction, SuM regulates exploration and theta activity. Since theta potently regulates hippocampal function, there is the concern that SuM manipulations could have broad effects beyond anxiety-like behavior.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

I am fine with the revised version of the manuscript.

Reviewer #3 (Public review):

Summary:

Sleep is typified by a behavioural attenuation of responsiveness to external stimuli (higher arousal thresholds). There are various mechanisms through which sensory perception could be dampened, and while thalamic and cortical gate points have been well studied, the focus here is on peripheral ones - at the level of the olfactory bulb (OB). While something conceptually similar has been shown in insects, this paper represents an important contribution to understanding attenuation of sensory perception during rodent sleep and anaesthesia.

This paper shows that respiration-locked potentials and gamma activity in the olfactory bulb, which are important for olfactory coding, are diminished during sleep and when under anaesthesia compared to wake. Further, this state-dependent activity in OB is likely to be locally generated. Using a tracheotomy procedure aimed to dissociate nasal airflow from natural inhalations, authors demonstrate that local field potentials (LFPs) in the OB phase lock with artificially generated air pulses (delivered into the nasal cavity) during the active phase of anaesthesia but not during a more passive state. LFPs did not synchronise with respiratory signals during either anaesthesia state. Lastly, the authors showed that as delta power increased (typical of slow-wave-sleep), the coherence between nasal inhalation rhythms and OB LFP coherence decreased, indicating that as rats experienced something akin to slow-wave-sleep (during anaesthesia), disconnection from the external environment could be augmented. Taken together, the authors argue that the change in activity observed in the olfactory bulb during sleep and anaesthesia provides a non-permissive state for sensory processing and manifests as sensory dissociation

Strengths:

The manuscript is well-written, and the experiments are thorough. Experiments examining coupling of nasal respiration with OB potentials and delta activity are particularly interesting as they point to augmented sensory disconnection during a sleep phase typically associated with higher arousal thresholds.

Weaknesses:

(1) An experiment addressing the following points, is missing:

Does odour stimulation that wakes up a subject restore gamma activity and respiration-locked potentials?

Is OB/respiration desynchrony maintained when presented with a non-rousing stimulus?

Is waking upon stimulus delivery less likely as delta activity increases and coherence between OB/respiratory rhythms weakens?

(2) Many of the experiments are performed under anaesthesia, which I understand is for practical reasons. While authors are forthcoming about limitations of using anaesthesia in lieu of natural sleep states, I would have preferred to see more experiments performed on sleeping animals.

Reviewer #3 (Public Review):

Summary:

72 subjects, and 144 hemispheres, from the Human Connectome Project had their parietal sulci manually traced. This identified the presence of previous undescribed shallow sulci. One of these sulci, the ventral supralateral occipital sulcus (slocs-v), was then demonstrated to have functional specificity in spatial orientation. The discussion furthermore provides an eloquent overview of our understanding of the anatomy of the parietal cortex, situating their new work into the broader field. Finally, this paper stimulates further debate about the relative value of detailed manual anatomy, inherently limited in participant numbers and areas of the brain covered, against fully automated processing that can cover thousands of participants but easily misses the kinds of anatomical details described here.

Strengths:

- This is the first paper describing the tertiary sulci of the parietal cortex with this level of detail, identifying novel shallow sulci and mapping them to behaviour and function.

- It is a very elegantly written paper, situating the current work into the broader field.

- The combination of detailed anatomy and function and behaviour is superb.

Reviewer #3 (Public review):

Summary:

Fan et al utilize large omics data sets to give an overview of proteomic and gene expression changes after 4 moths of intermittent fasting (IF) in liver, muscle and brain tissue. They describe common and district pathways altered under IF across tissues using different analysis approaches. Main conclusions presented are the variability in responses across tissues with IF. Some common pathways were observed, but there were notable distinctions between tissues.

Strengths:

(1) The IF study was well conducted and ran out to 4 months which was a nice long-term design.

(2) The multi omics approach was solid and additional integrative analysis was complementary to the illustrate the differential pathways and interactions across tissues.

(3) The authors did not over-step their conclusions and imply an overreached mechanism.

Weaknesses:

The weaknesses, which are minor, include use of only male mice and the early start (6 weeks) of the IF treatment. However, the authors have provided justification on why they chose male mice and the time points used in the study.

Reviewer #3 (Public review):

Summary:

The manuscript by Nishimura et al. examines the behavioural and neural mechanisms of stress-enhanced fear responding (SEFR) and stress-enhanced fear learning (SEFL). Groups of stressed (4 x shock exposure in a context) vs non-stressed (context exposure only) animals are compared for their fear of an unconditioned tone, and context, as well as their learning of new context fear associations. Shock of higher intensity led to higher levels of unlearned stress-enhanced fear expression. Immediate early gene analysis uncovered the PVT as a critical neural locus, and this was confirmed using fiber photometry, with stressed animals showing an elevated neural signal to an unconditioned tone. Using a gain and loss of function DREADDs methodology, the authors provide convincing evidence for a causal role of the PVT in SEFR.

Strengths:

(1) The manuscript uses critical behavioural controls (no stress vs stress) and behavioural parameters (0.25mA, 0.5mA, 1mA shock). Findings are replicated across experiments.

(2) Dissociating the SEFR and SEFL is a critical distinction that has not been made previously. Moreover, this dissociation is essential in understanding the behavioural (and neural) processes that can go awry in fear.

(3) Neural methods use a multifaceted approach to convincingly link the PVT to SEFR: from Fos, fiber photometry, gain and loss of function using DREADDs.

Weaknesses:

No weaknesses were identified by this reviewer; however, I have the following comments:

A closer examination of the Test data across time would help determine if differences may be present early or later in the session that could otherwise be washed out when the data are averaged across time. If none are seen, then it may be worth noting this in the manuscript.

Given the sex/gender differences in PTSD in the human population, having the male and female data points distinguished in the figures would be helpful. I assume sex was run as a variable in the statistics, and nothing came as significant. Noting this would also be of value to other readers who may wonder about the presence of sex differences in the data.

Comments on revisions:

Following revision, this reviewer felt all of the above comments were addressed.

Reviewer #3 (Public review):

In the manuscript, the authors generated several mutant plants defective in the eIF4E family proteins and detected cassava brown streak viruses (CBSVs) infection in these mutant plants. They found that CBSVs induced significantly lower disease scores and virus accumulation in the double mutant plants. Furthermore, they identified important conserved amino acid for the interaction between eIF4E protein and the VPg of CBSVs by yeast two hybrid screening. The experiments are well designed, however, some points need to be clarified:

(1) The authors reported that the ncbp1 ncbp2 double mutant plants were less sensitive to CBSVs infection in their previous study, and all the eIF4E family proteins interact with VPg. In order to identify the redundancy function of eIF4E family proteins, they generated mutants for all eIF4E family genes, however, these mutants are defective in different eIF4E genes, they did not generate multiple mutants (such as triple, quadruple mutants or else) except several double mutant plants, it is hard to identify the redundant function eIF4E family genes.

(2) The authors identified some key amino acids for the interaction between eIF4E and VPg such as the L51, it is interesting to complement ncbp1 ncbp2 double mutant plants with L51F form of eIF4E and double check the infection by CBSVs.

Comments on revisions:

The reviewer understand Cassava is not a model plant, it is hard for the authors to generate multiple genetic mutant plants for experiments, so nothing was done to respond to the comments raised by the reviewer.

Reviewer #3 (Public review):

Summary:

This study by Mercer et al. investigates how inhibitory modulation of basolateral amygdala neurons expressing Vglut1 and projecting to the nucleus accumbens (Vglut1BLA→NAc) influences motivated behavior in both appetitive and aversive tasks. Using a combination of whole-cell electrophysiology, chemogenetic inhibition and behavioral tests, the authors demonstrate that (1) reward conditioning increases inhibitory synaptic input and reduces intrinsic excitability of Vglut1BLA→NAc neurons, (2) chemogenetic inhibition of these neurons enhances the number of conditioned approaches in a Pavlovian task and the number of nosepoke responses in an instrumental task, elevates reward valuation, and increases fear discrimination and (3) these effects are linked to salience assignment and associative strength, rather than altered learning or reversal flexibility. The work challenges the classical excitatory function usually reported about the BLA projection to the NAc and highlights an interesting and thought-provoking result. Nevertheless, the study does not address the potential effect of their manipulation on motoric impulsivity, nor did they provide a theoretical framework explaining this unorthodox yet interesting effect.

Strengths:

The study establishes the initial finding with a correlational approach that informs a causal study. They find convincingly that Pavlovian conditioning induces an increase in inhibitory inputs onto Vglut1BLA→NAc neurons that leads to reduced excitability. Causality is studied using a powerful dual recombinase chemogenetic strategy to selectively inhibit this population of Vglut1BLA→NAc neurons and determine the effect on different behavioral tasks. The use of different tasks provides convergence on their effect. This surprising finding provokes interest and will stimulate further investigation into the mechanisms underlying these effects.

Weaknesses:

Several important aspects of the evidence remain incomplete.

(1) First, an important aspect of the underlying processes at play remains to be investigated. In all behavioral tasks, the authors find that their manipulation increases responding that they interpret as a facilitation of learning. However, none of the appetitive tasks include a control stimulus that could address the specificity of their effect. Given that on the Pavlovian task, responding to the CS is almost 100%, I suspect that their manipulation may induce motoric impulsivity. This aspect would clearly benefit from additional controls.

(2) Second, I have several interrogations about the time-resolved probability of port entries (PSTHs).

a) There is a mismatch between the results presented in Figure 1. Panel D shows a peak of responses on the PSTH at ~2s on day 5 (my remark applies to all days), suggesting that the average should lie around this value. However, panel C reports a latency to respond at ~4sec. Could the authors double-check their PSTHs?

b) More generally, the fact that in the Pavlovian task all PSTHs show a peak at almost exactly 2 sec is quite surprising and raises questions about how they are constructed. Sure, the most salient event is the water drop occurring 2s after cue onset. Yet, if mice responded only to these drops, the peak response should occur at 2s+reaction time, which is not the case. Figure 2 shows that on the first acquisition day, responding is already centered around 2s and does not decrease with learning, except for treated animals.

(3) Several methodological flaws are present.

a) The authors need to report clearly the statistics. In most cases, the statistical test used is mentioned in the figure caption with a single P-value. Thus, on two-way ANOVAs, I do not know whether the P-value relates to the interaction, the main effects, or the post-hoc tests.

b) Another important issue is related to the average time-resolved z-score probability of port entries. The bin size used, the smoothing (that is much too strong), and the baseline period used to calculate the z-score are absent from the methods.

(4) This study reports that manipulating 70% of the glutamatergic projection to the NAc induces an effect opposed to what has been previously reported in many different studies. Such a surprising finding deserves a more elaborate discussion about the mechanism that could be at play.

Reviewer #3 (Public review):

Summary

In this study, the authors investigated how inference about the current task context is encoded in the cortex, using MEG measurements. Using the same behavioral task that was initially developed for an fMRI study to identify the loci of task context representation, the current results complement and extend the previous study by identifying the candidate regions that are important for the inference process, not just for encoding the end product. They reported widespread modulation of cortical activity by uncertainty in evidence and volatility of task context changes. In comparison, modulation correlated with the decision variable underlying the task context inference process was more restricted to the parietal and visual cortices, particularly in alpha-band activity.

Strengths:

(1) The normative model provides a solid computational foundation for disambiguating quantities related to decision variables from those related to task factors (e.g., uncertainty and volatility).

(2) The MEG technique allows examination of cortical activity that is modulated by the temporally evolving decision variable.

(3) Rigorous modeling efforts, including comparisons of well-reasoned alternative/reduced models and examinations of diagnostic features using participant-matched simulations.

Weaknesses:

(1) There are two major surprises in the results that raise concerns about how to interpret these data. The first is the absence of modulation of prefrontal cortical activities by prior or posterior. As the authors acknowledged, there are extensive single-neuron recording data (e.g., from the Miller group) demonstrating the presence of task rule modulation in the monkey PFC and prior representation in the PFC in the mouse study that they cited. The second surprise is that the strongest modulation of prior/posterior/evidence was almost always observed in the visual cortex, in contrast to the common embodied cognition assumption. A more elaborated discussion about these discrepancies would help contextualize the current results.

(2) It is not clear why the effects in Figures 2D and E dipped before responses, which is not expected from any of the models. This could potentially affect the interpretation of the MEG signals in late-post-cue or pre-response periods.

(3) The definitions of the different periods (e.g., early/late post-cue) are vague, making it hard to assess the functional relevance of the signals. For example, is the difference between the early pre-response map in Figure 5B and the late evidence map in Figure 4B due to completely non-overlapping time periods? A diagram of the timing definitions for different task periods would be helpful.

(4) Perhaps related to #2, it is puzzling that evidence encoding is absent in the visual cortex during the early post-cue period.

(5) The presentation and discussion of results related to correlated variability assume that the readers have already read their previous paper. A little more elaboration of the significance of this measurement would be helpful.

Reviewer #3 (Public review):

Summary:

This paper develops a model to account for flexible and context-dependent behaviors, such as where the same input must generate different responses or representations depending on context. The approach is anchored in the hippocampal place cell literature. The model consists of a module X, which represents context, and a module H (hippocampus), which generates "sequences". X is a binary attractor RNN, and H appears to be a discrete binary network, which is called recurrent but seems to operate primarily in a feedforward mode. H has two types of units (those that are directly activated by context, and transition/sequence units). An input from X drives a winner-take-all activation of a single unit H_context unit, which can trigger a sequence in the H_transition units. When a new/unpredicted context arises, a new stable context in X is generated, which in turn can trigger a new sequence in H. The authors use this model to account for some experimental findings, and on a more speculative note, propose to capture key aspects of contextual processing associated with schizophrenia and autism.

Strengths:

Context-dependency is an important problem. And for this reason, there are many papers that address context-dependency - some of this work is cited. To the best of my knowledge, the approach of using an attractor network to represent and detect changes in context is novel and potentially valuable.

Weaknesses:

The paper would be stronger, however, if it were implemented in a more biologically plausible manner - e.g., in continuous rather than discrete time. Additionally, not enough information is provided to properly evaluate the paper, and most of the time, the network is treated as a black box, and we are not shown how the computations are actually being performed.