Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

This manuscript by Zhao et. al investigates the canonical hedgehog pathway in testis development of Nile tilapia. They used complementary approaches with genetically modified tilapia and transfected TSL cells (a clonal stem Leydig cell line) previously derived from 3-mo old tilapia. The approach is innovative and provides a means to investigate DHH and each downstream component from the ptch receptors to the gli and sf1 transcription factors. They concluded that Dhh binds Ptch2 to stimulate Gli1 to promote an increase in Sf1 expression leading to the onset of 11-ketotesterone synthesis heralding the differentiation of Leydig cells in the developing male tilapia.'

Strengths of the methods and results:

- The use of Nile tilapia is important as it is an important aquaculture species, it shares the genetic pathway for sex determination of mammalian species, and molecular differentiation pathways are highly conserved<br /> - The approach is rigorous and incorporates a novel TSL, clonal stem Leydig cell model that they developed that is relatively faithful in following endogenous developmental steps and can produce the appropriate steroid.<br /> - Tilapia are relatively amenable to CRISPR/Cas9 targeting and, with their accelerated developmental time frame, provide an excellent model system to interrogate specific signaling pathways.<br /> - The stepwise analysis from dhh-gli-sf1 is thoughtful and well done.

Achieved Aims: The authors set out to test the hypothesis that the canonical Dhh signaling pathway for Leydig cell differentiation and steroidogenic activity is mediated via ptch2 and gli1 regulation of sf1. The results are strong, there are additional steps needed to verify that redundancy/compensation is not contributing to the outcomes.

This work is important in better understanding of nuanced commonalities and differences in developmental pathways across species. Specific to Leydig cell differentiation and steroidogenesis, their work with tilapia supports conservation of the canonical Dhh pathway; however, there appear to be some differences in downstream mediators compared to mouse. Specifically, they conclude that ptch2/gli1 stimulates sf1 and steroidogenesis in tilapia where gli1 is dispensable in mouse. Instead, Gli3 has recently been shown to play an important role to stimulate Sf1 and support the hedgehog pathway.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

The authors describe co-regulated gene modules underlying stage differentiation in Leishmania donovani through a system-level analysis of multiple molecular layers. Using amastigotes isolated from infected hamster spleens and corresponding culture-derived promastigotes, they analyzed genomic variation, transcript abundance, protein levels, phosphorylation states, and metabolite profiles. By combining these, the study identified potential regulatory mechanisms associated with parasite differentiation and generated hypotheses regarding how gene expression is coordinated across different levels.

Strengths:

A major strength of the study is the breadth of the dataset generated. The integration provides an unusually comprehensive view of molecular changes associated with Leishmania differentiation in vitro. Such multi-layer datasets involving bona fide vertebrate host stages remain relatively rare in parasitology and will likely become a valuable resource for the molecular parasitology community. In addition, the use of amastigotes isolated from infected hamsters rather than relying on axenic models provided a biologically relevant framework for the analyses.

The revised manuscript improved several aspects of the original. The RNA-seq analysis is described with a clearer pipeline, and several claims regarding causal regulatory feedback associations have been appropriately toned down. Among the observations reported, the association between parasite differentiation and proteasome-mediated protein degradation is particularly remarkable. The combination of quantitative proteomics with pharmacological inhibition of the proteasome with lactacystin provides support for a role for protein turnover in developmental transitions and paves the way for future mechanistic studies.

Weaknesses:

Most regulatory interpretations remain largely inferential or indirect. The integration identifies correlations between different levels, but direct functional validation is limited/absent. Many of the descriptions should not be interpreted as validated. As highlighted by the authors in this revised version, the mechanistic studies will be part of future work and are beyond the scope of the current work. Of note, the attempt to confirm lactacystin-induced inhibition of proteasomal activity via anti-polyUb immunoblotting did not demonstrate the expected outcome of increase in overall poly-ubiquitylation.Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Reviewer #1 (Public review):

The manuscript by Tassan-Lugrezin et al. confirms the existence of the MICOS complex in the causative agent of malaria Plasmodium falciparum. Prior to this study, only one of the two core MICOS subunits, Mic60, was found by homology search to be encoded in the apicomplexan parasite's genome. This study demonstrates the absence of the other core subunit, Mic10. It also identifies another MICOS subunit, Mic19, which co-migrates with Mic60 in a very large molecular weight complex upon blue native polyacrylamide gel electrophoresis. The authors then demonstrate that expression of both Mic60 and Mic19 is considerably upregulated during the differentiation of P. falciparum from the pathogenic asexual blood stage (ABS) to gametocytes, which correlates with the activation of oxidative phosphorylation during this process. While gene deletion of Mic19, Mic60 and both simultaneously does not affect this transition, the crista are nevertheless deformed. More significantly, crista junctions are significantly reduced, indicating that MICOS serves the same function in apicomplexans as it does in opisthokonts: maintaining crista junctions. Furthermore, the genetic interaction of mic60 and mic19 observed by augmented crista deformation when both are deleted is further evidence of their biochemical interaction, further supporting their similar complexome profiles. This study represents an important contribution to our understanding of MICOS evolution. Furthermore, the study shows that proper cristae formation is not essential for Plasmodium life cycle progression under in vitro conditions. Moreover, mutant gametocytes are still able to mate in the mosquito vector, albeit with lower efficiency.

Strengths:

The study is a result of a lot of technically challenging work in the model Plamsodium. The technically difficult life cycle progression experiments are well performed as far as I can tell. The electron microscopy is very well done and rigorously analyzed to obtain information about crista parameters. In particular, the authors were able to quantify the occurrence and diameter of crista junctions, which is very challenging in small mitochondria with small cristae. Finally, the authors provide convincing support that Mic60 and the newly discovered Mic19 act to shape crista junctions and MICOS can apparently carry out this function without the core subunit Mic10.

Weaknesses:

In its current form, there are conceptual weaknesses. The authors focus on the development of crista from a highly likely acristate state. This is true. But there can be more insight by considering their result in light of discovering the first functioning MICOS complex without one of its two core proteins, Mic10. The surprisingly large size of is also not really considered by the authors. This brings me the second weakness in my opinion. While I think the study represents a lot of work utilizing appropriate and crucial experiments, it seems the Complexome data was not explored enough. This data revealed Mic19, but what other potential subunits are co-migrating with Mic60 and Mic19 that can explain the large size of Plasmodium MICOS? Also, what is the consequence of the loss of Mic60 and Mic19 on the mitoproteome? Perhaps other MICOS subunits can be identified by their depletion in the knockouts versus the parental cell line.

Comments on latest version:

I am reviewing this manuscript again after reviewing it for Reviewers Commons. I appreciate the author's responses to my comments. The new version is improved but, in my opinion, still needs more work.

These revisions are changes to text, interpretations and obtaining more data from existing data or databases. I do still think one experimental control is necessary to substantiate the authors claim about membrane potential.

Reviewer #1 (Public review):

Summary:

In this manuscript, the role of the insulin receptor and the insulin growth factor receptor was investigated in podocytes. Mice, where both receptors were deleted, developed glomerular dysfunction and developed proteinuria and glomerulrosclerosis over several months. Because of concerns about incomplete KO, the authors generated and studied podocyte cell lines where both receptors were deleted. Loss of both receptors was highly deleterious with greater than 50% cell death. To elucidate the mechanism of cell death, the authors performed global proteomics and found that spliceosome proteins were downregulated. They confirmed this directly by using long-read sequencing. These results suggest a novel role for insulin and IGF1R signaling in RNA splicing in podocytes.

This is primarily a descriptive study and no technical concerns are raised. The mechanism of how insulin and IGF1 signaling regulates splicing is not directly addressed but implicates potentially the phosphorylation downstream of these receptors. In the revised manuscript, it is shown that the mouse KO is incomplete potentially explaining the slow onset of renal insufficiency. Direct measurement of GFR and serial serum creatinines might also enhance our understanding of progression of disease, proteinuria is a strong sign of renal injury. An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful but may be masked by defects in other spliceosome genes. As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on.

Significance:

With the GLP1 agonists providing renal protection, there is great interest in understanding the role of insulin and other incretins in kidney cell biology. It is already known that Insulin and IGFR signaling play important roles in other cells of the kidney. So, there is great interest in understanding these pathways in podocytes. The major advance is that these two pathways appear to have a role in RNA metabolism.

Latest comments:

The new reviewer raised two major points, whether the KO effect on splicing is specific to IGF1 and whether the interpretation could be developmental rather than due to splicing. The reviewer raises some important issues but the evidence to suggest that this is specific is data in the literature that IR/IGF signaling is already known to regulate splicing and that splicing defects were not detected in other models that they have analyzed. I agree with the reviewer (and authors) that the incomplete floxing of the genes is a major complication. The point that there could be a developmental defect with mice being born with fewer podocytes and perhaps the authors should caveat this point. The fact that they mice are born with normal function, that renal function can be maintained with up to 80% loss of podocytes suggest that they are likely born with a good number of podocytes and the dysfunction that occurs at 6 months is due to a process, induced by the loss of IR/IGF signaling that is detrimental to the podocyte.

Reviewer #1 (Public review):

Summary:

Johnston and Smith used linear electrode arrays to record from small populations of neurons in the superior colliculus (SC) of monkeys performing a memory-guided saccade (MGS) task. Dimensionality reduction (PCA) was used to reveal low-dimensional subspaces of population activity reflecting the slow drift of neuronal signals during the delay period across a recording session (similar to what they reported for parts of cortex: Cowley et al., 2020). This SC drift was correlated with a similar slow-drift subspace recorded from the prefrontal cortex, and both slow-drift subspaces tended to be associated with changes in arousal (pupil size). These relationships were driven primarily by neurons in superficial layers of the SC, where saccade sensitivity/selectivity is typically reduced. Accordingly, delay-period modulations of both spiking activity and pupil size were independent of saccade-related activity, which was most prevalent in deeper layers of the SC. The authors suggest that these findings provide evidence of a separation of arousal- and motor-related signals. The analysis techniques expand upon the group's previous work and provides useful insight into the power of large-scale neural recordings paired with dimensionality reduction. This is particularly important with the advent of recording technologies which allow for the measurement of spiking activity across hundreds of neurons simultaneously. Together, these results provide a useful framework for comparing how different populations encode signals related to cognition, arousal, and motor output in potentially different subspaces.

Comments on revised manuscript:

The authors have done a very good job of responding to all of the reviewers' concerns.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the weaknesses noted above, which were raised in the previous round of review.]

Summary:

The manuscript investigates how exogenous attention modulates spatial frequency sensitivity within the foveola. Using high-precision eye-tracking and gaze-contingent stimulus control, the authors show that exogenous attention selectively improves contrast sensitivity for low- to mid-range spatial frequencies (4-8 cycles/degree), but not for higher frequencies (12-20 CPD). In contrast, improvements in asymptotic performance at the highest contrast levels occur across all spatial frequencies. These results suggest that, even within the foveola, exogenous attention operates through a mechanism similar to that observed in peripheral vision, preferentially enhancing lower spatial frequencies.

Strengths:

The study shows strong methodological rigor. Eye position was carefully controlled, and the stimulus generation and calibration were highly precise. The authors also situate their work well within the existing literature, providing a clear rationale for examining the fine-grained effects of exogenous attention within the foveola. The combination of high spatial precision, gaze-contingent presentation, and detailed modeling makes this a valuable technical contribution.

Weaknesses:

The manipulation of attention raises some interpretive concerns. Clarifying this issue, together with additional detail about statistics, participant profiles, other methodological elements, and further discussion in relation to oculomotor control in general, could broaden the impact of the findings.

Reviewer #1 (Public review):

Summary:

In this manuscript, Emperador-Melero et al. seek to determine whether recruitment of endocytic machinery to the periactive zone is activity-dependent or tethered to delivery of active zone machinery. They use genetic knockouts and pharmacological block in two model synapses - cultured mouse hippocampal neurons and Drosophila neuromuscular junctions - to determine how well endocytic machinery localizes after chronic inhibition or acute depolarization by super-resolution imaging. They find acute depolarization in both models have minimal to no effect on the localization of endocytic machinery at the periactive zone, suggesting that these proteins are constitutively maintained rather than upregulated in response to evoked activity. Interestingly, chronic inhibition slightly increases endocytic machinery levels, implying a potential homeostatic upregulation in preparation for rebound depolarization. Using genetic knockouts, the authors show that localization of endocytic machinery to periactive zones occurs independently of proper active zone assembly, even in the absence of upstream organizers like Liprin-α.

Overall, they propose that the constitutive deployment of endocytic machinery reflects its critical role in facilitating rapid and reliable membrane internalization during synaptic functions beyond classical endocytosis, such as regulation of the exocytic fusion pore and dense-core vesicle fusion. Although many experiments reveal limited changes in the localization or abundance of endocytic machinery, the findings are thorough, and data substantially supports a model in which endocytic components are organized through a pathway distinct from that of the active zone. This work advances our understanding of synaptic dynamics by supporting a model in which endocytic machinery is constitutively recruited and regulated by distinct upstream organizers compared to active zone proteins. It also highlights the utility of super-resolution imaging across diverse synapse types to uncover functionally conserved elements of synaptic biology.

Strengths:

The study's technical strengths, particularly the use of super-resolution microscopy and rigorous image analyses developed by the group, bolster their findings.

Weaknesses:

One limitation, acknowledged by the authors, is the persistence of spontaneous activity at these synapses, which could still impact the organization of these regions.

Comments on revisions:

The authors have addressed all of my previous comments.

Reviewer #1 (Public review):

Summary:

In their manuscript entitled "Terminal tracheal cells of Drosophila are immune privileged to maintain their Foxo-dependent structural plasticity", Bossen and colleagues determine that the terminal cells of the tracheal system differ from other larval tracheal cells in that they do not typically show an Imd-dependent immune response to fungal and viral infections. Authors reach this conclusion based on the expression of a reporter line, Drs-GFP. The authors speculate that this difference may reflect differential expression of an immune pathway component, as tracheal terminal cells (ttcs) do not respond to forced expression of PRGP-LS. The authors then go on to show that, unlike the other cells of the tracheal system, terminal cells do not express PGRP-LC as reported by a GAL4 enhancer trap. Forced expression of PGRP-LC in terminal cells resulted in reduced branching, cell damage and features of the cell death program. These effects could be suppressed by depletion of AP-1 or Foxo transcription factors. Authors show that Foxo plays a negative role in branching of ttcs, with ectopic branching occurring upon RNAi (or under hypoxic conditions). The authors speculate that immune privilege of the ttcs may have evolved to permit Foxo regulation of ttc branching.

Strengths:

The authors provide compelling genetic data that support their overall conclusions.

Weaknesses:

FC do not appear to express DRS reporter in Figure 1 or elsewhere, raising the question of whether fusion cells are also immune privileged.<br /> Fig 5, TRE_RFP expression, is convincing in wt ttc, but not in ttc o/x PGRP-LCx

Reviewer #3 (Public review):

Agarwal et al identified the small molecule semapimod from a chemical screen of repurposed drugs with specific antimycobacterial activity against a leucine-dependent strain of M. tuberculosis. To better understand the mechanism of action of this repurposed anti-inflammatory drug, the authors used RNA-seq to reveal a leucine-deficient transcriptomic signature from semapimod challenge. The authors then measured a decreased intracellular concentration of leucine after semapimod challenge, suggesting that semapimod disrupts leucine uptake as the primary mechanism of action. Unexpectedly however, resistant mutants raised against semapimod had a mutation in the polyketide synthase gene ppsB that resulted in loss of PDIM synthesis. The authors believe growth inhibition is a consequence of decreased accumulation of leucine as a result of an impaired cell wall and a disrupted, unknown leucine transporter. This study highlights the importance of branched-chain amino acids for M. tuberculosis survival and the chemical genetic interactions between semapimod and ppsB indicate that ppsB is a conditionally essential gene in a medium deplete of leucine.

The conclusions regarding the leucine and PDIM phenotypes are moderately supported by experimental data. The authors do not provide experimental evidence to support a specific link between leucine uptake and impaired PDIM production. Additional work is needed to support these claims and strengthen this mechanism of action.

A mechanistic gap still exists for the model of semapimod antitubercular activity. The basis for semapimod activity is that the leucine auxotroph strain cannot acquire leucine from its environment, and thus the bug ceases to grow. Under normal growth conditions, the leucine auxotroph strain produces PDIM and acquires exogenous leucine through some mechanism (either through a transporter or through PDIM). Semapimod binding to PpsB causes the cell to alter its PDIM profile (lacking experimental for this), and now with the altered PDIM profile the cell cannot acquire enough exogenous leucine to sustain growth (either because the altered PDIM profile interferes with the leucine transporter activity or through PDIM uptake). Acquiring a mutation in ppsB results in cells unable to produce PDIM (some evidence supporting this) but can now acquire enough exogenous leucine to sustain growth. I cannot find the connection between cells that have normal PDIM with normal leucine uptake and cells that are missing PDIM with normal leucine uptake.

(1) The manuscript would benefit from adding additional antibiotic controls to experiments. With the current experimental approaches, it is unclear if these signatures are the result of semapimod specifically or the effect of an antimicrobial agent. Adding additional strains to the 2D TLC experiments could provide more confidence in the absence or modifications of the PDIM band.

(2) The intriguing observation that wild-type H37Rv is resistant to semapimod but the leucine-auxotroph is sensitive should be further explored. If the authors are correct and semapimod does inhibit leucine uptake through a specific transporter or modified PDIM profiles, testing semapimod activity against the leucine-auxotroph in various concentrations of BCAAs could highlight the importance of intracellular leucine. Cells might recover growth in the presence of semapimod treatment if enough leucine is provided in the media and some fraction is able to enter the cell through the impaired PDIM barrier.

Reviewer #1 (Public review):

Summary:

The manuscript by Rayan et al. aims to elucidate the role of RNA as a context-dependent modulator of liquid-liquid phase separation (LLPS), aggregation, and bioactivity of the amyloidogenic peptides PSMα3 and LL-37, motivated by their structural and functional similarities.

Strengths:

The authors combine extensive biophysical characterization with cell-based assays to investigate how RNA differentially regulates peptide aggregation states and associated cytotoxic and antimicrobial functions.

Weaknesses:

While the study addresses an interesting and timely question with potentially broad implications for host-pathogen interactions and amyloid biology, some aspects of the experimental design and data analysis require further clarification and strengthening.

Space

Reviewer #2 (Public review):

Summary:

This study presents a detailed single-cell transcriptomic analysis of the post-natal development of mouse anterior chamber tissues. The dataset is robust, consisting of ~130,000 cells collected across seven time points from early post-natal development to adult. Analysis focused on the development of cells that comprise Schlemm's Canal (SC) and trabecular meshwork (TM).

Comments on revisions:

My critiques have been adequately addressed.

Reviewer #1 (Public review):

Summary:

The authors show that if they generate a weighted multi-conformer ensemble of structural models to fit crystallographic electron density data, the application of statistical mechanical methodologies to that ensemble can provide reasonable estimates of configurational entropy terms related to protein-ligand binding.

Strengths:

A fair range of proteins (12) and ligands (70) is included in the study. The analytical methodologies are well described. Both successful and less successful analytical approaches are discussed, and the latter are frequently as insightful as the former.

Weaknesses:

Compared to the universe of protein-ligand complexes, this dataset is inevitably very limited, so the generality of the observations made here remains speculative. Though a fair range of proteins is studied, the dynamic range in the binding affinity data is limited. The practical utility of the approach is never really commented on.

Reviewer #1 (Public review):

Matsumoto et al. identify Com2, a C2H2-type zinc finger transcription factor not previously linked to sphingolipid metabolism, as a regulator of this pathway in budding yeast. They show that depletion of sphingolipids by myriocin, an inhibitor of serine palmitoyl transferase, increases Com2 expression. This, in turn, promotes the expression of the protein kinase Ypk1 and enhances TORC2-dependent phosphorylation of Ypk1. The authors identify a Com2-binding site in the YPK1 promoter and provide evidence that Com2 functions upstream of Ypk1 to regulate its<br /> expression. They further report that Com2 abundance is controlled by the ubiquitin-proteasome system: degradation of Com2 is inhibited by myriocin treatment and enhanced by phytosphingosine. Mutational analyses of putative phosphorylation and ubiquitination sites support a role for these modifications in regulating Com2 stability. Based on these findings, the authors propose that Com2 acts as a transcriptional regulator of sphingolipid metabolism that responds to sphingolipid levels and promotes Ypk1 expression.

Strengths:

This study provides a valuable finding on the regulation of sphingolipid synthesis by the transcription factor Com2 in budding yeast. The evidence supporting the authors' claims is solid, although additional evidence clarifying the mechanisms and biological significance of ubiquitin-proteasome-mediated degradation of Com2 would strengthen the work. This work will be of interest to microbiologists studying budding yeast.

Weaknesses:

The biological significance of Com2 degradation is not sufficiently clear, which represents an important limitation of the study. It would also be important to determine whether Com2 is actively degraded under normal growth conditions, such as during logarithmic growth in the absence of drug treatment.

Reviewer #1 (Public review):

Summary:

This paper asks how the NK cell receptor KIR2DL4 binds HLA-G and undergoes endocytosis. The authors propose that an allosteric disulfide-bond switch controls whether the receptor is in a ligand-binding or non-binding state, and they support this model using mutagenesis, imaging, mass spectrometry, and structural prediction.

Strengths:

A major strength is the use of diverse, complementary approaches to validate the central claim. The authors combined unbiased random mutagenesis to identify key residues, confocal microscopy to track cellular localization , and mass spectrometry to quantify the redox states of specific disulfide bonds. These methods consistently support a single model: an allosteric disulfide switch. The transition between a Cys10-Cys28 bond and a Cys28-Cys74 bond serves as a functional switch that controls whether the receptor resides at the plasma membrane to bind ligand or remains inactive in endosomes.

Weaknesses:

The core model is interesting, but some of the strongest mechanistic claims still rely heavily on structure prediction rather than direct structural evidence, especially the proposed HLA-G contact surface in Figure 6.

The paper supports an effect of the disulfide state on trafficking and uptake, but the case for direct KIR2DL4-HLA-G binding still feels somewhat indirect. The manuscript itself notes that direct binding had not been previously shown, and the current explanation partly depends on inference about which disulfide state is present.

Most of the main experiments are done in transfected 293T cells, so it is still not fully clear how strongly this mechanism carries over to the more relevant NK-cell setting discussed in the paper.

The cellular evidence for the PDI story is not specific, since it depends a lot on inhibitor and blocking experiments that could affect the broader extracellular redox environment.

Reviewer #1 (Public review):

Summary:

Sun et al. generated germline-specific cKO mice for the Znhit1 gene and examined its effect on male meiosis. The authors found that the loss of Znhit1 affects the transcriptional activation of pachytene. Znhit1 is a subunit of the SRCAP chromatin remodeling complex and a depositor of H2AZ, and in cKO spermatocytes, H2AZ is not deposited into the gene region. The authors claim that this is why the PGA was not activated. These findings provide important insights into the mechanisms of transcriptional regulation during the meiotic prophase.

Strengths:

The authors used samples from their original mouse model, analyzing both the epigenome and the transcriptome in detail using diverse NGS analyses to gain new insights into PGA. The quality of the results appeared excellent.

Comments on revisions:

Sun et al. have responded to each comment with great care and sincerity, and substantial improvements are evident.

In particular, the addition of scRNA-seq data from P35 samples appears to play an important role in supporting the authors' claims.

However, there is still room for improvement in the reanalysis of the data and in the Discussion section.

From the data perspective, for example, the authors state in line 347 of the revised manuscript that "We found that Znhit1-deficient spermatocytes phenocopied abnormal meiotic phenotypes observed in A-MYB mutants." However, the corresponding descriptions in the main text and figure legends are not sufficiently detailed, and therefore do not fully support or substantiate this interpretation. Incorporating a statistical comparison between DEGs in Znhit1-sKO and A-myb KO would likely strengthen this point.

Regarding the overall structure of the Discussion, the connections among delayed DSB repair, MSCI, and PGA regulation via H2A.Z remain somewhat descriptive and difficult to follow. This may reflect a lack of direct evidence linking these processes; however, a more logically structured and clearly articulated Discussion would improve clarity.

Reviewer #3 (Public review):

In this manuscript, the authors use HiC to study the 3D genome of CD14+ CD16+ monocytes from the blood of healthy and those from patients with Alcohol-associated Hepatitis.

Overall, the authors perform a cursory analysis of the HiC data and conclude that there are a large number of changes in 3D genome architecture between healthy and AH patient monocytes. They highlight some specific examples that are linked to changes in gene expression. The analysis is of such a preliminary nature that I would usually expect to see the data from all figures in just one or two figures.

In addition, I have a number of concerns regarding the experimental design and the depth of the analyses performed that I think must be addressed.

(1) There is a myriad of literature that describes the existence of cell-type-specific 3D genome architecture. In this manuscript, there is an assumption by the authors that the CD14+ CD16+ monocytes represent the same population from both the healthy and diseased patients. Therefore, the authors conclude that the differences they see in the HiC data are due to disease-related changes in the equivalent cell types. However, I am concerned that the AH patient monocytes may have differentiated due to their environment so that they are in fact akin to a different cell type and the 3D genome changes they describe reflect this. This is supported by published articles, for example: Dhanda et al., Intermediate Monocytes in Acute Alcoholic Hepatitis Are Functionally Activated and Induce IL-17 Expression in CD4+ T Cells. J Immunol (2019) 203 (12): 3190-3198, in which they show an increased frequency of CD14+ CD16+ intermediate monocytes in AH patients that are functionally distinct.

I suggest that if the authors would like to study the specific effects of AH on 3D genome architecture then they should carefully FACsort the equivalent monocyte populations from the healthy and AH patients.

(2) The analysis of the HiC data is quite preliminary. In the 3D genome field, it is usual to report the different scales of genome architecture, for example, compartments, topologically associated domains (TADs) and loops. I think that reporting this information and how it changes in AH patients in the appropriate cell types would be of great interest to the field.

Comments on revisions:

In the revision the authors did not respond to my concerns which I believe still remain valid and compromise the author's conclusions of AH-specific effects on genome architecture.

Reviewer #1 (Public review):

Summary:

Al Asafen and colleagues here apply a set of scanning fluorescence correlation spectroscopic approaches (Raster Image Correlation Spectroscopy (RICS), cross-correlation RICS, and pair correlation function spectroscopy) to address the nucleo-cytoplasmic kinetics of the Dorsal (Dl) transcription factor in early Drosophila embryos. The Toll/Dl system has long been appreciated to establish dorsal-ventral polarity of the embryo through Toll-dependent control of Dl nuclear localization, and represents one of a handful of model morphogen gradients produced with high enough precision to yield robust biophysical measurements of general transcription factor activity and function. By measurement of GFP-tagged Dl protein, either in wild-type embryos, or in mutant embryos with low/medium/high levels of Toll signaling, the authors report diffusivity of Dl in nuclear and cytoplasmic compartments, as well as the fraction of mobile and immobile Dl, which can be correlated with DNA binding through cross-correlation RICS. A model is presented where Cactus/IkB is implicated in preventing Dl from binding to DNA.

Strengths:

The study uses raster image correlation spectroscopy approaches to measure biophysical components of the Dl gradient in Drosophila embryos. It convincingly demonstrates a positive correlation between Toll pathway activity and the fraction of bound Dl in the nucleus. RICS methodology has widespread potential applications in cell and developmental biology, and this study will contribute to its adoption.

Weaknesses:

The study seeks to test a hypothesis for how the Toll pathway may limit Dl DNA binding in the nucleus. This experiment, while producing initial support for a role of nuclear Cactus, is confounded by co-expression of wild-type Dl, thus limiting the interpretation of the experimental results.

Reviewer #1 (Public review):

Summary:

In this manuscript, Chen, Tu, and Lu focused on how brain-wide dopamine release dynamically changes during sleep/wake state transitions. Using multi-site fiber photometry to monitor DA release, alongside simultaneous EEG and EMG recordings, the authors show distinct DA dynamics during transitions from NREM to WAKE, REM to WAKE, WAKE to NREM, and NREM to REM. Next, they analyze temporal coordination between regions using cross-correlation analysis. Finally, chemogenetic activation of VTA or DRN but not SNc dopamine neurons is shown to promote wakefulness.

Strengths:

The manuscript addresses an interesting question: how brainwide dopamine activity evolves across sleep/wake transitions. The combination of multi-site DA recordings with simultaneous EEG/EMG monitoring is technically sophisticated. The experimental logic is generally clear, and the dataset is rich. The result has several interesting observations.

Weaknesses:

The authors used the GRAB-DA2m sensor to monitor dopamine release. Although DA2m exhibits higher affinity for dopamine compared to NE (around 15-fold difference in EC50 in HEK cell assays), it is still possible that NE contributes to the recorded signals, particularly during sleep/wake transitions when locus coeruleus activity is strongly modulated. Given the widespread and state-dependent dynamics of NE, this potentially needs to be addressed.

Similarly, the chemogenetic experiments rely on CNO to activate hM3Dq-expressing dopamine neurons. However, it is well established that CNO can be converted to clozapine in rodents, and clozapine itself is known to influence sleep/wake. Although the authors included non-hM3Dq-expressing mice as controls, the potential confounding effects of clozapine on sleep regulation remain a concern.

Midbrain dopamine neurons exhibit both tonic and phasic firing patterns. In Figure 1, most reported dopamine transitions appear relatively slow. However, some faster, phasic-like components are observable. For example, in NAc-L during REM-to-WAKE transitions, there are 2 phasic-like decreases between −20 and 0 s. The authors used laser-evoked stimulation experiments in the VTA and DRN and showed that 2 s versus 10 s stimulation produces distinct dopamine kinetics, suggesting that different firing patterns generate distinct DA dynamics. Moreover, the temporal profiles vary not only across regions but also across transitions within the same region. For example, in CeA, the NREM-to-WAKE transition shows a relatively rapid decrease, whereas REM-to-WAKE displays a much slower decline. Similarly, some regions (e.g., NAc-L NREM-to-WAKE, DRN REM-to-WAKE) show faster changes, while others (e.g., mPFC WAKE-to-NREM, VTA NREM-to-WAKE) show slower kinetics. These observations argue against a simple region-specific explanation and instead suggest that distinct firing modes may differentially contribute depending on transition type.

While cross-correlation analysis provides insight into the temporal coordination of DA signals across regions, several limitations should be considered. Sleep/wake transitions are inherently non-stationary events, whereas cross-correlation assumes relatively stable signal properties within the analysis window. This mismatch may bias lag estimates and obscure transient lead-lag relationships. Moreover, the temporal resolution of fiber photometry and the kinetics of genetically encoded DA sensors limit the precision with which timing relationships can be interpreted, particularly for sub-second lags.

In the Introduction, the authors state that they aim to address 'which dopaminergic populations causally drive these patterns.' However, the chemogenetic approach used operates on a relatively slow timescale: CNO-induced activation takes 15-30 minutes to produce effects, and the induced changes are long-lasting. In contrast, the dopamine transitions described in Figure 1 occur on a much faster timescale compared to CNO manipulation. Thus, while chemogenetic activation demonstrates that stimulating VTA or DRN dopamine neurons promotes wakefulness, it does not directly establish that these populations causally drive the rapid transition-related DA dynamics observed in the photometry recordings.

Reviewer #1 (Public review):

Summary:

Pecak et al have deciphered the conformational dynamics of a heterodimeric model ABC transporter, TmrAB, a functional homolog of the human antigen transporter TAP, using single-molecule Forster resonance energy and fluorophores attached to residues at either nucleotide binding domains or periplasmic gate. The analysis not only differentiated ATP-free and bound states but also enabled the real-time monitoring of protein conformational changes, precisely dissecting transport cycles and resolving transient intermediates. This study is absolutely significant in providing and establishing a general pipeline delineating the conformational dynamics in heterodimeric ABC transporters.

Strengths:

The scientific study is very well documented for experimental design, results, and conclusions supported by the experimental data. The authors have determined the conformational dynamics of TmrAB across different ATP concentrations, including physiological ones, and resolved an outward open state and other conformational states consistent with previous cryoEM and DEER studies.

Weaknesses:

The scientific study needs a bit of in-depth analysis with respect to consistency in Kd and its implications on the mechanism.

Reviewer #1 (Public review):

Summary:

This is an interesting study describing intensity changes of lamellipodial Arp2/3 complex incorporation dependent on the substratum the cells are spreading on (PLL vs fibronectin), but also on manipulation of either contractility or osmotic pressure or even external mechanical load exerted onto cells, e.g., by increasing medium viscosity. The authors use quite fancy cell systems for their studies, first of all, a CRISPR-engineered fibroblast cell line in which both endogenous loci of the Arp2/3 complex subunit Arpc2 are tagged with mScarlet, but at the same time, conditionally removable using tamoxifen. These lines, optionally also harboring Pxn-GFP and Lifeact-miRFP670, have previously been described by the authors (Chandra et al, 2022, PMID: 34861242). In addition, they use cells allowing local photoactivation of Rac signalling through a Tiam1 activation module combined with Halo-tagged Arpc2, apparently stably co-expressed in tamoxifen-treated Arpc2-KO fibroblasts. These cells may or may not have been published previously.

Overall, the study provides convincing evidence that Arp2/3 complex accumulation in the lamellipodium negatively correlates with its width and perhaps the mechanical load these actin networks are exposed to at the leading edge membrane, shown initially through allowing cells to spread on substrates in which the formation of integrin-based adhesions is poor (PLL) or stimulated (through fibronectin). In the latter case, lamellipodia are comparably narrow, perhaps reasonably well clutched, and thus feel sufficient counter-force at the leading edge membrane to build a dense, Arp2/3-dependent actin network. Albeit interesting and important to show as the authors did, these results are not entirely surprising given the literature published on actin remodeling in cells in conditions similar to those used by the authors (i.e., on PLL). Thus, the results should be better embedded into the context of this previous literature to more precisely reveal which aspects are new and interesting and which ones are more or less intuitive and expected.

However, the authors also show yet another result, which is quite spectacular indeed, revealing dramatic local protrusion of a Rac-dependent lamellipodium on PLL only in the presence of methylcellulose, but not on PLL alone. Although the authors cannot fully explain the mechanisms causing these results, they are thought-provoking and will certainly stimulate future, relevant research on this topic and new insights. Altogether, I think this is an interesting study that can be shared rapidly, given that the authors provide more experimental detail and transparency concerning their used cell model systems. Aside from a few other suggestions for amendments and corrections, I would also recommend citing classical literature that has provided the basis for the interpretation of the results shown here, as specified below.

Specific criticism and comments:

(1) I feel the paper is interesting for actin remodeling and Arp2/3 complex aficionados, but quite difficult to read and to understand in places for non-experts in the field, so I think the text requires more detailed explanation of specific terms, model systems used, and overall correction of either grammatical or semantic errors, or colloquial language.

(2) In general, I think the characterization of Arp2/3 complex incorporation into the lamellipodia of cells spreading on PLL versus FN is interesting, as it has not been done previously in such a systematic fashion to my knowledge. However, I think the authors could emphasize better how this relates to previously established structural features of actin filament networks, published on PLL. So more than 3 decades ago, Hotchin & Hall published clear evidence that starved fibroblasts can only form focal complexes or adhesions downstream of PDGF or LPA-stimulation if seeded on FN, but not on PLL (see Figure 1 in PMID: 8557752). Around the same time, Flinn and Ridley showed this virtual absence of classical, Rac-dependent focal complexes to be accompanied by the formation of beautiful, broad lamellipodia (see Fig. 1A in PMID: 8743960), which only formed in the absence of excess RhoA activity and thus contractility by the way (see also below). A few years later, Small et al summarized all these phenotypes in a comprehensive review and also showed that cells on PLL (similar to the rapidly migrating keratocytes) combined large, flat lamellipodia with tiny, nascent adhesions scattered throughout these structures (see Figure 2 in PMID: 10047522). These authors also noted that the sole inhibitor-mediated reduction of contractility could switch FN-phenotypes with narrow, ruffling lamellipodia and peripheral focal complexes back to a PLL-type phenotype of broad lamellipodia (see Figure 1 in PMID: 10047522). In the following decade then, different labs (Verkhovsky, Bershadsky, Vavylonis, Watanabe et al) showed beautiful phase contrast or fluorescence movies illustrating that the broad lamellipodial phenotype of cells plated on PLL was accompanied by low frequency membrane ruffling and instead a rapid, continuous rearward flow of continuously assembling actin filament networks, partly also directly shown with actin networks labeled with both LifeAct and Arp2/3 complex subunits (see e.g. PMIDs 18800171 and 22500749). In Alexandrova et al, 2008 (PMID 18800171), authors showed that the formation of adhesions in spreading cells triggers the transition from fast to slow flow (which is of course relevant to the current study and conclusions), whereas Ryan et al, 2012 (PMID 22500749) already established the broad incorporation of actin and Arp2/3 complex into the very broad lamellipodia formed on PLL by Xenopus fibroblasts and the rapid flow of both components from distal to proximal lamellipodial regions. None of these seminal studies has been cited, although they are highly relevant for the interpretation and conclusions of the results presented. I would strongly recommend specifically referring to these studies, as this will actually support the conclusions and interpretations drawn.

(3) On the subject of literature, on the second page of the intro, end of 2nd paragraph, the authors describe Rac signaling to Arp2/3 complex through WRC considered essential for Arp2/3-mediated actin assembly at lamellipodial leading edges, but aside from one of their own papers cite none of the seminal studies by Insall, Scita, Stradal, Rottner, Bogdan labs having published seminal aspects on this pathway.

Considering the rapid F-actin flow in lamellipodia, obviously accompanied by admittedly sparse but continuous Arp2/3 complex incorporation, it is not so surprising that the latter will be obligatory here, and also the accumulation of its prominent activator WRC, as well as the branch stabilizer cortactin. Thus, the data described on page 3 of the Results section could also be framed in the context of all this previously published knowledge, providing a more comprehensive and realistic view of the relevance and novelty of the described data.

(4) In the abstract, the authors state in the context of the force-feedback mechanism established in vitro for the formation of Arp2/3 complex-dependent actin networks that "this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells". In my view, this is not correct, as in their prominent Cell paper, the Sixt laboratory has done exactly that (Mueller et al, 2017, PMID: 18800171). Although Mueller et al have not looked at Arp2/3 complex dynamics as far as I recall, they have still connected the extent and hence intensity of actin networks at the leading edges of keratocyte lamellipodia with the forces exerted onto them, including direct experimental manipulation of those forces. Although the study has been cited in an independent context, this point should be made clear, and the corresponding sentence in the abstract should be amended.

(5) One point that struck me a little bit was the authors' detailed description of cell spreading on PLL and the quite strong variability of Arp2/3 incorporation dependent on the timing after spreading (as for instance the very strong and quite narrow Arp2/3 leading edge intensity at 2 hours post-seeding in Figure 3S2D). In the authors' view, they have worked with a very clean system, as they emphasized to even have eliminated the FN-locus in their cells, excluding the secretion of endogenous FN (PMID: 34861242), but how about ECM components potentially present in serum, such as, for instance, vitronectin? Indeed, it looks like the authors have done all experiments in the presence of 10% serum as far as I can see, although most of the classical PLL-experiments mentioned above have been performed with starved cells in the absence of serum. I think it would generate a more complete picture of the phenotypes and results as compared to the literature if the authors performed a subset of the key experiments on PLL without serum. I don't think the starving of cells as such is important and could be counteracted by simply lamellipodia-inducing growth factors adding into the spreading medium, traditionally perhaps PDGF or EGF (dependent on the receptor distribution of those fibroblasts), but the absence of serum would have two advantages: it would not only exclude any potential impact of serum-containing ECM components, but also alleviate the hyperstimulation of the Rho-pathway through LPA-bound BSA, the major serum-protein, which has previously been shown to counteract the "undisturbed" formation of PLL-type lamellipodia (see Figure 1B in Flinn & Ridley, PMID: 8743960).

(6) Regarding the scanning EM-images shown in the Supplement, currently called Figure 3S2A and -B (in the text erroneously termed Figures 3S1A and-B, see above). I am not sure how representative these individual EM-images of the cell plated on PLL are, given the data of rapid rearward flow of actin and Arp2/3 complex subunits, at least at early stages of spreading. Again, the classical literature on PLL-type lamellipodia and, in particular, previously published movies of such lamellipodia suggest broad lamellipodia with few ruffles, and the opposite with cells plated on FN. So in this context, the scanning EM-data shown on both PLL and FN do neither fit the authors' own data very well nor the literature, and I would recommend making sure that the individual cells selected were (i) correctly annotated and (ii) representative of a specific time point of spreading actually fitting the previously described data.

(7) It also surprised me to see that the authors describe the spreading process on PLL to actually be much slower than on FN (see Figure 3S2C - in the text Figure 3S1C). It is tempting to speculate that this might change if plating the cells in serum-free medium, as traditionally, full spreading and lamellipodia formation downstream of PDGF-stimulation (at least in 3T3 fibroblasts) is described to occur in the range of 10-30 minutes at maximum, and not several hours as shown here. This point could also be considered, or at least discussed.

(8) The movies are of very high quality and beautiful to look at, but it would help the reader to get a bit more information in the legends (like the meaning of the time-stamps, which will display elapsed time in minutes:seconds I assume, but this info is missing from the legends as far as I can see. Also, it would help the reader to better mark in the movies when a specific treatment kicks in. For instance, in movie 10, the legend states treatment starts at 10:00 (minutes:seconds?), but it would help very much if the authors could paste the term "blebbistatin" directly into the movie, beginning with the frame of treatment start.

Reviewer #1 (Public review):

Summary:

Yuan and colleagues present a thorough study of gene activation before and during metamorphosis in sponge larvae, combining in-depth analyses of staged transcriptomes and chromatin accessibility profiling (ATACseq). Amongst several very interesting findings, the study reveals that the acquisition of settlement competence, which arises in response to decreasing light at sunset, is characterized by changes in chromatin accessibility that anticipate strong transcriptional shifts occurring as metamorphosis starts. Another notable finding is a set of transcription factors amongst the genes strongly up-regulated at the onset of metamorphosis. In addition, larvae exposed to constant light, a condition that stalls metamorphosis, were found to activate metabolic pathways that are not normally expressed in swimming larvae. Together, the findings provide a rare level of understanding into how environmental conditions can promote deployment of alternative developmental programs in planktonic larvae.

Strengths:

This is a very comprehensive, well-documented and rigorous study of a phenomenon of wide interest. It will inspire researchers working on other species to look for similar, environmentally-driven "anticipatory" epigenetic mechanisms. It also provides a wealth of detailed information on genes, notably transcription factors, that are candidates for involvement in regulating specific metamorphosis transitions - and beyond. The data presented here are thus undoubtedly a rich and valuable resource.

Weaknesses:

I see no significant weaknesses; however, the documentation of the data is very compressed, with all the findings contained in 4 multi-panel figures with succinct legends. It is not always straightforward to connect the conclusion statements in the text to the figures. Although the relevant data is available in supplementary files, I would appreciate more help in navigating the data to assess the support for key conclusions, if possible, illustrating each text conclusion explicitly in the main figures.

Reviewer #1 (Public review):

Objectives of the study and impact of the work:

The authors of this article primarily aim to reconstruct the evolutionary history of the insect odorant receptor (OR) family, which is responsible for the detection of odorant signals by olfactory neurons. Due to the lack of phylogenetic signal present in the sequences of this multigene family, which evolves very rapidly, phylogenetic analyses have so far never made it possible to precisely retrace how ORs diversified prior to the appearance of present-day insect orders, and what the drivers of this diversification were. For example, one may suspect that the adaptation of ORs to odors emitted by plants constituted a critical step in insect evolution during the "angiosperm terrestrial revolution," which occurred at the end of the Cretaceous, but nothing currently allows this to be asserted.

There are very nice examples, notably in Drosophilids, derived from comparisons between closely related species and documenting mechanisms of OR adaptation to certain signals. However, what the authors attempt to do in this work is to produce a macroevolutionary analysis at the scale of insects as a whole, based almost exclusively on bioinformatic analyses. To do this, they annotated OR genes in about one hundred insect species and developed pipelines for analyzing sequence similarity, structural similarity, and functional similarity, the latter being estimated through a molecular docking approach. An important feature in the evolution of insect ORs is the emergence of a unique co-receptor, called Orco, which appears to be an OR that has lost the ability to bind odorants. In addition to the large-scale bioinformatic analysis, the authors also aim to explore more specifically the factors that favored the emergence of Orco and the selective advantage conferred by the existence of OR-Orco complexes.

Given the importance of odorant receptors in insect biology and in their adaptation to different environments and lifestyles, retracing their evolutionary history is indeed a major question in evolutionary biology. In principle, this type of work therefore has the potential to become a reference in the field and to provide a basis for significant scientific advances.

Major strengths and weaknesses:

The sampling chosen for collecting OR sequences is very impressive, with more than 100 insect families represented, covering most of the major orders. This sampling appears appropriate for the question being addressed. The analysis pipeline used to collect the sequences makes sense, relying on homology-based annotation tools coupled with a structure-based filter. Nevertheless, one can note aberrant numbers of ORs for certain species (much lower than reality), which indicates that the pipeline probably did not function correctly for all genomes. In the absence of a validation step comparing the results with already known OR repertoires, it is difficult to estimate the overall quality of the data. The authors chose to apply a fairly stringent filter on sequence quality (based on predicted 3D structure), which reduces the number from 14,000 to 9,000. This choice seems logical given the subsequent use of these data, but it inevitably leads to data loss. The fact that some OR genes may be missing and that the total number may not be exact for each species is not prohibitive for studying the evolution of the family at a broad scale; however, it calls into question certain results that rely on this total number, such as the correlation between the number of ORs and genome size, lifestyle, and diet.

From the dataset collected, the authors attempted to categorize ORs in several ways, starting with the reconstruction of sequence similarity networks. The approach is interesting, but in the end, the results do not seem to be sufficiently exploited, and it is not obvious what the advantage of this approach is compared with the "classical" phylogenetic approach, which generally fails to reveal homology relationships between ORs from species belonging to different insect orders. Here again, the majority of the clusters identified are "order-specific," and when this is not the case, the authors did not attempt to exploit the results. For example, clusters SeqC26 or SeqC28, which appear to be shared by many insects, are potentially very interesting. It might have been relevant to combine this similarity-based clustering approach with phylogenetic reconstructions within each shared cluster.

The clustering based on structure also leads to the identification of a majority of "order-specific" clusters, but once again, the clusters shared by several orders are not truly exploited, which does not provide new insight into the evolution of ORs. However, the authors highlight a group of ORs in flies that appear to possess an unusual intracellular region. This is interesting, although it is a result more relevant to OR structure than to their evolution. The function of these ORs in Drosophila melanogaster, if it is known, is not discussed.

The analysis of structural diversity then leads the authors to focus on the Orco co-receptors, which are characterized by modifications of the binding pocket and the emergence of an extracellular loop that could explain the loss of the ability to bind odorant molecules. This part, which relies on in vitro experiments, is interesting and constitutes the most striking result of the study, which could in itself have been the subject of a separate manuscript. However, the molecular dynamics modelling does not add anything in the way it is conducted (5 ns is too short).

The rest of the manuscript is based on the prediction of OR response spectra using molecular docking. The work that has been carried out is extremely substantial, and the objective of linking clusters based on sequence similarity or 3D structural similarity with functional categories is entirely relevant. Nevertheless, I see two major problems with this in silico functional analysis:

(1) The docking score threshold used was chosen thoughtfully, which is very good, and according to the calculation performed, should ensure a true positive rate of more than 20%, which is excellent in such a docking analysis. But in the absence of functional validation, this 20% true positive rate is not sufficient to extrapolate OR function as the authors do in the remainder of the manuscript. The risk of error remains too high to compare in such detail the function of ORs from insects with different lifestyles or diets.

(2) The six functional clusters identified are only slightly different from one another, with similar detection of all chemical families except acids and amines (which was expected, given that these families are a priori detected by IRs rather than ORs). This shows that even though the approach is relevant and deserves to be tested, it cannot be used to establish a link between groups/lineages of ORs and response spectra at the scale of insects as a whole. This is reflected in the final analysis by the fact that there is no visible link between sequence or structural clusters and functional clusters. Given the uncertainty surrounding the docking results, the entire subsequent analysis of the relationship between the Binding Breadth Index and ecological variables is highly questionable.

Finally, the evolutionary analysis proposed to conclude that the work suffers from an incorrect interpretation: ORs of non-holometabolous insects cannot be considered equivalent to those of species that existed before the Permian-Triassic extinction. The fact that a locust or a cockroach has more narrowly tuned ORs than holometabolous insects does not mean that this was also the case for ancestral insects. To advance this type of conclusion, it would be necessary to conduct a phylogenetic analysis and reconstruct ancestral states, which is not the case here.

In summary, despite the large number of analyses performed, the authors do not succeed in achieving the stated objective of reconstructing the evolutionary history of insect ORs, and the results obtained do not sufficiently support the conclusions regarding the links between OR repertoires and environment or lifestyle.

Reviewer #1 (Public review):

Summary:

Dancausse et al. investigate behavioral responses to nicotine exposure in Drosophila larvae. They discover that high concentrations of nicotine lead to less movement and twitching, which recover slowly after several hours. Exposure to lower concentrations, however, increases locomotion and leads to hyperactive behavior. The authors also perform pharmacological and genetic manipulations to address the role of dopamine for these behavioral changes. Additionally, they test the role of MB intrinsic neurons by genetic silencing. Both Dopamine and MB manipulations affect responses to nicotine exposure. Finally, they investigate how larvae respond to repeated exposures to nicotine and find that they do not habituate. Additionally, repeated exposure to nicotine leads to a preference towards higher concentrations in a gradient assay.

Strengths:

The authors use rigorous behavioral analysis and discover interesting concentration and experience-dependent effects of nicotine exposure on locomotion in fly larvae, which will be worth investigating in the future to decipher the underlying neural mechanism.

Weaknesses:

As the manuscript currently stands, the results of genetic manipulations are hard to interpret and rather inconclusive. The genetic manipulations have been performed using broadly expressing genetic driver lines, which weakens the conclusions drawn by the authors. Thus, no specific neural populations or brain regions have been discovered, and there is little insight into the underlying neural mechanism.

Based on gradient experiments, the authors suggest that fly larvae could serve as a model organism for addiction. This claim is quite strong, but no control experiments are shown for shorter exposure or a single exposure with a longer resting period before the gradient test. To compare this to addiction-like behaviors, more control experiments should be performed.

The authors should clarify better how experiments were performed in Materials and Methods. Generally, the authors perform novel behavioral analysis, which is not explained in enough detail. The nicotine concentration that has been used for most experiments is this a relevant concentration comparable to other studies? This information would be useful to put into context with other findings.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate how the anterior claustrum may integrate temporally separated task-relevant signals to guide behavior in a delayed escape paradigm. Because in vivo neural recordings from claustrum during this task are extremely limited-comprising single-trial data with small neuronal samples-the authors adopt a modeling-driven approach. They train recurrent neural networks (RNNs) using only behavioral data (escape latency) to reproduce task performance and then analyze the internal dynamics of the trained networks. Within these networks, they identify a subset of units whose activity exhibits persistent responses and strong correlations with behavior, which the authors label as "claustrum-like." Using dimensionality reduction, decoding, and information-theoretic analyses, they argue that these units dynamically integrate conditioned stimulus (CS) and door-opening signals via nonlinear, trajectory-based population dynamics rather than fixed-point attractor states.

To bridge model predictions and biology, the authors complement the modeling with in vitro slice experiments demonstrating recurrent excitatory connectivity and prolonged activity in the anterior claustrum that depends on glutamatergic transmission. They further compare latent neural trajectories derived from previously published in vivo claustrum recordings to those observed in the RNN, reporting qualitative similarities. Based on these results, the authors propose that the claustrum implements temporal signal integration through recurrent excitatory circuitry and dynamic population trajectories, potentially supporting broader theories of integrative brain function.

Strengths:

This study addresses an important and challenging problem: how to infer population-level computation in a brain structure for which in vivo data are sparse and experimentally constrained. The authors are commendably transparent about these limitations and seek to overcome them through a principled modeling framework. The integration of behavioral modeling, RNN analysis, and slice electrophysiology is ambitious and technically sophisticated.

Several aspects stand out as strengths. First, the behavioral RNN is carefully trained and interrogated using a rich set of modern analytical tools, including cross-temporal decoding, trajectory analysis, and partial information decomposition, providing multiple complementary views of network dynamics. Second, the slice experiments convincingly demonstrate recurrent excitatory connectivity in anterior claustrum, lending biological plausibility to the model's reliance on recurrent dynamics. Third, the manuscript is clearly written, logically organized, and conceptually engaging, and it offers a coherent mechanistic hypothesis that could guide future large-scale recording experiments.

Importantly, the work has significant heuristic value: rather than merely fitting data, it attempts to generate testable computational ideas about claustral function in a regime where direct empirical access is currently limited.

Weaknesses:

Despite these strengths, the manuscript suffers from a recurring and substantial conceptual issue: systematic over-interpretation of model-data correspondence. While the modeling results are potentially insightful, the extent to which they are presented as recapitulating real claustral neural mechanisms goes beyond what the available data can support.

A fundamental limitation is that the RNN is trained solely on behavioral output, without being constrained by neural data at either single-unit or population levels. As a result, the internal network dynamics are underdetermined and non-unique. Many distinct internal solutions could plausibly generate identical behavior. However, the manuscript frequently treats the specific internal solution discovered in the RNN as if it were a close approximation of the actual claustrum circuit.

This issue is compounded by the sparse nature of the in vivo data used for comparison. The GPFA-based trajectory analyses rely on pseudo-populations and single-trial recordings, yet are interpreted as evidence for robust population-level dynamics. Because neurons were not recorded simultaneously, the inferred trajectories necessarily lack true population covariance and shared trial-to-trial variability, limiting their interpretability as genuine population dynamics. Similarly, conclusions about trajectory-based versus attractor-based computation are drawn almost exclusively from model analyses and then generalized to the biological system.

Overall, while the modeling framework is appropriate as a hypothesis-generating tool, the manuscript repeatedly crosses the line from proposing plausible mechanisms to asserting explanatory or even causal equivalence between the model and the brain. This undermines the otherwise strong contributions of the work.

Below are several specific points that warrant further clarification or revision:

(1) Tone of model-data correspondence

Numerous statements describe the RNN as "closely mimicking," "recapitulating," or being "nearly identical" to claustral neural dynamics, sometimes extending to claims about causal relationships between neural activity and behavior. Given that neural data were not used to train the model, and that only a small subset of trained networks showed the reported dynamics, these statements should be substantially softened throughout the manuscript. The RNN should be framed as providing one possible computational realization consistent with existing data, not as a close instantiation of the biological circuit.

(2) Non-uniqueness of RNN solutions

The fact that only a small fraction of trained networks exhibited "claustrum-like" clusters deserves deeper discussion. This observation raises the possibility that the identified solution is fragile or highly specific rather than canonical. The authors should explicitly discuss the non-uniqueness of internal solutions in behavior-trained RNNs, including the range of alternative network dynamics that can reproduce the same behavior. In particular, it should be clarified why the specific network exhibiting "claustrum-like" clusters is informative about claustral computation, rather than representing one arbitrary solution among many.

(3) GPFA trajectory comparisons

The qualitative similarity between RNN trajectories and GPFA-derived trajectories from sparse in vivo data is interesting but insufficient to support claims of robustness or population-level structure. Statements suggesting that these patterns are unlikely to arise from noise or random fluctuations are not justified given the single-trial, pseudo-population nature of the data. Either additional quantitative controls should be added, or the interpretation should be substantially tempered.

(4) Scope of functional claims

The discussion connecting the findings to broad theories of claustral function, global workspace, or consciousness extends well beyond the data presented. These speculative links should be clearly labeled as such and significantly reduced in strength and prominence.

The manuscript repeatedly describes the delayed escape task as an "inference-based behavioral paradigm" and states that animals "infer that a value-neutral alternative space is likely to be safer" when the CS is presented in a novel environment. While I appreciate that the US-CS association was established in a different context and that the CS is then presented in a new environment, I am not convinced that the current behavioral evidence uniquely supports an inference interpretation.

First, it is not clear that this task is widely recognized in the literature as a canonical inference task, in the sense of, for example, sensory preconditioning, transitive inference, or model-based inference paradigms. Rather, the observed effect-that CS animals escape faster to a neutral compartment than neutral-CS controls-can be parsimoniously interpreted in terms of generalized threat value, heightened fear/anxiety, or a bias toward avoidance/escape under elevated threat, without requiring an explicit inferential step about the specific safety of the alternative compartment. The fact that no prior training is needed is compatible with flexible generalization, but does not by itself demonstrate inference in a more formal computational sense.

Second, the inference claim becomes central to the manuscript's conceptual framing (e.g., the idea that rsCla supports "inference-based escape"), yet the behavioral analyses presented here and in the cited prior work do not clearly rule out simpler accounts. Clarifying this distinction would help avoid overstating both the inferential nature of the behavior and the specific role of rsCla and the RNN's "claustrum-like" cluster in supporting inference per se, as opposed to more general integration of threat-related signals with an opportunity for escape.

This manuscript presents an interesting and potentially valuable modeling-based framework for thinking about temporal integration in the claustrum, supported by solid slice physiology. However, in its current form, it overstates the degree to which the proposed RNN dynamics reflect actual claustral neural mechanisms. With substantial revision-especially a more cautious interpretation of model-data similarity and a clearer articulation of modeling limitations-the study could make a meaningful contribution as a hypothesis-generating work rather than a definitive mechanistic account.

Comments on revisions:

The authors have carefully addressed the concerns raised in the initial review. In particular, the manuscript has been substantially improved in terms of tone, conceptual clarity, and the interpretation of the modeling results. The revised version now presents a well-balanced and appropriately framed account of the work.

The study offers a compelling and useful hypothesis-generating framework for understanding temporal integration in the claustrum, and I support its publication. As a minor point, given the acknowledged limitations of pseudo-population and single-trial data, it would be preferable to slightly soften a few remaining statements that describe trajectory structure as directly "reflecting" population-level dynamics (e.g., using "consistent with" instead).

Reviewer #1 (Public review):

Summary:

This study presents a novel toolkit for visualizing and manipulating neurotransmitter-specific vesicles in C. elegans neurons, addressing the challenge of tracking neurotransmitter dynamics at the level of individual synapses. The authors engineered endogenously tagged vesicular transporters for glutamate, GABA, acetylcholine, and monoamines, enabling cell-specific labeling while maintaining physiological function. Additionally, they developed conditional knockout strains to disrupt neurotransmitter synthesis in single neurons. The study reveals that over 10% of neurons in C. elegans exhibit co-transmission, with a detailed case study on the ADF sensory neuron, where serotonin and acetylcholine are trafficked in distinct vesicle pools. The approach provides a powerful platform for studying neurotransmitter identity, synaptic architecture, and co-transmission.

Strengths:

(1) This toolkit offers a generalizable framework that can be applied to other model organisms, advancing the ability to investigate synaptic plasticity and neural circuit logic with molecular precision.

(2) The use of this toolkit, the authors uncover molecular heterogeneity at individual synapses, revealing co-transmission in over 10% of neurons, and offers new insights into neurotransmitter trafficking and synaptic plasticity, advancing our understanding of synaptic organization.

Weaknesses:

(1) While the article introduces valuable tools for visualizing neurotransmitter vesicles in vivo, the core techniques are based on previously established methods. The study does not present significant technological breakthroughs, limiting the novelty of the methodological advancements.

(2) The article does not fully explore the potential implications or the underlying mechanisms governing this process, while the discovery of co-transmission in over 10% of neurons is an intriguing finding. A deeper investigation into the functional uniqueness and interactions of neurotransmitters released from individual co-transmitting neurons-perhaps through case study example-would strengthen the study's impact.

Comments on revisions:

I have no further questions regarding this work. I would like to congratulate the authors on the forthcoming publication of their manuscript. This study presents a versatile methodological framework with strong potential to advance the field of neuroscience, particularly in dissecting neural circuit function and neurotransmission dynamics in vivo.

Reviewer #1 (Public review):

Summary:

Ma et al. show that melanoma cells induce an EMT-like state in nearby keratinocytes and that when this state is induced experimentally by Twist-overexpression the resulting alteration in keratinocytes is inhibitory for melanoma invasion. These conclusions are based on experiments in vivo with zebrafish and, in vitro, with human cells. The work is carefully done and provides new insights into the interactions between melanoma cells and their environment.

Strengths:

Use of both zebrafish and human cells adds confidence that findings are relevant to human melanomas while also further demonstrating utility of the zebrafish system for discovering important new features of melanoma biology that could ultimately have clinical impacts. The work also combines a nice suite of approaches including different models for induced melanomagenesis in zebrafish, single cell RNA-sequencing, and more. Some of the final observations are intriguing as well, especially the possibility of EMT induced melanocyte-keratinocyte interactions via Jam3 expression; it will be interesting to see if these is indeed a mechanism for restraining melanoma invasion. The paper is clearly written and the inferences appropriate for the results obtained. Overall the work makes a solid contribution to our understanding of important, but too often neglected, roles of the tumor microenvironment in promoting or inhibiting tumor progression and outcome.

Weaknesses:

No critical weaknesses noted.

Comments on revisions:

The authors have adequately addressed my comments and concerns.

Reviewer #1 (Public review):

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

Strengths:

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

Weaknesses:

One minor weakness is the mismatch between the neural analyses and behavioral data. Neural analyses (i.e. population activity trajectories) indicate a separation of the neural activity associated with each mixture. Given this analysis, one might expect the psychometric curve to have a significantly steeper slope. One potential explanation is the concentration of the stimuli utilized in the mixture discrimination task. The authors utilize equivalent concentrations, rather than intensity matched concentrations. In this case, a single stimulus can (theoretically) dominant the perception of a mixture resulting in a biased behavioral response despite accurate concentration coding. Given the difficulty of iso-intensity matching concentrations, this concern is not paramount.

Reviewer #1 (Public review):

Summary:

This paper characterises the physiological and computational underpinnings of the accumulation of intermittent glimpses of sensory evidence, with a focus on the centroparietal positivity and motor beta lateralization. The main finding is that the centroparietal positivity builds up during evidence accumulation but falls back to baseline during gaps, while motor beta lateralization maintains a continuous a sustained representation throughout the gap and until response.

Strengths:

- Elegant combination of electroencephalography and computational modelling.

- Innovative task design, including parametric manipulation of gap duration.

- The authors describe results of two separate experiments, with very similar results, in effect providing an internal replication.

Weaknesses:

- A direct characterization of how the centroparietal positivity and motor beta lateralization interact is missing, which limits the novelty. In their reply to reviewers, the authors argue that the signal-to-noise ratio of EEG signals is insufficient for such analyses at the single-trial level. If so, a binned or trial-averaged approach could still be attempted.

- An exhaustive characterisation of sensors and frequency bands is also missing. In their reply to reviewers, the authors suggest that this would detract from their hypothesis-driven focus. I disagree: the main hypothesis and figures could remain centred on the centroparietal positivity and motor beta lateralization, with a more comprehensive mapping of sensors and frequencies placed in supplementary material. Since the purpose of the paper is to examine EEG-based decision signals in a novel behavioural context, a broader characterisation of the underlying EEG landscape would seem appropriate.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

Overall, I think the authors made many improvements to their manuscript. There are, however, still a number of concerns that first need to be addressed, since it is still not currently possible to fully evaluate the analyses, results, and conclusions presented in the paper. I list these points below:

(1) The authors still use causal language where they must not use causal language. This is true for many places in the manuscript; I am highlighting here just a few places, but the authors nevertheless have to go carefully through the whole manuscript to change these instances.

Some examples:

(a) In response to my comment (1) in the previous round, where the authors adjusted their text, the authors still use causal language in their last sentence "... procrastination behavior has been observed to impair general health..." Unless the cited study truly allowed causal conclusions, the causal language should be removed here as well.

(b) The authors still make (causal) claims about the involvement of self-control in their observed results. To reiterate from the previous round of revisions: The authors cannot make any strong claims about the role of self-control processes because they do not directly measure self-control nor do they directly manipulate self-control or have a design that would rule out alternative mechanisms other than self-control. Therefore, their claims about self-control have to be toned down. It is laudable that the authors have added a statement towards the end of their discussion about not being able to make strong conclusions about the role of self-control. But the authors need to use similar careful wording not just at the end of the discussion but throughout the manuscript.

(i) In the abstract, the authors use the formulation "...conceptualized roles of self-control on procrastination..." -- this wording is still too strong, suggesting that you actually studied self-control.

(ii) In the introduction (page 4, lines162-169), the way the authors formulate these sentences suggests that they directly measured self-control. Again, the authors need to make it explicit that they are not directly measuring self-control but its hypothesized down-stream consequences on valuations/behavior.

(iii) In the discussion, for example, on page 11, lines 555 and following, the authors write:

"One major contribution this study has made is to disentangle the neurocognitive mechanism of procrastination by demonstrating that self-control could increase task-outcome value so as to reduce procrastination."

Again, please be aware that you are NOT demonstrating that self-control does anything, since you only measure procrastination rates, outcome values, and task aversiveness. It is possible that mechanisms other than self-control might be relevant for this. Perhaps neuromodulation directly increases outcome values, without involvement of self-control processes. You simply cannot know that and therefore you cannot make those claims in the form that you are making them. You can write that the observed results are consistent with the idea that neuromodulation might have had an effect on self-control and this in turn might have affected outcome values. But you also need to make it explicit that, to substantiate these claims, you would need more direct evidence that indeed self-control was involved. These more careful formulations would not at all reduce the value of your work, but indeed they would rather demonstrate your carefulness in interpreting the results you obtained.

(2) I am still puzzled by the power analysis. In the text, you write that a sample size of 18 participants (i.e., 9 per group) would be sufficient to achieve 80% power. I still feel this seems far too optimistic and hard to believe, but that is not my point here. While in the text, you write that you need 18 participants, the G*power output seems to suggest a sample size of 34, not 18. Why this contradiction? Or is it not contradictory? If it is not, then please explain it more fully.

(3) I have several comments about the mixed-effects analysis.

First of all, I want to thank the authors for adding more details, things have become much clearer now. However, I still have a few questions and comments related to these analyses:

(a) The variable Emotions was within-subjects, as far as I understood. Accordingly, Emotions should most likely be modelled with random slopes varying over participants (in addition to being modelled as a fixed effect).

(b) The analyses still cannot fully be evaluated as I cannot access the scripts and data. The authors mention that the scripts and data should be available via a link they provide (https://doi.org/10.57760/sciencedb.35140). However, when I try to access these materials via this link, no page opens; it seems the link is dead?

(c) What are the results and conclusions if you do not include the covariates of no interest? I.e., please re-run your main models without age, gender, SES, Emotions.

(d) The authors mention that they use GLMMs, which would suggest generalized mixed-effects models, but they do not describe what family/distribution they used. Since they mention lmerTest and seem to report F-tests, my guess is that they used Gaussian models. However, both their DVs (procrastination rates and their ratings) are bounded variables and at least procrastination rates hit the lower boundary. That can mean that their analyses suffer from inflated Type 1 and/or Type 2 rates. Therefore, please repeat the analyses with an appropriate generalized mixed-effects model (perhaps a beta regression type of model?).

(e) When reporting the results of the mixed-effects models, the authors report the regression coefficient, standard error, DFs and p value, but not the actual test statistic. Please add the information about the test statistic and report all degrees of freedom (in case of F tests that would be the degrees of freedom of the test and the residual degrees of freedom).

(f) Thank you for adding the analysis where you remove the last two sessions. But currently you present them in the manuscript without explaining/motivating why you do this. Please add this motivation, as otherwise it will be puzzling for the reader why you conduct these analyses.

(4) Mediation analysis

In your manuscript, you present some mediation analyses. Please be aware that such mediation analyses cannot establish causality and they suffer from extremely high Type 1 error rates (see, e.g., https://datacolada.org/103).

My suggestion would be to completely remove all mediation analyses. However, if you want to keep them, then you need to be extremely careful in how you present the results. You need to explicitly mention that you cannot derive any causal conclusions from them and that simulation studies have shown that such mediation analyses suffer from extremely high Type 1 errors.

As an example (but the mediation results are mentioned in several places, for example, also in the abstract):

On page 10, lines 501-503: What you can causally conclude is that neuromodulation affects your measured variables (outcome values, procrastination rates, task aversiveness), but you cannot conclude that the effect of neuromodulation on procrastination rates causally operates via outcome values. Thus, please adjust the formulation accordingly. The same applies to the mediation section that follows right afterwards (page 10, lines 505-522).

(5) In the introduction, the authors introduce several theoretical procrastination frameworks (TMT, mood repair, TDM). Do the results of the current paper help to decide which framework might be the most appropriate, at least for the authors data set? It might be of interest to address this explicitly.

(6) The language is sometimes hard to understand and seems in quite some places grammatically incorrect. Thus, I think the paper would profit very much from thorough English proofreading.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

This study addresses the important question of how top-down cognitive processes affect tactile perception in autism - specifically, in the Fmr1-/y genetic mouse model of autism. Using a 2AFC tactile task in behaving mice, the study investigated multiple aspects of perceptual processing, including perceptual learning, stimulus categorization and discrimination, as well as the influence of prior experience and attention.

Strengths:

The experiments seem well performed, with interesting results. Thus, this study can/will advance our understanding of atypical tactile perception and its relation to cognitive factors in autism.

Reviewer #1 (Public review):

Summary:

It is well known that neurons in the medial prefrontal cortex (mPFC) are involved in higher cognitive functions such as executive planning, motivational processing and internal state mediated decision-making. These internal states often correlate with the emotional states of the brain. While several studies point to the role of mPFC in regulating behavior based on such emotional states, the diversity of information processing in its sub-populations remains a less explored territory. In this study, the authors try to address this gap by identifying and characterizing some of these sub-populations in mice using a combination of projection-specific imaging, function-based tagging of neurons, multiple behavioral assays and ex-vivo patch clamp recordings.

Strengths:

The authors targeted mPFC projections to the nucleus accumbens (NAc) and basolateral amygdala (BLA). Using the open field task (OFT), the authors identified four relevant behavioral states as well as neurons active while the animal was in the center region ("center-ON neurons"). By characterizing single unit activity and using dimensionality reduction, the authors show differentiated coding of behavioral events at both the projection and functional levels. They further substantiate this effect by showing higher sensitivity of mPFC-BLA center-ON neurons during time spent in the open arms of the elevated plus maze (EPM). The authors then pivoted to the three-chamber social interaction (SI) assay to show the different subsets of neurons encode preference of social stimulus over non-social. This reveals an interesting diversity in the function of these sub-populations on multiple levels. Lastly, the authors used the tube test as a manipulation of the anxiety state of mice and compared behavioral differences before/after in the OFT and social interaction tasks. This experiment revealed that "losers" of the tube test spend less time in the center of the open field while "winners" show a stronger preference for the familiar mouse over the object. Using patch-clamp experiments, the authors also found that "winners" exhibit stronger synaptic transmission in the mPFC-NAc projection while "losers" exhibit stronger synaptic transmission in the mPFC-BLA projection. Given the popularity of the tube test assay in rank determination, this provides useful insights into possible effects on anxiety levels and synaptic plasticity. Overall, the many experiments performed by the authors reveal interesting differences in mPFC neurons relative to their involvement in high or low anxiety behaviors, social preference and social rank.

Weaknesses:

The authors have addressed all comments.

Reviewer #1 (Public review):

Summary:

This manuscript examines the evolution of molluscan shells using single-cell analyses of the adult mantle of Crassostrea gigas and compares these data with previous datasets from embryonic and larval stages of this species and other spiralians. The authors provide support for a scenario in which secretory cells are broadly conserved across spiralians, and the incorporation of lineage-restricted genes contributes to the evolution of molluscan shells.

Strengths:

High-quality datasets for mantle tissue in Crassostrea gigas and thorough comparisons with existing datasets for this species and other spiralians. Balanced discussion.

Weaknesses:

No major weaknesses. The analyses follow fairly standard approaches in the field that have been previously applied and developed in similar systems.

Reviewer #1 (Public review):

This paper reports a previously unrecognized mechanism by which platelets compact fibrin fibers during clot retraction. Rather than simply pulling on fibers, the authors propose that platelets generate swirling motions that wind and loop fibrin into dense structures.

While the results are intriguing, the underlying physical mechanism remains unexplained. In particular, it is unclear how platelets generate swirling motion capable of inducing fibrin coiling, especially when suspended in 3d fibrin mesh. This raises concerns about the conclusions. Also, does fibrin have inherent chirality or structural asymmetry that could promote coiling independently of platelet activity? Furthermore, platelet retraction typically involves platelet aggregation rather than isolated cells, and it is unclear how fibrin coiling would proceed in clustered platelets.

DOI: 10.3389/fcimb.2026.1792069

Resource: (ZFIN Cat# ZDB-GENO-130722-1,RRID:ZFIN_ZDB-GENO-130722-1)

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-GENO-130722-1

What is this?

Reviewer #1 (Public review):

Summary:

The authors introduce the Training Village (TV), an open-source and modular system that allows group-housed rodents to live in enriched home cages while individually accessing a single shared operant box for automated cognitive training. The paper reported the animals' activity both in the operant box and in the home cages, which is novel.

Strengths:

A major strength of the work is that it moves beyond a proof-of-concept and demonstrates sustained box usage, long-term trial accumulation, and compatibility with different task designs.

(1) The platform provided a technical contribution in rodent cognitive neuroscience: obtaining large amounts of behavioral data from complex tasks while reducing experimenter intervention and preserving social housing.

(2) The authors demonstrate that the system can sustain prolonged task engagement (up to 12 months), maintain efficient use of a single operant box.

(3) The manuscript opens interesting opportunities for studying behavior outside standard session-based training. Because animals self-initiate training while remaining in a group-housed setting, the platform has the potential to illuminate relationships among motivation, spontaneous activity, and task engagement that are hard to access in conventional paradigms.

Weaknesses:

(1) One area that would benefit from further clarification is the manuscript's core advance relative to prior automated group-housed training systems, particularly Mouse Academy (Qiao et al., 2018). The authors listed some advantages in the Discussion section; however, those were some minor engineering improvements, and what is more interesting is the scientific question or results that can be asked or obtained from this study. The current study clearly presents a functional and carefully documented platform, but it would help the reader if the authors more explicitly distinguished the present system from earlier related approaches, both in terms of system design and in terms of experimental validation.

(2) At the system level, several of the claimed advantages could be supported more directly with quantitative data. For example, if the double-detection corridor and alarm system are important distinguishing features, it would be valuable to report measures such as detection accuracy, missed detections, co-entry failures, alarm frequency, and the degree of manual intervention required in practice. Similarly, the welfare-related arguments are plausible and important, but would be strengthened by more direct evidence, such as longitudinal body weight data, water intake, or comparison with group-housed no-task controls.

(3) At the experimental level, the manuscript would also benefit from a more detailed characterization of training performance. Although three behavioral paradigms are presented, the data currently shown provide a stronger demonstration of feasibility than of training optimization. For a study focused on automated cognitive training, it would be critical to include more information on learning speed, progression across stages, success and failure rates, and variability across animals. Along the same lines, the comparison with manual training is a useful addition, but a broader benchmark including learning curves, time to criterion, and between-animal variability would make the practical value of the system easier to assess.

(4) The authors claimed that they conducted 3 complex cognitive tasks (3AFC, 2AFC, 2AB) in their setup. However, those 3 tasks are quite basic for rodents and have been demonstrated in many studies, especially comparing tasks implemented in Yu et al., eLife 2025. Therefore, lowering this 'complex' statement is necessary.

(5) The authors claimed that they have successfully implemented the so-called hybrid mode, but it is only briefly described and not supported by citations or data. Since this may be one of the most broadly applicable use cases of the platform, a more detailed explanation of how the system can be integrated with recording workflows would strengthen the manuscript.

(6) The manuscript highlights the opportunity to relate task behavior to home-cage activity and to study individualized behavioral patterns. To better support these aspects, it would be helpful to include more subject-level analyses, rather than relying predominantly on population averages, or alternatively to discuss in more concrete terms which features of the dataset may be especially informative for studying individuality. More generally, the manuscript would benefit from clarifying whether different parameter settings within this group-housed framework may be better suited for maximizing training efficiency versus preserving more naturalistic or socially modulated behavior, and what the implications of these choices may be for interpretation.

(7) In Table S1, 'Touch screen' is task-specific and is not necessarily a metric. 'Testing outside home cage' is also not necessarily an advantage (please clarify if it is). Many other systems implemented different levels of 'Alarm system', which is not reflected in the table.

(8) Table S3 shows important data that help the reader to evaluate the paper's work, thus is deserved to move to the main text.

Reviewer #1 (Public review):

Summary:

This paper examines whether humans use protracted temporal integration in a noise-free, deferred-response contrast discrimination task, using a covert evidence-duration manipulation combined with EEG (SSVEP, CPP, Mu/Beta). The key finding is that evidence for protracted sampling is behaviorally and neurally supported, but even joint CPP + behaviour fitting cannot fully discriminate a standard integration (DDM) model from a novel "extremum-flagging" non-integration model. The paper is transparent about this outcome.

Strengths:

This is a well-conducted and well-written study that makes a genuine contribution to the perceptual decision-making literature by introducing a clean experimental design for probing temporal integration without participants adapting their strategy and demonstrating for the first time that a non-integration model (extremum-flagging) can replicate CPP waveform dynamics that have long been considered hallmarks of evidence accumulation. The transparent treatment of equivocal modelling outcomes is commendable.

Weaknesses:

My main concerns relate to statistical power, the under-specification of the and the extremum-flagging mechanism. Addressing these would greatly strengthen the paper.

(1) The sample of 16 participants (15, after the exclusion of one participant) is described as "close to similar EEG studies" with no formal power analysis. Given that the paper's core claim rests on subtle quantitative differences between two model classes - differences that are, by the authors' own admission, not sufficient to declare a winner - even a modest increase in sample size might yield a more decisive outcome. At a minimum, the authors should report a sensitivity analysis or post-hoc power calculation to indicate what effect sizes the current N could reliably detect, particularly for the rmANOVA comparisons and the neural constraint fitting.

(2) The Extremum-flagging model is the paper's most novel contribution, yet its physiological basis is underspecified. The model posits that each decision-terminating bound-crossing triggers a stereotyped, half-sine-shaped centroparietal signal, but no neural circuit or computational mechanism is proposed for how the brain could detect the first bound-crossing event in a non-accumulating evidence stream or generate a temporally precise, fixed-amplitude signal in response. Possible connections to P3b theories of context updating and response facilitation are acknowledged, but these are vague functional descriptions rather than mechanistic accounts. I think the discussion should engage more directly with potential neural substrates that could generate this flagging signal, and whether these are consistent with the known generators of the CPP/P3b. Without this, the extremum-flagging model risks being viewed as a mathematical convenience rather than a biologically plausible alternative.

(3) The Integration model at the preferred neural weighting estimates a high-to-low contrast drift rate ratio of 8.7, whereas the empirical Mu/Beta lateralization slopes suggest a ratio of approximately 3.5. The authors attribute this discrepancy to the nonlinear contrast response function of early visual cortex and the salience of the high-contrast evidence onset, but these explanations are speculative. These outcomes are arguably the most quantitatively damaging result for the integration model, so they deserve more than a brief discussion. I would recommend that the authors (a) estimate what range of contrast response nonlinearities would be required to close this gap, (b) test whether an alternative drift rate parameterization (e.g., scaling drift rates directly by SSVEP amplitude rather than contrast) reduces the discrepancy, or (c) be more explicit about treating this as a point against the Integration account.

(4) The sensitivity analysis over neural constraint weightings (w = 0.1 to 1000) is thoughtful, but the paper ultimately acknowledges that the preferred weighting is w=10, chosen because it achieves "a good fit to CPP dynamics without substantively sacrificing behavioral fit" - a qualitative criterion. No principled statistical framework is used to select the optimal weighting or to compare models at a given weighting. A Bayesian model comparison could provide a more formal framework for combining behavioral and neural fit components, and would allow a clearer statement about the relative posterior probability of each model.

Reviewer #1 (Public review):

Vasilevskaya and Keller test different models of cortical function through the lens of predictive processing, a powerful framework for the brain to learn and predict the statistics of the world via generative internal models. The authors use a clever combination of behavioral perturbations in closed-loop and open-loop visuomotor virtual reality assays, a paradigm the Keller lab pioneered and used effectively in the past decade, in conjunction with two-photon imaging of neuronal calcium responses and targeted optogenetic perturbations of activity. They specifically put to test proposed hierarchical vs. non-hierarchical circuit implementations of predictive processing by analyzing the logic of inter-lamina interactions (superficial vs. deep; L2/3 vs. L5/6).

The authors conclude that both versions of predictive processing architectures they analyze are likely invalid, and instead formulate an alternative novel model of cortical function based on a recently developed machine learning algorithm for self-supervised learning (joint embeddings of predictive architectures, JEPA) and its further refinements. JEPA borrows elements from predictive processing, engaging two encoder networks and training the output of one network to predict the output of the other. In their new model of cortical computations, prediction error neurons in L2/3 compare the deep layers (L5/6) activity, which is taken as a teaching signal, to a local, L2/3 prediction of this latent representation.

Specifically, the authors build on their previous work and reports from other groups that different sets of L2/3 neurons compute positive prediction errors (fire when sensory stimuli appear unexpectedly with respect to the movements of the animal; e.g., grating onsets in the absence of locomotion) and respectively negative prediction errors (fire when sensory stimuli are absent, while the brain expected them to be present; e.g. mice locomote but visual flow is suddenly halted - visuomotor mismatches). These L2/3 positive and negative prediction error neurons exchange messages with neurons in the deeper cortical layers that, the authors propose, build an internal representation (R) of the sensory stimuli given the animals' movements.

In the hierarchical model, internal representation neurons (R) are supposed to act as a teaching signal for both types of prediction error neurons; the output of the positive prediction error neurons is assumed to suppress activity of R such that the error between the teaching signal and the prediction is minimized; similarly, in the non-hierarchical version, R serves as a prediction for the prediction error neurons, and in turn it receives excitatory drive from the positive prediction error neurons and negative input from the negative prediction error neurons.

The authors find that the functional impact of L5 neurons on L2/3 neurons is not compatible with the non-hierarchical architecture they and other groups proposed, but rather in accordance with the hierarchical model. At the same time, the functional impact of L2/3 neurons (positive vs. negative prediction error neurons) on L5 neurons (internal representation) appears not compatible with the hierarchical model, but rather in accordance with the non-hierarchical implementation.

They further hypothesize that L2/3 prediction error neurons don't use sensory input, but rather the L5 activity as a teaching signal, and test it using perturbations (halts) of optogenetic stimulation of L5 neurons coupled with locomotion (Figure 7).

All in all, the question is topical, and the new model addresses a decades-long quest to develop a unifying model of cortical function. The findings reported here transform our understanding of cortical computations, opening new, exciting avenues for future investigation. The experimental design and execution are rigorous; the arguments are clearly laid out (in spite of ample potential for confusion given the numerous loops and sign flips). These include a discussion of why the non-hierarchical model proposed by the same group does not hold, as well as potential caveats in interpreting the results and novel testable proposed experiments emerging from the JEPA-like model.

I have several questions about the interpretations of some of the claims and suggestions for potential additional experiments and analyses.

(1) Some of the pieces of the puzzle remain to be identified and demonstrated: the existence of internal representation neurons in L2/3 and ascertaining that the L5/6 neurons analyzed function indeed as internal representation neurons. The authors find that stimulation of L2/3 positive prediction error neurons enhances activity of L5 neurons...If L5 neurons hold a latent representation that serves as a teaching signal for L2/3 neurons (as the authors posit), wouldn't one expect that the input they receive from the positive prediction neurons be suppressive, such that the error is further minimized?

(2) Do the authors envision any specific differences between the representations of the two encoder networks posited to exist in L2/3 and L5 in the JEPA-like implementation? Are they synchronous/offset in their temporal representations, or any other features?

(3) Where is the prediction coming from onto L2/3 neurons? Is it emerging locally in L2/3 from the putative internal representation neurons, or is it long-range - as work from the authors previously proposed? Or a mix of both?

(4) What is the role of the indiscriminate L4 input that appears to enhance activity of both positive and negative prediction error neurons in L2/3?

(5) Does Figure 7D change in a meaningful manner if the authors plot the correlation between optomotor mismatch response and visuomotor mismatch response specifically for the negative prediction error neurons in L2/3 (Adamts-2) rather than for all L2/3 cells sampled?

(6) Do the optomotor mismatch responses in L2/3 neurons depend on how long the closed-loop coupling of optogenetic stimulation of Tlx3 L5 neurons and locomotion speed has been in place for?

Reviewer #1 (Public review):

Summary:

This manuscript investigates mutations and expression patterns of zinc finger proteins in Kenyan breast cancer patients. Whole-exome sequencing and RNA-seq were performed on 23 breast cancer samples alongside matched normal tissues.

Strengths:

Whole-exome sequencing and RNA-seq were performed on 23 breast cancer samples alongside matched normal tissues in Kenyan breast cancer patients. The authors identified mutations in ZNF217, ZNF703, and ZNF750.

Weaknesses:

(1) Research scope:

The results primarily focus on mutations in ZNF217, ZNF703, and ZNF750, with limited correlation analyses between mutations and gene expression. The rationale for focusing only on these genes is unclear. Given the availability of large breast cancer cohorts such as TCGA and METABRIC, the authors should compare their mutation profiles with these datasets. Beyond European and U.S. cohorts, sequencing data from multiple countries, including a recent Nigerian breast cancer study (doi: 10.1038/s41467-021-27079-w), should also be considered. Since whole-exome sequencing was performed, it is unclear why only four genes were highlighted, and why comparisons to previous literature were not included.

(2) Language and Style Issues

There are many typos and clear errors in the main text (e.g. (ref)).

Additionally, several statements read unnaturally. For example:

"Investigators uncovered 170 mutations ..." should instead be phrased as "We identified 170 mutations ...."

"The research team ..." should be rephrased as "Our team ...."

(3) Methods and Data Analysis Details

The methods section is vague, with general descriptions rather than specific details of data processing and analysis. The authors should provide:

(a) Parameters used for trimming, mapping, and variant calling (rather than referencing another paper such as Tang et al. 2023).

(b) Statistical methods for somatic mutation/SNP detection.

(c) Details of RNA purification and RNA-seq library preparation.

Without these details, the reproducibility of the study is limited.

(4) Data Reporting

This study has the potential to provide a valuable resource for the field. However, data-sharing plans are unclear. The authors should:

a) Deposit sequencing data in a public repository.

b) Provide supplementary tables listing all detected mutations and all differentially expressed genes (DEGs).

c) Clarify whether raw or adjusted p-values were used for DEG analysis.

d) Perform DEG analyses stratified by breast cancer subtypes, since differential expression was observed by HER2 status, and some zinc finger proteins are known to be enriched in luminal subtypes.

(5) Mutation Analysis

Visualizations of mutation distribution across protein domains would greatly strengthen interpretation. Comparing mutation distribution and frequency with published datasets would also contextualize the findings.

Comments on revisions:

The revised manuscript hasn't addressed any of these concerns. Careful proofreading is recommended, even if the authors do not intend to make further modifications to the manuscript.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

Here, the authors have addressed the recruitment and firing patterns of motor units (MUs) from the long and lateral heads of triceps in the mouse. They used their newly developed Myomatrix arrays to record from these muscles during treadmill locomotion at different speeds, and they used template-based spike sorting (Kilosort) to extract units. Between MUs from the two heads, the authors observe differences in their firing rates, recruitment probability, phase of activation within the locomotor cycle and interspike interval patterning. Examining different walking speeds, the authors find increases in both recruitment probability and firing rates as speed increases. The authors also observed differences in the relation between recruitment and the angle of elbow extension between motor units from each head. These differences indicate meaningful variation between motor units within and across motor pools, and may reflect the somewhat distinct joint actions of the two heads of triceps.

Strengths:

The extraction of MU spike timing for many individual units is an exciting new method that has great promise for exposing the fine detail in muscle activation and its control by the motor system. In particular, the methods developed by the authors for this purpose seem to be the only way to reliably resolve single MUs in the mouse, as the methods used previously in humans and in monkeys (e.g. Marshall et al. Nature Neuroscience, 2022) do not seem readily adaptable for use in rodents.

The paper provides a number of interesting observations. There are signs of interesting differences in MU activation profiles for individual muscles here, consistent with those shown by Marshall et al. It is also nice to see fine scale differences in the activation of different muscle heads, which could relate to their partially distinct functions. The mouse offers greater opportunities for understanding the control of these distinct functions, compared to the other organisms in which functional differences between heads have previously been described.

The Discussion is very thorough, providing a very nice recounting of a great deal of relevant previous results.

Reviewer #1 (Public review):

Summary:

The manuscript by Bohra et al. describes the indirect effects of ligand-dependent gene activation on neighboring non-target genes. The authors utilized single-molecule RNA-FISH (targeting both mature and intronic regions), 4C-seq, and enhancer deletions to demonstrate that the non-enhancer-targeted gene TFF3, located in the same TAD as the target gene TFF1, alters its expression when TFF1 expression declines at the end of the estrogen signaling peak. Since the enhancer does not loop with TFF3, the authors conclude that mechanisms other than estrogen receptor or enhancer-driven induction are responsible for TFF3 expression. Moreover, ERα intensity correlations show that both high and low levels of ERα are unfavorable for TFF1 expression. The ERa level correlations are further supported by overexpression of GFP-ERa. The authors conclude that transcriptional machinery used by TFF1 for its acute activation can negatively impact the TFF3 at peak of signaling but once, the condensate dissolves, TFF3 benefits from it for its low expression.

Strengths:

The findings are indeed intriguing. The authors have maintained appropriate experimental controls, and their conclusions are well-supported by the data.

Weaknesses:

There are some major and minor concerns that related to approach, data presentation and discussion. But the authors have greatly improved the manuscript during the revision work.

Comments on latest version:

The authors have done a lot of work for the revision. The manuscript has been greatly improved.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors present comprehensive experimental observations and a theoretical framework to explain the heterogeneous behaviour of sarcomeres in cardiomyocytes. They show that a stochastic component exists in their contractile activity, which may act as a feedback mechanism regulating physiological function.

Strengths:

Experiments and data analysis are robust and valid. The rigorous statistical analysis and unbiased methods enable the authors to draw well-supported conclusions that go beyond the existing literature. Their outcomes inform about cellular activity at the individual level and the authors explain how the transient dynamics of single sarcomeres are governed by a force-velocity relationship and lead to the complex contractile patterns. The similarity of the results to the study cited in [24] demonstrates the validity of the in vitro setup for answering these questions and the feasibility of such in-vitro systems to extend our knowledge of out-of-equilibrium dynamics in cardiac cells.

Very interesting the suggestion that the interplay between intrinsic fluctuations and the dynamic instability are part of a feedback mechanism for maintaining structural and functional homeostasis.

The addition of the theoretical model and the new text of the manuscript improves the clarity of the study.

Reviewer #1 (Public review):

Actin filaments and their kinetics have been the subject of extensive research, with several models for filament length control already existing in the literature. The work by Rosario et al. focuses instead on bundle length dynamics and how their fluctuations can inform us on the underlying kinetics. Surprisingly, the authors show that irrespective of the details, typical "balance point" models for filament kinetics give the wrong scaling of bundle length variance with mean length compared to experiments. Instead, the authors show that if one considers a bundle made of several individual filaments, length control for the bundle naturally emerges even in the absence of such a mechanism at the individual filament level. Furthermore, the authors show that the fluctuations of the bundle length display the same scaling with respect to the average as experimental measurements from different systems. This work constitutes a simple yet nuanced and powerful theoretical result that challenges our current understanding of actin filament kinetics and helps relate accessible experimental measurements such as actin bundle length fluctuations to their underlying kinetics. Finally, I found the manuscript to be very well written, with a particularly clear structure and development, which made it very accessible.

Comments on revisions:

I maintain my original favorable assessment of this manuscript.

I thank the authors for considering my comments and for their thoughtful replies. It would have been helpful to see some of the comments reflected in the text and discussion. I leave this to the authors.

I appreciate that the authors replaced the figures with higher-resolution versions, but I maintain my assessment that the graphical and aesthetic quality of the figures, especially the size of the legends (which are often tiny and difficult to read), labels, colors, etc., could be improved. Again, I leave this to the authors.

Reviewer #1 (Public review):

Summary:

The factors that create and maintain diversity in host-associated microbiomes remain poorly understood. A better understanding of these factors will help in the efforts to leverage the adaptive potential of the microbiome to help solve pressing problems in health and agriculture.

Experimental evolution provides a promising path forward as we can track the causes and consequences in the emergence of novel variants, but experimental evolution remains underutilized in host-microbiome interactions. Here, Gracia-Alvira utilizes a long-term experimental evolution study in Drosophila simulans under hot and cold temperature regimes to identify strain-level variation in an important fly bacterium, Lactiplantibacillus plantarum. They identify three strains of L. plantarum, which are most prevalent in their respective three temperature regimes, suggesting that these are locally adapted bacteria. Then, using a combination of genomics, in vitro, and in vivo, Gracia-Alvira et al attempt to understand the factors that led to the differentiation of the hot and cold L. plantarum and their impacts on the fly host.

Strengths:

This is an excellent use of experimental evolution to track the emergence of novelty in the microbiome. The genomic analyses are all solid and appropriate for the data sets. It is especially striking that the comparisons with the other, independent experimental evolution studies in different labs (and across continents between Portugal and South Africa) show a consistent response to temperature. Many have disregarded the microbiome as it is something that is too sensitive to seemingly innocuous variables (particularly in the fly microbiome), such that we cannot find generalities. However, this finding highlights the potential for experimental evolution to uncover these dynamics. The question of how strains emerge and are maintained is timely and is one of the key open questions in host-microbiome evolution currently.

Weaknesses:

(1) The framing in the title and throughout the discussion about "subspecies competition" does not match the data that was collected. The subspecies competition requires actually tracking the competitive outcomes between the hot, cold, and unevolved L. plantarum. In the in vivo work, I can see that mixes of the strains were made, but they did not track whether the cold strain outcompeted the hot strain in vivo under cold conditions, for example. While Figure 4 is suggestive that there is ongoing competition in the hot temperature regime, this is not necessarily shown in the cold, which is dominated by the C clade. It could also be that the bacteria cannot survive in the flies at the different temperatures. The growth curve assays hint that the bacteria can grow, but the plate reader couldn't actually maintain the 18 {degree sign}C temperature (line 455). So all of this evidence is very indirect and insufficient to say that strain competition is driving these patterns.

(2) The in vivo results are interesting in that there appears to be a fitness cost of clade C, but the explanation is underdeveloped. I say under-developed because in Figure 4, the cold L. plantarum remains much higher throughout adaptation to the hot temperature regime than the hot L. plantarum in the cold regime. The hot L. plantarum is low abundance throughout the cold regime. I felt like this observation was not explained, but it seems relevant to understanding the strain dynamics.

I will also note that this is not the first time that L. plantarum or other Lactobacillus have been shown to exert fitness costs to Drosophila. Gould, PNAS, 2018, shows that both Lactobacillus plantarum and Lactobacillus brevis in mono-association have lower fitness (measured through Leslie matrix projections using lifespan and fecundity) than axenic flies. Many studies of wild Drosophila fail to find Lactobacillus, or it is low abundance (e.g., Chandler, PLoS Genetics, 2014; Wang, Environmental Microbiology Reports, 2018; Henry & Ayroles, Molecular Ecology, 2022; Gale, AEM, 2025). This might help provide useful context for the in vivo results.

(3) The data in Figure 4 are compelling to focus on the L. plantarum variants. However, I can see from the methods that the competitive mapping included only other strains of Wolbachia. It is not clear how other members of the microbiome changed in response to the temperature regimes. As I note in point #2, given that Lactobacillus is often rare, it is not clear what the rest of the microbiome looks like over the course of adaptation. Indeed, it seems like Mazzucco & Schlotterer, PRSB, 2021 did a broader analysis of the microbiome and found that Acetobacter is by far the most common bacterium (I think this data is also part of the data shown here?). Expanding on why or why not in this context is important and will improve this study, particularly if the focus is on connecting these evolutionary dynamics to ecological competition to explain the emergence of strain diversity.

Reviewer #1 (Public review):

Giordano et al. demonstrate that yeast cells expressing separated N- and C-terminal regions of Tfb3 are viable and grow well. Using this creative and powerful tool, the authors effectively uncouple CTD Ser5 phosphorylation at promoters and assess its impact on transcription. This strategy is complementary to previous approaches, such as Kin28 depletion or the use of CDK7 inhibitors. The results are largely consistent with earlier studies, reinforcing the importance of the Tfb3 linkage in mediating CTD Ser5 phosphorylation at promoters and subsequent transcription.

Notably, the authors also observe effects attributable to the Tfb3 linker itself, beyond its role as a simple physical connection between the N- and C-terminal domains. These findings provide functional insight into the Tfb3 linker, which had previously been observed in structural studies but lacked clear functional relevance. Overall, I am very positive about the publication of this manuscript and offer a few minor comments below that may help to further strengthen the study.

Page 4 PIC structures show the linker emerging from the N-terminal domain as a long alpha-helix running along the interface between the two ATPase subunits, followed by a turn and a short stretch of helix just N-terminal to a disordered region that connects to the C-terminal region (see schematic in Fig. 1A).

The linker helix was only observed in the poised PIC (Abril-Garrido et al., 2023), not other fully-engaged PIC structures.

Page 8 Recent structures (reviewed in (Yu et al., 2023)) show that the Kinase Module would block interactions between the Core Module and other NER factors. Therefore, TFIIH either enters into the NER complex as free Core Module, or the Kinase Module must dissociate soon after.

To my knowledge, this is still controversial in the NER field. I note the potential function on the kinase module is likely attributed to the N-terminal region of Tfb3 through its binding to Rad3. Because the yeast strains used in Fig. 6 retain the N-terminal region of Tfb3, the UV sensitivity assay presented here is unlikely to directly address the contribution of the kinase module to NER.

Page 11. Notably, release of the Tfb3 Linker contact also results in the long alpha-helix becoming disordered (Abril-Garrido et al., 2023), which could allow the kinase access to a far larger radius of area. This flexibility could help the kinase reach both proximal and distal repeats within the CTD, which can theoretically extend quite far from the RNApII body.

Although the kinase module was resolved at low resolution in all PIC-Mediator structures, these structural studies consistently reveal the same overall positioning of the kinase module on Mediator, indicating that its localization is constrained rather than variable. This observation suggests that the linker region may help position the kinase module at this specific site, likely through direct interactions with the PIC or Mediator. This idea is further supported by numerous cross-links between the linker region and Mediator (Robinson et al., 2016).

Comments on revisions:

Revised ms clarified all my points, including those I previously misunderstood.

Reviewer #1 (Public review):

Summary:

The authors address whether theta/beta ratio /TBR) can be used as a clinical biomarker for ADHD.

Strengths:

The data were acquired independently from 2 separate datasets, and there are sufficient subjects for adequate statistical power. The authors applied up-to-date EEG data preprocessing, state-of-the-art feature extraction, and statistical analyses, using a multiverse approach. By testing and comparing all meaningful approaches, defined a priori in the previous meta-analysis, the author convincingly demonstrates that TBR cannot be used as a clinical biomarker, and previous positive results can be explained by interactions between different factors (alpha peak frequency, aperiodic component, age).

Weaknesses:

There are no apparent issues with data, separate datasets, large sample sizes, and state-of-the-art data analysis.

Reviewer #1 (Public review):

Summary:

The authors set out to conduct a behavioral comparison of macaque and human vision across a wide range of visual properties. Such a comparison is critical for evaluating the use of macaques as a model system for understanding human vision and the underlying neural mechanisms. This goal represents a unique endeavour since prior studies have typically focused on only highly specific tasks. While the authors found consistent coarse representational structure for objects, evidence for Weber's Law and amodal completion, there was divergence for mirror image confusion and the use of global or local image properties.

Strengths:

There are three major strengths of the study. First, the authors employed a behavioral paradigm (oddball search) that allowed them to test multiple perceptual phenomena without having to train the macaques on the specific type of stimuli tested. Second, humans and macaques could be tested in an identical manner. Third, the authors tested a range of different visual properties and phenomena, allowing a broader comparison between species.

There are also some weaknesses to the study (described below), but that doesn't change the fact that the authors have demonstrated and validated a novel approach for systematic and comprehensive comparisons of vision across species.

Weaknesses:

The weaknesses of the study arise in part because of the breadth of the work. In cases where there are divergences between the two species, it would be helpful to know what might account for such divergence, to have more depth. Is it really a species difference, or could there be a different account? For example, does the difference in mirror image confusion arise because the stimuli were objects that would have been highly familiar to the humans but not the macaques? Further, the authors often used small sets of stimuli (e.g. 8 objects only in the test of object similarity; a small set of highly specific occlusion stimuli), and how well the findings will generalize beyond those stimuli is unclear.

The authors discuss the implications of training macaques to perform specific tasks on specific stimuli in comparing across species. While I agree that extensive training in monkeys could change perception, it is important to also consider that humans have been extensively trained through the types of visual tasks we conduct throughout our lives, so I'm not sure it is universally true that the best comparison is between humans and untrained monkeys. But this just consideration just highlights the general problem of comparing across species.

Reviewer #1 (Public review):

Summary:

This paper describes a complex series of studies that measure and explain object recognition in mice. The authors demonstrate that mice are capable of solving an object recognition task, that object identity is decodable in different regions of cortex, and the decodability, to some extent, can be captured by extant theory on object manifolds in deep neural networks. The authors further add some correlational analysis of the time courses of object discriminability to bolster their claim of an object processing hierarchy in the mouse cortex.

The behavioral and neural data described in this paper are likely to be of interest to the general neuroscience community. That said, I have some issues with the analyses, modeling, and image dataset that I'll detail below.

Strengths:

(1) The behavioral work is incredibly cool. Getting mice to solve this task is a real achievement and opens up new avenues for mice as a model for complex visual tasks.

(2) Similarly, the neural recordings are astounding in their scale.

(3) This could be the most complete demonstration of a primate-analogous object processing network in the mouse.

Weaknesses:

No new weaknesses were noted by this reviewer.

Reviewer #1 (Public review):

Summary:

Liao et al. performed a large-scale integrative analysis to explore the function of two cancer genes (BRCA1 and BRCA2) in lung cancer, which is one of the cancers with an extremely high mortality rate. The detailed genetic analysis demonstrated new roles of BRCA1/2 in causing the tumor microenvironment in lung cancer. In particular, the discovery of different mechanisms of BRCA1 and BRCA2 provides an essential foundation for developing drugs that target BRCA1 or BRCA2 in lung cancer therapy.

Strengths:

(1) This study leveraged large-scale genomic and transcriptomic datasets to investigate the prognostic implications of BRCA1/2 mutations in LUAD patients (~2,000 samples). The datasets range from genomics to single-cell RNA-seq to scTCR-seq.

(2) In particular, the scTCR-seq offers a powerful approach for understanding T cell diversity, clonal expansion, and antigen-specific immune responses. Leveraging these data, this study found that BRCA1 mutations were associated with CD8+ Trm expansion, whereas BRCA2 mutations were linked to tumor CD4+ Trm expansion and peripheral T/NK cell cytotoxicity.

(3) This study also performed a comprehensive analysis of genomic variation, gene expression, and clinical data from the TCGA program, which provides an independent validation of the findings from LUAD patients newly collected in this study.

(4) This study provides an exemplary integration analysis using both computational biology and wet bench experiments. The experimental testing in the A549 cell line further supports the robustness of the computational analysis.

(5) The findings of this study offer a comprehensive view of the molecular mechanisms underlying BRCA1 and BRCA2 mutations in LUAD. BRCA1 and BRCA2 are two well-known cancer-related genes in multiple cancers. However, their role in shaping the tumor microenvironment, particularly in lung cancer, is largely unknown.

(6) By focusing on PD-L1-negative LUAD patients, this study demonstrated the molecular mechanisms underlying resistance to immune therapy. These new insights highlight new opportunities for personalized therapeutic strategies to BRCA-driven tumors. For example, they found histone deacetylase (HDAC) inhibitors consistently downregulated 4-R genes in A549 cells.

(7) The deposition of raw single-cell sequencing (including scRNA-seq and scTCR-seq) data will provide an essential data resource for further discovery in this field.

Comments on revisions:

The author has revised accordingly. I have no further comments.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed most of the comments raised in the previous round of review.]

Summary:

This study addresses the emerging role of fungal pathogens in colorectal cancer and provides mechanistic insights into how Candida albicans may influence tumor-promoting pathways. While the work is potentially impactful and the experiments are carefully executed, the strength of evidence is limited by reliance on in vitro models, small patient sample size, and the absence of in vivo validation, which reduces the translational significance of the findings.

Strengths:

(1) Comprehensive mechanistic dissection of intracellular signaling pathways.

(2) Broad use of pharmacological inhibitors and cell line models.

(3) Inclusion of patient-derived organoids, which increases relevance to human disease.

(4) Focus on an emerging and underexplored aspect of the tumor microenvironment, namely fungal pathogens.

Reviewer #1 (Public review):

In this revision the authors address some of the points, but they also make some technical errors. My overall view of the manuscript hasn't changed since the original evaluation.

Previously I noted that SC sparsity presents an issue when comparing to full FC matrices. They authors misinterpreted the Honey et al paper. They resampled ALL entries of the SC matrix (including zeros) from a Gaussian distribution. In effect, this assigns zeros small (but uniform) weights. In Honey et al, the authors resampled only existing edge weights from a gaussian distribution (the rationale at the time was that there might be pushback against the extremely heavy-tailed edge weight distribution). In other words, the zeros are still zeros following this resampling procedure.

That said, I agree that the log transform is likely useful or necessary given edge weight distributions.

In short, I still think that the approach is interesting and meritorious, I just don't think the execution is correct.

Reviewer #1 (Public review):

In this manuscript, the authors aim to determine the ligand on Plasmodium falciparum infected erythrocytes for the NK cell integrin, LFA-1, following up on previous evidence that LFA-1 is important for immune cell-mediated recognition of iRBCs.

They start by incubating LFA-1 with iRBCs and show by flow analysis that a substantial population of these iRBCs binds to the LFA-1 (Fig 1C). They do conduct the control with uninfected RBCs, but put this in the supplementary material. As this is a critical control, I think that it should be moved to Figure 1C as it is essential to allow interpretation of the iRBC data. The authors also do not state which strain of P. falciparum they used (line 144). This is critical information, as different strains have different variant surface antigens and should be included. With these changes, this data seems convincing.

They next incubated LFA-1 with the iRBCs, cross-linked and conducted a pulldown, identifying GP130 as a binding partner. Using cross-linkers is a dangerous strategy as it risks non-specific cross-linking. Did they try without cross-linking and find an interaction?

They raised antibodies to PfGBP and showed IFA, which reveals that these antibodies stain iRBCs (Figure 2Ciii). This experiment lacks a critical control of uninfected RBCs, which needs to be included to show that the staining is specific. Without this, it is not possible to conclude that there is iRBC-specific staining with PfGBP.

They then conduct a pulldown using LFA-Fc, which does show GP130 only in the presence of the LFA-Fc, but not when empty beads are used. This is convincing. BLI measurements are also used to study this interaction (Figure 2Ci). The BLI data is presented in such a way that any association phase is obscured by the y-axis, which makes it impossible to know whether there is binding here. I think that the data needs to be shown with some baseline before the addition of the ligand so that association can be seen. The data is also a bit messy with a downward drift and the curves showing different shapes, for example, with the 1.0uM curve seeming to have a different association rate. As this is the only data which shows a direct interaction between LFA1 and GBP, as pulldowns are done with lysates, which might mean bridging components. I think that it is important to repeat the BLI, or use additional biophysical methods to assess binding, to obtain more convincing data.

The authors next do some modelling of the putative complex. This is done by homology modelling and docking, which is not the most up-to-date method and is overinterpreted. Personally, I would remove this data as I did not find it convincing and it is not important for the story. If the authors wish to include it, then I think that they should validate the modelling by mutagenesis to show that the residues which the models indicate might bind are involved in the interaction.

They next made GP130 and tested the binding of this to THP-1 cells, which are often used as a model for macrophages. They observe greater binding of PfGBP-Fc to these cells when compared with hIgG and show that LFA-1 siRNA reduces this binding. I was a little confused about how the flow plots related to the graph in the bottom right corner of Figure 3Bii. In the flow plots, hIgG control shows 12.8% of cells in the gated region, while the unstained cells has 5.63%, but the MFI data shows a decrease in binding for hIgG vs unstained cells. How is this consistent? Also the siRNA reduces the number of cells in gated region from 66.6% to 25.9%, which is still substantially more that 5.63% in the unstained control. This also doesn't seem quite consistent with the MFI data. Could the authors explain this? Also perhaps an additional experiment would be to add soluble LFA-1 into this assay as an additional control to determine whether this blocks PfGBP binding to the THP-1 cells? It could. Be that there are additional mechanisms of binding which indicate why the siRNA has a partial effect. The same is true for the NK cell experiments in Figure 3Ci in which the siRNA has a partial effect. The authors also test binding to HEK, HepG2 and 'stem' cells and claim 'only background levels of binding', but in each case, there is more binding to these cells by PfGBP-Fc than by hIgG, albeit less than in THP-1 and NK cells. Why have the authors decided that these increases are not significant? All in all, these experiments do indicate a role for the GBP-LFA1 interaction in the binding of immune cells to iRBCs, but perhaps not as absolutely as is suggested.

The authors next produce CHO cells with PfGBP on the surface. These cells bind to LFA-1 specifically. When these cells were incubated with primary NK cells, they did see increases in activation markers, which were reduced by addition of antiCD11a, suggesting these to be specific. They also conduct the same experiment with anti-GBP with iRBCs but this is in a different figure. It would be easier for the reader if Figure 5B were in the same figure as Figure 4B as it is related data using the same method. I found this data convincing, showing that the LFA1:GBP interaction does contribute to immune cell recognition and activation.

The authors next conduct an experiment in which they assess parasite growth in the presence of NK cells and in the presence of anti-GBP. They use Heochst staining as a measure of parasite growth and claim that NK cells reduce the number of parasites, but that anti-GBP abolishes this effect (Figure 5A). I found this experiment very unconvincing as there are small effects and no demonstration of significance. More commonly used approaches to study parasite growth are lactate dehydrogenase GIA assays or calcein-AM labelling. I did not find this experiment convincing and would either remove or supplement with additional data using a more robust assay, with repeats and tests of statistical significance.

In summary, the authors present a set of data which comes together to indicate an interaction between LFA1 and PfGBP on the Plasmodium infected erythrocyte surface. Pulldown studies show convincingly that these two proteins co-precipitate and BLI data suggest that this is direct. Also convincing is that NK cell activation can be reduced using antibodies against either LFA1 or PfGBP, indicating that this interaction does play a role in immune cell recognition of iRBCs.

Comments on revised version:

The authors made some minor changes in response to my review, but did not present any substantial new data to demonstrate a direct interaction between PfGBP and LFA1 or to convincingly show differences in NK cell-mediated killing.

Reviewer #1 (Public review):

In this study, the authors investigate responses to methionine in the olfactory system of the Xenopus tadpole. They show that the LFP response is local to the glomerular layer, arises ipsilaterally, and is blocked by pharmacological blockade of AMPA and NMDA receptors, with little modulation during blockade of GABA-A receptors. They then show that this response is translently enlarged following transection of the contralateral olfactory nerve, but not the optic lobe nerve. Measurement of ROS- a marker of inflammation- was not affected by contralateral nerve transection, and LFP expansion was not affected by pharmacological blockade of ROS production. Imaging biased towards presynaptic terminals suggests that the enlargement of the LFP has a presynaptic component. A D2 antagonist increases the LFP size and variability in intact tadpoles, while a GABA-B antagonist does not. Finally, the authors provide anatomical and physiological evidence that the contralateral dopamine signal may arise from the lateral pallium. Overall, I found the array of techniques and approaches applied in this study to be creatively and effectively employed.

Reviewer #1 (Public review):

Summary:

The authors convincingly demonstrate that when PKMzeta is genetically deleted from the hippocampus, the related atypical PKC, PKClambda is upregulated and compensates both neurophysiologically and behaviorally for the missing PKMzeta. Specifically, the upregulatiion of PKClambda supports late-phase hippocampal long-term potentiation (L-LTP) and long-term spatial memory in the PKMzeta knockout mice.

Strengths:

The study uses up-to-date transgenic techniques to alter the expression of the two atypical PKCs. The synaptic and behavioral experiments are well-controlled and appear to have been carefully executed.

Weaknesses:

None

Reviewer #1 (Public review):

The manuscript by Tang et al. characterizes the expression dynamics and functional roles of aldehyde dehydrogenase 1 activity in uterine physiology. Using a combination of in vivo lineage tracing and cell ablation coupled with organoid culture, the authors propose that Aldh1a1 lineage-marked cells contribute to uterine gland development and epithelial regeneration. The descriptive data will be of interest to reproductive biologists and clinicians and will build on established hypotheses in the field. The manuscript is well written and scientifically sound; however, several experimental limitations and interpretation caveats should be addressed.

The methods surrounding the passage number and duration of culture following sorting prior to transcriptomic profiling should be clarified in the figure legends. Related to this, the representative images in Figures 1D and 1E do not appear consistent with the quantification presented in Figures 1F-H and should be reconciled.

The conclusion that ALDH1A1+ cells are enriched in populations with stem cell characteristics relies primarily on transcriptomic analysis. Protein-level co-localization should be performed to strengthen this claim.

The overlap of 19 genes between the data set here and AXIN2 HI data is presented as evidence of shared stemness identity, but no statistical assessment of this overlap is provided. A hypergeometric test should be performed to determine whether this overlap is greater than expected by chance.

The impact of tamoxifen injection on Aldh1a1 expression should be characterized in the neonatal uterus, as tamoxifen itself has known estrogenic activity that could confound interpretation of the lineage tracing results at early postnatal timepoints. Related to this, while low-dose tamoxifen is shown to label individual cells within 24 hours of injection, the translation dynamics of the label following Cre-mediated recombination can require up to 72 hours. The presence of only a few labeled clones at PND8 but multiple separate clones per cross-section at later timepoints warrants discussion and may reflect labeling kinetics rather than clonal expansion.

It would strengthen the in vivo ablation data to validate the degree of cell death following diphtheria toxin treatment directly. It is possible that a general decrease in cell number rather than specific loss of a stem cell population is responsible for the observed reduction in gland number and FOXA2 expression (Tongtong et al 2017).

The lineage tracing data in the postpartum endometrium demonstrate that Aldh1a1-marked cells are present during regeneration, but it remains unclear whether these cells are preferentially activated or expanded in response to tissue injury. Coupling these studies with diphtheria toxin-mediated ablation during active regeneration would more directly test the proposed regenerative role of this population.

The contribution of stromal Aldh1a1 lineage-positive cells is underexplored in the discussion, given the lineage tracing data showing stromal labeling across multiple timepoints and its potential relevance to mesenchymal-to-epithelial transition.

Finally, the word 'control' may overstate the functional evidence presented. 'Contribute' may be more accurate given the partial and context-dependent nature of the phenotypes observed.

Reviewer #1 (Public review):

Summary:

The authors investigate how methicillin-resistant (MRSA) and sensitive (MSSA) Staphylococcus aureus adapt to a new host (C. elegans) in the presence or absence of a low dose of the antibiotic oxacillin. Using an "Evolve and Resequence" design with 48 independently evolving populations, they track changes in virulence, antibiotic resistance, and other fitness-related traits over 12 passages. Their key finding is that selection from both the host and the antibiotic together, rather than either pressure alone, synergistically results in the evolution of the most virulent pathogens. Genomically, they find that this adaptation repeatedly involves parallel mutations in a small number of key regulatory genes, most notably codY, agr, and saeRS.

Strengths:

The main advantage of the research lies in its strong and thoroughly replicated experimental framework, enabling significant conclusions to be drawn based on the concept of parallel evolution. The study successfully integrates various phenotypic assays (virulence, growth, hemolysis, biofilm formation) with whole-genome sequencing, offering an extensive perspective on the adaptive landscape. The identification of certain regulatory genes as common targets of selection across distinct lineages is an important result that indicates a level of predictability in how pathogens adapt. Furthermore, the detailed mapping of specific parallel mutations provides a highly useful genomic resource for the microbiology community.

Revisions and Re-Appraisal:

In the initial version of the manuscript, a primary limitation was the use of causal language to link specific mutations to phenotypes, despite the evidence from the evolution experiment being correlational. In this revised version, the authors have excellently addressed this limitation. They have meticulously revised the text to accurately reflect these relationships as strong, statistically significant genetic associations rather than confirmed facts. Furthermore, they explicitly acknowledge that future ancestral reconstruction experiments will be required to confirm direct causality. The authors have also appropriately clarified the visual interpretations of their data (such as the PCA clustering) and refined their discussion of mutation rates. With these revisions, the claims made are fully supported by the data presented.

Impact and Context:

The authors successfully achieve their aims, demonstrating that the combined effects of host and antibiotic pressures collaboratively propel the evolution of heightened virulence. While the nematode model does not perfectly mimic human or mammalian infection, the evolutionary principles uncovered here are highly relevant to both evolutionary biology and infectious disease management. The evidence presented is compelling, and the strong correlational hypotheses generated by this study offer a robust and significant basis for upcoming mechanistic research into pathogen adaptation.

Comments on revisions:

I commend the authors for their thorough, thoughtful, and highly constructive revision. You have successfully addressed all of my major and minor comments. The addition of Table S2 and the careful revisions to the causal language have significantly strengthened the manuscript and clarified the data interpretation. I have no further recommendations. Great work!

Reviewer #1 (Public review):

Summary:

This paper examines whether action potentials (APs) reliably propagate to the distal axon in neocortical parvalbumin-expressing interneurons (PV-Ins) during prolonged high-frequency activity, as occurring during epileptiform activity. The authors use dual soma and axon-attached patch-clamp recordings from mouse and human PV-INs and show that axon AP amplitude declines when the firing frequency exceeds ~200 Hz and fails during seizure-like bursts. Finally, they show that elevation of external K+ to 10 mM also reduces AP amplitude. Taken together, these data strongly suggest that the reduction in transmitter release observed during intense PV-INs activity or during seizure-like events is mainly mediated by the reduction in the presynaptic AP amplitude in PV-INs.

Strengths:

This paper is very interesting, well-written and technically impressive. It provides new and important results. The paper will have a great impact in the field of both axon physiology and epilepsy.

Weaknesses:

I did not find any significant weakness in the methods, data analysis and results.

Reviewer #1 (Public review):

This study investigates how astrocyte metabolic state influences astrocyte-synapse interactions and the organization of the dopaminergic circuit in the Drosophila CNS. Using a creative split-GFP-based contact reporter ("PEAPOD"), combined with genetic perturbations of glycolytic enzymes, synaptic labeling, EM, transsynaptic tracing, single-cell transcriptomics, and behavioral assays, the authors propose that disruption of astrocyte glycolysis enhances astrocyte-dopamine neuron contacts, promotes synaptogenesis, and biases dopaminergic-motor circuit connectivity through a mechanism involving altered Neuroligin 2 trafficking.

The work is conceptually ambitious and technically broad. The development and application of a contact reporter for astrocyte-neuronal interfaces is potentially valuable to the field, and the convergence of multiple glycolytic perturbations on similar phenotypes is a notable strength. However, several central conclusions currently extend beyond the direct evidence presented. Clarification and calibration of these claims would substantially strengthen the manuscript.

Major Points:

(1) Astrocyte glycolytic impairment is inferred rather than directly demonstrated

The central premise of the manuscript is that reduced astrocyte glycolysis drives the observed phenotypes. While multiple glycolytic enzymes (e.g., pfk, eno, pyk) are genetically perturbed and produce similar increases in PEAPOD signal, the manuscript does not directly demonstrate altered glycolytic flux or metabolic state in astrocytes under these conditions. Reduced enzyme levels or genetic mutation do not necessarily establish functional metabolic deficiency, particularly given potential compensatory mechanisms.

Because glycolytic impairment is foundational to the proposed mechanism, the conclusions should either be supported by direct metabolic readouts in astrocytes or framed more cautiously as perturbations of glycolytic enzymes rather than confirmed metabolic deficiency.

(2) Interpretation of the PEAPOD signal requires clearer calibration

The PEAPOD system is an innovative tool to detect membrane proximity between astrocytes and dopamine neurons. However, the manuscript frequently interprets increased PEAPOD intensity and volume as increased "ensheathment" or increased synaptic contact. A split-GFP-based reporter measures membrane apposition within a defined spatial range but does not directly quantify structural ensheathment, synapse number, or functional synaptic engagement.

Although the authors show an association of the PEAPOD signal with presynaptic markers in some regions, the distinction between increased membrane contact, altered membrane organization, and true changes in perisynaptic coverage should be more explicitly discussed. Several conclusions would benefit from clearer wording that distinguishes contact proximity from ultrastructural or functional synapse remodeling.

(3) Evidence for biased dopaminergic-motor circuit wiring is indirect

The manuscript proposes that disruption of astrocyte glycolysis biases dopaminergic-motor connectivity. This conclusion relies heavily on trans-Tango labeling intensity and downstream cell-type composition analysis via FACS and single-cell RNA sequencing.

Transsynaptic labeling approaches can be influenced by expression levels, reporter trafficking, labeling efficiency, and differential recovery during dissociation and FACS. Changes in labeled cell abundance or reporter intensity do not necessarily equate to altered synaptic wiring. Given that this conclusion represents a major conceptual advance of the study, the manuscript should either provide additional orthogonal support or temper the claim to reflect that altered labeling efficiency or synaptic engagement, rather than definitive rewiring, has been demonstrated.

(4) Mechanistic claims regarding Neuroligin 2 trafficking are suggestive but not definitive

The authors propose that astrocyte glycolytic disruption alters Neuroligin 2 (Nlg2) trafficking, leading to ER retention and downstream synaptogenic effects. The observation of Nlg2-positive intracellular bodies colocalizing with ER markers is intriguing. However, quantitative analysis, additional compartment markers, and/or biochemical support would be necessary to firmly establish altered ER exit or glycosylation status.

At present, the mechanistic model is plausible but should be presented more explicitly as a working model supported by suggestive evidence rather than a fully established trafficking defect.

(5) Behavioral phenotypes are not yet causally linked to dopaminergic circuit changes

The locomotor phenotypes observed upon astrocyte glycolytic perturbation are clear. However, the manuscript attributes these changes to altered dopaminergic-motor connectivity. A direct causal linkage between astrocyte metabolic state, dopaminergic circuit remodeling, and behavior is not conclusively demonstrated. The discussion should either clarify the inferential nature of this link or provide additional evidence supporting dopamine-specific dependence.

Minor Points:

(1) Statistical analyses across multi-group comparisons should be more clearly justified, particularly where multiple pairwise tests are performed. A clarification of the multiple-comparison correction and the exact comparison strategy would improve rigor.

(2) The temporal interpretation of activity-dependent remodeling experiments would benefit from a clearer explanation of what timescale is being tested.

(3) Developmental compensation versus the acute effects of glycolytic perturbation are not fully distinguished and should be discussed.

(4) The orthology and functional equivalence of Drosophila Nlg2 should be described with greater precision to avoid potential confusion.

Reviewer #1 (Public review):

Summary:

In this manuscript, Kong et al. conduct a systematic analysis of the multi-cancer risk locus at 2q33. The authors start with a careful analysis of co-localization between the melanoma risk SNPs and several other cancers and conclude that a subset of credible causal SNPs is shared among different cancers, including breast cancer. Next, they define a starting list of 27 SNPs as potential credible causal SNPs and analysis of TADs (topologically associating domains) to zoom in on CASP8 and FLA CC1 as potential target genes. They then systematically rule out coding and splicing variants in the set and focus on a smaller set of three SNPs constituting a melanocyte enhancer element. Using a combination of mass spectrometry, reporter assays, and electrophoretic mobility shift assays, the authors define a role for transcription factors IRF2 and E4F1 in the regulatory network driving risk at the locus.

This work represents a high-quality tour de force, using multiple tools, to zoom in on a gene expression regulatory network associated with risk for multiple cancers. It provides a detailed framework for analyses of other multi-cancer risk loci. Limitations of the work, which is rather a current limitation of the field, is the lack of a model to study how the identified network of regulatory elements, transcription factors, and target genes mechanistically drive risk at the organismal level. Advances such as those described in this manuscript contribute significantly to our knowledge of how common risk variants drive risk.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the major comments raised in the previous round of review. Public Reviews below refer to the version submitted to Review Commons.]

Summary:

Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

Major comments:

(1) Lack of direct in vitro binding measurements:

The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And do the sybodies affect the interaction of ScpAB with SMC?

It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

(2) Many modes of sybody binding to Smc are plausible

The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

(3) Sybody expression in vivo

Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

(4) Sybodies should phenocopy ATP hydrolysis mutant of Smc

The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

Significance:

Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins has been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Original review:

Summary:

Lumen formation is a fundamental morphogenetic event essential for the function of all tubular organs, notably the vertebrate vascular network, where continuous and patent conduits ensure blood flow and tissue perfusion. The mechanisms by which endothelial cells organize to create and maintain luminal space have historically been categorized into two broad strategies: cell shape changes, which involve alterations in apical-basal polarity and cytoskeletal architecture, and cell rearrangements, wherein intercellular junctions and positional relationships are remodeled to form uninterrupted conduits. The study presented here focuses on the latter process, highlighting a unique morphogenetic module, junction-based lamellipodia (JBL), as the driver for endothelial rearrangements.

Strengths:

The key mechanistic insight from this work is the requirement of the Arp2/3 complex, the classical nucleator of branched actin filament networks, for JBL protrusion. This implicates Arp2/3-mediated actin polymerization in pushing force generation, enabling plasma membrane advancement at junctional sites. The dependence on Arp2/3 positions JBL within the family of lamellipodia-like structures, but the junctional origin and function distinguish them from canonical, leading-edge lamellipodia seen in cell migration.

Weaknesses:

The study primarily presents descriptive observations and includes limited quantitative analyses or genetic modifications. Molecular mechanisms are typically interrogated through the use of pharmacological inhibitors rather than genetic approaches. Furthermore, the precise semantic distinction between JAIL and JBL requires additional clarification, as current evidence suggests their biological relevance may substantially overlap.

Reviewer #1 (Public review):

Summary:

In their paper, Shimizu and Baron describe the signaling potential of cancer gain-of-function Notch alleles using the Drosophila Notch transfected in S2 cells. These cells do not express Notch or the ligand Dl or Dx, which are all transfected. With this simple cellular system, the authors have previously shown that it is possible to measure Notch signaling levels by using a reporter for the 3 main types of signaling outputs, basal signaling, ligand-induced signaling and ligand-independent signaling regulated by deltex. The authors proceed to test 22 cancer mutations for the above-mentioned 3 outputs. The mutation is considered a cluster in the negative regulatory region (NRR) that is composed of 3 LNR repeats wrapping around the HD domain. This arrangement shields the S2 cleavage site that starts the activation reaction.

The main findings are:

(1) Figure 1: the cell system can recapture ectopic activation of 3 existing Drosophila alleles validated in vivo.

(2) Figure 2: Some of the HD mutants do show ectopic activation that is not induced by Dl or Dx, arguing that these mutations fully expose the S2 site. Some of the HD mutants do not show ectopic activation in this system, a fact that is suggested to be related to retention in the secretory pathway.

(3) Figure 3: Some of the LNR mutants do show ectopic activation that is induced by Dl or Dx, arguing that these might partially expose the S2 site.

(4) Figure 4-6: 3 sites of the LNR3 on the surface that are involved in receptor heterodimerization, if mutated to A, are found to cause ectopic activation that is induced by Dl or Dx. This is not due to changes in their dimerization ability, and these mutants are found to be expressed at a higher level than WT, possibly due to decreased levels of protein degradation.

Strengths and Weaknesses:

The paper is very clearly written, and the experiments are robust, complete, and controlled. It is somewhat limited in scope, considering that Figure 1 and 5 could be supplementary data (setup of the system and negative data). However, the comparative approach and the controlled and well-known system allow the extraction of meaningful information in a field that has struggled to find specific anticancer approaches. In this sense, the authors contribute limited but highly valuable information.

Comments on revised version:

I reviewed the changes and response to criticism, and it seems to me that all has been reasonably addressed.

Reviewer #1 (Public review):

Summary:

The authors have studied how a virus (EMCV) uses its RNA (Type 2 IRES) to hijack the host's protein-making machinery. They use cryo-EM to extract structural information about the recruitment of viral Type 2 IRES to ribosomal pre-IC. The authors propose a novel interaction mechanism in which the EMCV Type 2 IRES mimics 28S rRNA and interacts with ribosomal proteins and initiator tRNA (tRNAi).

Strengths:

(1) Getting structural insights about the Type 2 IRES-based initiation is novel.

(2) The study allows a good comparison of other IRES-based initiation systems.

(3) The manuscript is well-written and clearly explains the background, methods, and results.

Comments on revised version:

I have gone through the revised manuscript by Das and Hussain along with the rebuttal comments. While the poor resolution of the ribosomal complex limits detailed analysis of the molecular interactions, addition of the luciferase reporter assay in the supplementary has enriched the paper.

Reviewer #1 (Public review):

Summary:

The authors dissected the ears with some surrounding tissue from 600 embryos at 4 developmental time points of wild-type larvae, as well as from an lmx1bb mutant, performed scRNA-seq analyses, and subclustered the ear/neuromast clusters. They identified cluster markers and performed PAGA pseudotime analyses to build developmental timelines of lineages. They validated some of the cluster markers with HCRs. Many of the clusters are not annotated in detail, but the data sets are still valuable for the community.

Strengths:

Using scRNA-Seq, the authors identified cluster markers for tissues of the developing zebrafish ear and validated some of them with HCRs. The data they compiled and submitted to public databases is a valuable resource for the community.

Weaknesses:

Many of the clusters have not been annotated or rely on published data. For the ones for which no HCRs or UMAPs are shown, it is therefore difficult to estimate which of the markers are indeed the most cell type/state-specific ones.

Major comments:

(1) It would be very useful if the cluster numbers in the Excel files also had the associated cell type annotations as a second column (at least for the ones that are known). E.g., in Supplemental Table 2, the text states which clusters represent which neuromast and ear cell type, but these are not mentioned in the Excel table.

(2) Many of the clusters have not been annotated or rely on published data. For the ones for which no HCRs or UMAPs are shown, it is therefore difficult to estimate which of the markers are indeed the most cell-type/state-specific ones.

(3) Uploading the data to gEAR (https://umgear.org/dataset_explorer.html), a web-based, publicly available ear database, would further increase the usefulness of this study to the broader community.

Method:

The authors should provide the details about how many cells were sequenced for each ear developmental stage, how many cells were present per cluster (page 8), and how many cells were present in each subcluster of ear and lateral line clusters (page 10).

Reviewer #1 (Public review):

Summary:

In this work, the authors revisit a well-defined experimental system for studying temporal gene expression mechanisms in TNF-alpha-stimulated macrophages, bringing new tools to the process. Using a hybrid-capture approach, they are able to obtain deeper RNA sequencing of target genes, which allows them to identify potential differences in splicing kinetics of individual introns. Further implementing transcriptional blocks to measure intron half-lives, and predictive machine learning models to identify potential contributing cis-acting RNA elements, they define a group of 'bottleneck' introns whose delayed splicing is a rate-limiting step in mRNA maturation.

Strengths:

(1) The hybrid-capture approach enables deeper RNA sequencing of target transcripts.

(2) The neural network application to identify motifs outside of splice sites could be related to intron removal kinetics.

(3) The paper uses splicing reporters with modulation of 5' splice sites to test the effect on reporter gene expression in the context of 'bottleneck' introns.

Weaknesses:

(1) While evidence is provided that these introns are distinct from previously published splicing kinetics studies, 'bottleneck' introns are not adequately placed in context for assessment of how they are similar or different.

(2) Splicing reporters are a good approach, but the complexities of post-transcriptional gene expression regulation are not adequately addressed

(3) Deep learning models are a potentially powerful tool for identifying novel regulatory sequences; however, their use here is underdeveloped.

Reviewer #1 (Public review):

Summary:

In this manuscript, Matsuda and collaborators present a model of how tracheal tubulogenesis is controlled in Drosophila embryos. Some of the results backing the model are new, but others are based on information already published by the authors. However, the results in this manuscript present different molecular markers not published before, which agree with previous conclusions. The manuscript also analyses the requirement of the dpp and EGFR signalling pathways for trachealess (trh) maintenance, one of the main tracheal transcription factors.

Strengths:

The two most interesting novel points of the manuscript are:

(1) Its contribution to the analysis of how the dpp and EGFR pathways contribute to the maintenance of trh expression.

(2) The experimental evidence showing that mechanical invagination is not a requirement for trh maintenance in the tracheal cells, an intriguing hypothesis previously suggested by (Kondo Hayashi 2019 eLife 8:e45145) that can now be discarded by the data presented in this work.

Weaknesses:

Because of the mixture of new and already published data, this manuscript can be considered as a review/experimental paper.

Already known data:<br /> - The results showing that hh and vvl drive tracheal invaginaton independently of trh are reported in Figure 5 of (Matsuda et al. 2015 eLife 4:e09646).<br /> - The results showing dpp requirement for trh maintenance are partially reported in Figure 6 of (Matsuda 2015 eLife 4:e09646).

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

Zacharia and colleagues investigate the role of the C-terminus of IFT172 (IFT172c), a component of the IFT-B subcomplex. IFT172 is required for proper ciliary trafficking and mutations in its C-terminus are associated with skeletal ciliopathies. The authors begin by performing a pull-down to identify binding partners of His-tagged CrIFT172968-C in Chlamydomonas reinhardtii flagella. Interactions with three candidates (IFT140, IFT144, and a UBX-domain containing protein) are validated by AlphaFold Multimer with the IFT140 and IFT144 predictions in agreement with published cryo-ET structures of anterograde and retrograde IFT trains. They present a crystal structure of IFT172c and find that a part of the C-terminal domain of IFT172 resembles the fold of a non-canonical U-box domain. As U-box domains typically function to bind ubiquitin-loaded E2 enzymes, this discovery stimulates the authors to investigate the ubiquitin-binding and ubiquitination properties of IFT172c. Using in vitro ubiquitination assays with truncated IFT172c constructs, the authors demonstrate partial ubiquitination of IFT172c in the presence of the E2 enzyme UBCH5A. The authors also show a direct interaction of IFT172c with ubiquitin chains in vitro. Finally, the authors demonstrate that deletion of the U-box-like subdomain of IFT172 impairs ciliogenesis and TGFbeta signaling in RPE1 cells.

However, some of the conclusions of this paper are only partially supported by the data, and presented analyses are potentially governed by in vitro artifacts. In particular, the data supporting autoubiquitination and ubiquitin-binding are inconclusive. Without further evidence supporting a ubiquitin-binding role for the C-terminus, the title is potentially misleading.

Strengths:

(1) The pull-down with IFT172 C-terminus from C. reinhardtii cilia lysates is well performed and provides valuable insights into its potential roles.

(2) The crystal structure of the IFT172 C-terminus is of high quality.

(3) The presented AlphaFold-multimer predictions of IFT172c:IFT140 and IFT172c:IFT144 are convincing and agree with experimental cryo-ET data.

Reviewer #1 (Public review):

Summary:

This manuscript asks how the uterine lumen is remodeled across the peri-implantation window and whether this remodeling is functionally linked to embryo attachment and subsequent pregnancy establishment. The authors combine whole-organ three-dimensional imaging of optically cleared mouse uteri with single-cell and spatial transcriptomic profiling, conditional deletion of p38α at the uterine-wide versus epithelial-restricted level, and rescue experiments using progesterone and leukemia inhibitory factor. Based on these datasets, the authors propose that the luminal epithelium undergoes a previously underappreciated phase of organ-scale architectural reorganization before attachment, and that a p38α-dependent stress-responsive program coordinates epithelial remodeling together with epithelial-stromal communication required for implantation competence.

Strengths:

By moving beyond local attachment-site morphology to a horn-level representation of luminal topology, the work provides anatomical context that is difficult to reconstruct from conventional section-based approaches and should be broadly useful to the implantation community. The integration of organ-scale morphology with single-cell and spatial transcriptomic datasets, together with genetic perturbation and rescue experiments, adds breadth and increases the potential long-term utility of the dataset for investigators interested in uterine receptivity and embryo-uterine interactions.

Weaknesses:

(1) The whole-uterus analysis of luminal folds and creases requires stronger methodological support. Given the mechanical compliance of the uterine lumen, it is difficult to evaluate from the current description whether dissection, fixation, clearing, and/or mounting could influence the observed luminal topography. This issue is particularly important because several key conclusions depend on the spatial distribution of folds across the uterine horn. A fuller account of tissue handling and reconstruction, together with validation that the preparation preserves native morphology, would substantially strengthen confidence in the organ-scale conclusions.

(2) Several of the central morphological claims are supported primarily only by representative reconstructions. This includes the proposed flattening/creasing dynamics, alternating stretched and shrunken regions, persistence of abnormal folding in the mutant uterus, and the extent of structural rescue following progesterone supplementation. The authors could extract objective measures from the reconstructed luminal surface and provide more statistical analysis to demonstrate the reproducibility of the results.

(3) The manuscript appears to over-reach in concluding that luminal remodeling zones embryos before attachment from day 4 to 5. As presented, the data support a correlation between luminal architecture and embryo position, but do not discriminate between (i) luminal remodeling directing embryo positioning, (ii) embryos locally shaping the lumen, or (iii) parallel regulation of both. The evidence is based on observations of the uterus and the inside blastocysts at certain time points around implantation. Without the time-lapse analysis within the uterus, the dynamic interactions between embryos and the uterus couldn't be determined.

(4) The key conclusion of the manuscript is that uterine p38α regulates luminal epithelial remodeling required for embryo attachment, as shown in the title. Against this background, the finding that epithelial-restricted loss of p38α does not overtly impair fertility is notable, as it suggests that the major function of p38α may not be epithelial cell-autonomous but instead may arise through other uterine compartments that secondarily influence the epithelium. At present, however, this conclusion remains insufficiently supported: the epithelial-specific model is not characterized in sufficient depth during the peri-implantation period, and the transcriptomic evidence for altered epithelial-stromal communication does not by itself explain the phenotypic difference between uterine-wide and epithelial-specific deletion. If stromal p38α is proposed as the critical upstream regulator, more direct testing, such as stromal-specific deletion, would be needed.

Reviewer #1 (Public review):

Summary:

The authors provide high-resolution cryoEM structures to map and functionally characterize human antibodies against SARS-CoV-2 elicited by a standard mRNA vaccine. Here, they report high-resolution structural information on seven previously documented neutralizing antibodies from this response, which were produced from early plasmablasts and which engage diverse targets on the viral spike glycoprotein. This structural information is then integrated with functional assays to define how antibody epitope specificity, geometry, and conformational dynamics may shape neutralization outcomes.

Strengths:

A core strength of the study is a technically-well executed analysis of multiple 'ectopically balanced' mAbs elicited by early B cell plasmablast responses. These antibodies engage different neutralizing targets on the S-trimer of SARS-CoV-2, including the RBD and NTD domains. This has resolved a core distinction in terms of how nAbs engaging these features (and subfeatures, e.g., more conserved hydrophobic pocket within NTD) neutralize the virus.

Weaknesses:

A general weakness is that these antibody classes have been structurally characterized already (albeit individually), and much of this work has been done in the context of understanding susceptibility to escape mutations (delta, omicron, and subvariants therein; class I-IV antibody crossreactivity on Wuhan SARS-CoV-2 to present). It is exceptionally fine technical work presenting the antibodies in a collection like this, but perhaps the new predictive power of this analysis is somewhat overstated.

The early plasmablast angle seems like it could be better fleshed out. Many of the known SARS-CoV-2 nAbs are from the plasmablast pool, but how does this predict the antibody profile at latter stages, as per the stated goal and claim of the current study? Does the paratope pattern of plasmablast antibodies then change within the immune sera at later time points? New or existing cryoEMPEM data could shed light on this.

Reviewer #1 (Public review):

Summary:

The authors set out to evaluate how accurately direct sequencing of RNA can identify and quantify several chemical modifications on RNA molecules, focusing primarily on m6A. A central goal of the work is to compare this approach with an independent chemical-based method (glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI), using the same RNA samples, in order to assess reproducibility, false-positive signals, and sensitivity across a range of detection strategies. The authors further aim to demonstrate the biological utility of this approach by applying it to two human cell types, primary human fibroblasts and HD10.6 neurons. While the manuscript also reports detection of additional RNA modifications (pseudouridine and m5C, the depth of analysis and strength of controls are greatest for m6A, which forms the primary focus of the study

Strengths:

A strength of this work is the direct comparison of two distinct measurement approaches performed on the same RNA input material; this has not been done in other recently published benchmarking studies evaluating the utility of the recent direct RNA sequencing for calling m6A. The authors systematically test multiple analysis models and show that, when appropriate filtering is applied, detection of modified sites is reproducible across software versions. The use of synthetic RNA standards and METTL3 inhibitors as negative controls helps to reinforce the overall results.

The data show good agreement between the two methods at higher m6A modification levels, supporting the conclusion that direct RNA sequencing can reliably detect high-confidence modification sites. The authors also demonstrate that this approach can, in principle, provide information at the level of individual RNA variants (although only one example was provided), which is difficult to achieve with short-read methods. The methodology described here is likely to be useful to others seeking to apply similar approaches to identify and quantify m6A. The study also explores the detection of other RNA modifications, which highlights the broader potential of the approach, although these analyses are necessarily more exploratory given the more limited controls and data available.

Weaknesses:

Despite these strengths, several issues limit the interpretation of the results and should be clarified for readers.

First, the authors appropriately address false-positive signals by estimating expected false-positive rates and by quantitatively comparing sequence motif enrichment before and after filtering. These analyses provide important support for the use of stoichiometry-based thresholds and demonstrate that filtering substantially improves specificity. However, even after filtering, a subset of detected sites remains outside the expected sequence context. It therefore remains unclear to what extent these non-canonical sites reflect genuine biology versus residual technical artifacts.

Second, claims regarding the ability of direct RNA sequencing to resolve modification patterns across different RNA variants are supported by very limited evidence. The conclusion that this approach provides superior isoform-level quantification relative to short-read methods is based largely on a single gene example. While this case is interesting, it does not establish how widespread or general this advantage is. A broader analysis indicating how many genes show isoform-specific modification patterns detectable by this method, and how often these are missed by the comparison approach, would be necessary to support a general claim.

Third, the biological interpretation of cell type-specific differences in modification levels remains underdeveloped. Although differences in modification stoichiometry are reported between fibroblasts and neuron-derived cells, the functional consequences of these differences are not addressed. It is unclear whether changes in modification levels are associated with differences in RNA abundance, stability, or translation. As a result, statements suggesting that these modifications fine-tune core cellular pathways are speculative and should either be supported with additional analyses or framed more cautiously.

Related to this point, differences in gene expression between the two cell types are a potential confounding factor. The pathway enrichment patterns presented appear biased toward particular functional categories, but without clear control for differential gene expression, it is difficult to determine whether the observed enrichment reflects cell type-specific regulation of RNA modification or simply differences in which genes are expressed. Clarifying how background gene sets were defined for these analyses would help readers interpret the results.

The manuscript also suggests broader differences in overall modification levels between cell types, but this is not validated using an independent global assay. An orthogonal measurement of total modification levels on polyadenylated RNA (for example, dot blot) would help place site-specific stoichiometry differences in a clearer biological context.

Finally, the effects of the METTL3 inhibitor on these cell types are not fully characterized. While changes in m6A modification patterns are reported following treatment, the manuscript does not address whether the treatment affects cell growth or viability.

Appraisal of conclusions and impact:

Overall, the study provides an informative technical assessment of direct RNA sequencing for modification detection and establishes clear conditions under which the method performs well. The evidence strongly supports conclusions related to technical benchmarking, reproducibility, and the importance of filtering and controls, particularly for m6A. In contrast, conclusions regarding isoform-specific regulation and cell type-specific biological roles of RNA modification are less well supported by the data currently presented, and would benefit from either additional analysis or more restrained interpretation.

The work is likely to have a meaningful impact as a practical reference for researchers using direct RNA sequencing, particularly by clarifying sources of false positives and the value of appropriate controls. With clearer limits placed on biological interpretation or more data presented in support of the biological interpretation, the study would serve as a valuable reference for users seeking to apply these technologies reliably.

Reviewer #1 (Public review):

Summary:

This study aims to investigate the development of infants' responses to music by examining neural activity via EEG and spontaneous body kinematics using video-based analysis. The authors also explore the role of musical pitch in eliciting neural and motor responses, comparing infants at 3, 6, and 12 months of age.

Strengths:

A key strength of the study lies in its analysis of body kinematics and modeling of stimulus-motor coupling, demonstrating how the amplitude envelope of music predicts infant movement, and how higher musical pitch may enhance auditory-motor synchronization.

EEG data provide evidence for enhanced neural responses to music compared to shuffled auditory sequences. These findings ecourage further investigation of the proposed developmental trajectory of neural responses to music and their link to musical behavior in infants.

Comments on revisions:

The authors have addressed my questions in their revision. I have no other questions. Thank you for the opportunity to read and evaluate this interesting study and also for all the work carried out in response to the comments.

Reviewer #1 (Public review):

Summary:

This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's co-factor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Fig. 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Fig. 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Fig. 2). They present data suggesting that in 73% of SGCs BMP signaling is low (assessed by dad-lacZ) (Fig. 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Fig. 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Fig. 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Fig. 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on non-mutant cells (i.e., SGCs) to prevent their differentiation, similar to what in seen in the ovarian stem cell niche. This preprint from Shaowei Zhao and colleagues presents results that suggest tumorous germline stem cells (GSCs) in the Drosophila ovary mimic the ovarian stem cell niche and inhibit the differentiation of neighboring non-mutant GSC-like cells. The authors use FRT-mediated clonal analysis driven by a germline-specific gene (nos-Gal4, UASp-flp) to induce GSC-like cells mutant for bam or bam's co-factor bgcn. Bam-mutant or bgcn-mutant germ cells produce tumors in the stem cell compartment (the germarium) of the ovary (Fig. 1). These tumors contain non-mutant cells - termed SGC for single-germ cells. 75% of SGCs do not exhibit signs of differentiation (as assessed by bamP-GFP) (Fig. 2). The authors demonstrate that block in differentiation in SGC is a result of suppression of bam expression (Fig. 2). They present data suggesting that in 73% of SGCs BMP signaling is low (assessed by dad-lacZ) (Fig. 3) and proliferation is less in SGCs vs GSCs. They present genetic evidence that mutations in BMP pathway receptors and transcription factors suppress some of the non-autonomous effects exhibited by SGCs within bam-mutant tumors (Fig. 4). They show data that bam-mutant cells secrete Dpp, but this data is not compelling (see below) (Fig. 5). They provide genetic data that loss of BMP ligands (dpp and gbb) suppresses the appearance of SGCs in bam-mutant tumors (Fig. 6). Taken together, their data support a model in which bam-mutant GSC-like cells produce BMPs that act on non-mutant cells (i.e., SGCs) to prevent their differentiation, similar to what in seen in the ovarian stem cell niche.

Strengths:

(1) Use of an excellent and established model for tumorous cells in a stem cell microenvironment

(2) Powerful genetics allow them to test various factors in the tumorous vs non-tumorous cells

(3) Appropriate use of quantification and statistics

Weaknesses:

(1) What is the frequency of SGCs in nos>flp; bam-mutant tumors? For example, are they seen in every germarium, or in some germaria, etc or in a few germaria.

This concern was addressed in the rebuttal. The line number is 106, not line 103.

(2) Does the breakdown in clonality vary when they induce hs-flp clones in adults as opposed to in larvae/pupae?

This concern was addressed in the rebuttal. However, these statements are no on lines 331-335 but instead starting on line 339. Please be accurate about the line numbers cited in the rebuttal. They need to match the line numbers in the revised manuscript.

(3) Approximately 20-25% of SGCs are bam+, dad-LacZ+. Firstly, how do the authors explain this? Secondly, of the 70-75% of SGCs that have no/low BMP signaling, the authors should perform additional characterization using markers that are expressed in GSCs (i.e., Sex lethal and nanos).

The authors did not perform additional staining for GSC-enriched protein like Sex lethal and nanos.

(4) All experiments except Fig. 1I (where a single germarium with no quantification) were performed with nos-Gal4, UASp-flp. Have the authors performed any of the phenotypic characterizations (i.e., figures other than figure 1) with hs-flp?

In the rebuttal, the authors stated that they used nos>flp for all figures except for Fig. 1I. It would be more convincing for them to prove in Fig. 1 than there is not phenoytpic difference between the two methods and then switch to the nos>FLP method for the rest of the paper.

(5) Does the number of SGCs change with the age of the female? The experiments were all performed in 14-day old adult females. What happens when they look at young female (like 2-day old). I assume that the nos>flp is working in larval and pupal stages and so the phenotype should be present in young females. Why did the authors choose this later age? For example, is the phenotype more robust in older females? or do you see more SGCs at later time points?

The authors did not supply any data to prove that the clones were larger in 14-day-old flies than in younger flies. Additionally, the age of "younger" flies was not specified. Therefore, the authors did not satisfactorily answer my concern.

(6) Can the authors distinguish one copy of GFP versus 2 copies of GFP in germ cells of the ovary? This is not possible in the Drosophila testis. I ask because this could impact on the clonal analyses diagrammed in Fig. 4A and 4G and in 6A and B. Additionally, in most of the figures, the GFP is saturated so it is not possible to discern one vs two copies of GFP.

In the rebuttal, the authors stated that they cannot differential one vs two copies of GFP. They used other clone labeling methods in Fig. 4 and 6. I think that the authors should make a statement in the manuscript that they cannot distinguish one vs two copies of GFP for the record.

(7) More evidence is needed to support the claim of elevated Dpp levels in bam or bgcn mutant tumors. The current results with dpp-lacZ enhancer trap in Fig 5A,B are not convincing. First, why is the dpp-lacZ so much brighter in the mosaic analysis (A) than in the no-clone analysis (B); it is expected that the level of dpp-lacZ in cap cells should be invariant between ovaries and yet LacZ is very faint in Fig. 5B. I think that if the settings in A matched those in B, the apparent expression of dpp-lacZ in the tumor would be much lower and likely not statistically significantly. Second, they should use RNA in situ hybridization with a sensitive technique like hybridization chain reactions (HCR) - an approach that has worked well in numerous Drosophila tissues including the ovary.

The HCR FISH in Fig.5 of the revised manuscript needs an explanation for how the mRNA puncta were quantified. Currently, there is no information in the methods. What is meant but relative dpp levels. I think that the authors should report in and unbiased manner "number" of dpp or gbb puncta in TFs. For the germaria, I think that they should report the number of puncta of dpp or gbb divide by the total area in square pixels counted. Additionally, the background fluorescence is noticeably much higher in bamBG/delta86 germaria, which would (falsely) increase the relative intensity of dpp and gbb in bam mutants. Although, I commend the authors for performing HCR FISH, these data are still not convincing to me.

(8) In Fig 6, the authors report results obtained with the bamBG allele. Do they obtain similar data with another bam allele (i.e., bamdelta86)?

The authors did not try any experiments with the bamdelta86 allele, despite this allele being molecularly defined, where the bamBG allele is not defined.

Comments on second revision:

The authors have adequately addressed several points. However, there is still no information in the material and methods for how they measured and quantified the HCR-FISH probe signal. They have the same size region that they use for each genotype, but they do not control for the number of nuclei in each square. I would also be helpful if they provided a different image for the gbb probe stained in the mutant background. It is the only panel that does not have other germaria in very close proximity. I am still not fully convinced of the HCR data, esp for gbb.

Reviewer #1 (Public review):

This manuscript makes a significant contribution to the field by exploring the dichotomy between chemical synaptic and gap junctional contributions to extracellular potentials. While the study is comprehensive in its computational approach, adding experimental validation, network-level simulations, and expanded discussion on implications would elevate its impact further.

Strengths:

Novelty and Scope:

The manuscript provides a detailed investigation into the contrasting extracellular field potential (EFP) signatures arising from chemical synapses and gap junctions, an underexplored area in neuroscience.<br /> It highlights the critical role of active dendritic processes in shaping EFPs, pushing forward our understanding of how electrical and chemical synapses contribute differently to extracellular signals.

Methodological Rigor:

The use of morphologically and biophysically realistic computational models for CA1 pyramidal neurons ensures that the findings are grounded in physiological relevance.<br /> Systematic analysis of various factors, including the presence of sodium, leak, and HCN channels, offers a clear dissection of how transmembrane currents shape EFPs.

Biological Relevance:

The findings emphasize the importance of incorporating gap junctional inputs in analyses of extracellular signals, which have traditionally focused on chemical synapses.<br /> The observed polarity differences and spectral characteristics provide novel insights into how neural computations may differ based on the mode of synaptic input.

Clarity and Depth:

The manuscript is well-structured, with logical progression from synchronous input analyses to asynchronous and rhythmic inputs, ensuring comprehensive coverage of the topic.

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

The authors Hall et al. establish a purification method for snake venom metalloproteinases (SVMPs). By generating a generic approach to purify this divergent class of recombinant proteins, they enhance the field's accessibility to larger quantity SVMPs with confirmed activity and, for some, characterized kinetics. In some cases, the recombinant protein displayed comparable substrate specificity and substrate recognition compared to the native enzyme, providing convincing evidence of the authors' successful recombinant expression strategy. Beyond describing their route towards protein purification, they further provide evidence for self-activation upon Zn2+ incubation. They further provide initial insights on how to design high throughput screening (HTS) methods for drug discovery and outline future perspectives for the in-depth characterization of these enzyme classes to enable the development of novel biomedical applications.

Strengths:

The study is well presented and structured in a compelling way and the universal applicability of the approach is nicely presented.<br /> The purification strategy results in highly pure protein products, well characterized by size exclusion chromatography, SDS page as well as confirmed by mass spectrometry analysis. Further, a significant portion of the manuscript focuses on enzyme activity, thereby validating function. Particularly convincing is the comparability between recombinant vs. native enzymes; this is successfully exemplified by insulin B digestion. By testing the fluorogenic substrate, the authors provide evidence that their production method of recombinant protein can open up possibilities in HTS. Since their purification method can be applied to three structurally variable SVMP classes, this demonstrates the robust nature of the approach.

Weakness

The product obtained from the purification protocol appears to be a heterogenous mixture of self-activated and intact protein species. The protocol would benefit from improved control over the self-activation process. The authors explain well why they cannot deplete Zn2+ in cell culture or increase the pH to prevent autoactivation during the current purification steps. However, this leads me to the suggestion, if the His tag could be exchanged to a different tag that is less pH sensitive and not dependent on divalent ions (Strep-Tactin XT?) to allow for removal of divalent ions and low pH during purification steps. Another suggestion would be if they could replace the endogenous protease cleavage site in their expression construct design to a TEV protease recognition site, for example, to have more control over activation of the recombinant proteins.

The graphic to explain the universal applicability of the approach, Figure S1, has some mistakes, like duplication of text, an arrow without a meaning and should be revised.

Overall, the authors successfully purified active SVMP proteins of all three structurally diverse classes in high quality and provided convincing evidence throughout the manuscript to support their claims. The described method will be of use for a broader community working with self-activating and cytotoxic proteases.

Comment on the revised version:

I find that the clarity and overall structure of the manuscript have improved. However, the weakness I previously highlighted has neither been addressed experimentally nor convincingly explained. Therefore, the assessment stayed unchanged from my side.

Reviewer #1 (Public review):

Summary:

This study identifies HSD17B7 as a cholesterol biosynthesis gene enriched in sensory hair cells, with demonstrated importance for auditory behavior and potential involvement in mechanotransduction. Using zebrafish knockdown and rescue experiments, the authors show that loss of hsd17b7 reduces cholesterol levels and impairs hearing behavior. They also report a heterozygous nonsense variant in a patient with hearing loss. The gene mutation has a complex and somewhat inconsistent phenotype, appearing to mislocalize, reduce mRNA and protein levels, and alter cholesterol distribution, supporting HSD17B7 as a potential deafness gene.

The study presents an interesting deafness candidate with a complex mechanism, and highlights an underexplored role for cholesterol (and lipids) in hair cell function.

The authors were very responsive to the initial reviews, and the manuscript is now significantly stronger.

Strengths:

- HSD17B7 is a new candidate deafness gene with plausible biological relevance.

- Cross-species RNAseq convincingly shows hair-cell enrichment.

- Lipid metabolism, particularly cholesterol homeostasis, is an emerging area of interest in auditory function.

- The connection between cholesterol levels and MET is potentially impactful and, if substantiated, would represent a significant advance.

- The localization of HSD17B7 is reasonably convincing, despite the lack of a KO control: In HEI-OC1 cells, HSD17B7 localizes to the ER, as expected. In mouse hair cells, the staining pattern is cytosolic. The developmental increase between P1 and P4, and the higher expression in OHCs aligns nicely with RNAseq data.

Weaknesses:

- The pathogenic mechanism of the E182STOP variant is unclear: The mutant protein presumably does not affect WT protein localization, arguing against a dominant-negative effect. Yet, overexpression of HSD17B7-E182* alone causes toxicity in zebrafish and it binds and mislocalizes cholesterol in HEI-OC1 cells, suggesting some gain-of-function or toxic effect. In addition, the mRNA of the variant has low expression level, suggesting nonsense-mediated decay. The mechanistic conclusions of the study are therefore not as clear cut as one would had hoped, but it might just be a reflection of real biological complexity.

- The link to human deafness is based on a single heterozygous patient with no syndromic features. Given that nearly all known cholesterol metabolism disorders are syndromic, this raises concerns about causality or specificity. HSD17B7 is therefore, at this point, a candidate deafness gene, and not a fully established "novel deafness gene". This is acknowledged by the authors.

- This study does not directly investigate how reduced cholesterol levels affect MET. However, this is not a significant limitation given the study's scope, and it is reasonable that such detailed functional analyses are left to specialists in hair cell physiology.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors describe the use of BindCraft computational protein design to create a series of binders to the chemokine CCL25. This chemokine normally mediates CCR9-dependent trafficking of immune cells to the gut, making it a potential target for the treatment of inflammatory bowel disease and related conditions. Importantly, CCL25 also binds a scavenging receptor, ACKR4. The computational protein design approach used does not involve defining particular epitopes to be targeted, allowing a free search for any potential interaction surface.

Among four designs tested, three were predicted to interact at a similar site on the chemokine, while a fourth clone, VUP25111, was predicted to bind to a different site. All four designs showed binding to CCL25, with similar high-nM KD values in all cases. The first three clones showed evidence of direct competition with the receptor for CCL25 binding, while VUP25111 showed incomplete inhibition of binding. In functional assays, all clones acted as antagonists except for VUP25111, which inhibited arrestin recruitment by CCR9, but did not affect G protein activation by CCR9 or arrestin recruitment by ACKR4 (which signals exclusively through arrestin and not G protein).

Strengths:

The work is completed to a high technical standard, and the functional diversity of the clones is intriguing. It is exciting to see pathway-selective modulation of signaling, and this basic paradigm is likely to generalize to other chemokine/receptor systems. The exceptional complexity of chemokine signaling makes this an excellent area to explore the development of modulators that can restrict signaling to a specific subset of receptors.

Weaknesses:

No major weaknesses were noted by this reviewer.

Reviewer #1 (Public review):

This manuscript makes a significant contribution to the field by exploring the dichotomy between chemical synaptic and gap junctional contributions to extracellular potentials. While the study is comprehensive in its computational approach, adding experimental validation, network-level simulations, and expanded discussion on implications would elevate its impact further.

Strengths:

Novelty and Scope:<br /> The manuscript provides a detailed investigation into the contrasting extracellular field potential (EFP) signatures arising from chemical synapses and gap junctions, an underexplored area in neuroscience.<br /> It highlights the critical role of active dendritic processes in shaping EFPs, pushing forward our understanding of how electrical and chemical synapses contribute differently to extracellular signals.

Methodological Rigor:<br /> The use of morphologically and biophysically realistic computational models for CA1 pyramidal neurons ensures that the findings are grounded in physiological relevance.<br /> Systematic analysis of various factors, including the presence of sodium, leak, and HCN channels, offers a clear dissection of how transmembrane currents shape EFPs.

Biological Relevance:<br /> The findings emphasize the importance of incorporating gap junctional inputs in analyses of extracellular signals, which have traditionally focused on chemical synapses.<br /> The observed polarity differences and spectral characteristics provide novel insights into how neural computations may differ based on the mode of synaptic input.

Clarity and Depth:<br /> The manuscript is well-structured, with a logical progression from synchronous input analyses to asynchronous and rhythmic inputs, ensuring comprehensive coverage of the topic.

Weaknesses and Areas for Improvement:

Generality and Validation:<br /> The study focuses exclusively on CA1 pyramidal neurons. Expanding the analysis to other cell types, such as interneurons or glial cells, would enhance the generalizability of the findings.<br /> Experimental validation of the computational predictions is entirely absent. Empirical data correlating the modeled EFPs with actual recordings would strengthen the claims.

Role of Active Dendritic Currents:<br /> The paper emphasizes active dendritic currents, particularly the role of HCN channels in generating outward currents under certain conditions. However, further discussion of how this mechanism integrates into broader network dynamics is warranted.

Analysis of Plasticity:<br /> While the manuscript mentions plasticity in the discussion, there are no simulations that account for activity-dependent changes in synaptic or gap junctional properties. Including such analyses could significantly enhance the relevance of the findings.

Frequency-Dependent Effects:<br /> The study demonstrates that gap junctional inputs suppress high-frequency EFP power due to membrane filtering. However, it could delve deeper into the implications of this for different brain rhythms, such as gamma or ripple oscillations.

Visualization:<br /> Figures are dense and could benefit from more intuitive labeling and focused presentations. For example, isolating key differences between chemical and gap junctional inputs in distinct panels would improve clarity.

Contextual Relevance:<br /> The manuscript touches on how these findings relate to known physiological roles of gap junctions (e.g., in gamma rhythms) but does not explore this in depth. Stronger integration of the results into known neural network dynamics would enhance its impact.

Suggestions for Improvement:

Broader Application:<br /> Simulate EFPs in multi-neuron networks to assess how the findings extend to network-level interactions, particularly in regions with mixed synaptic connectivity.

Experimental Correlation:<br /> Collaborate with experimental groups to validate the computational predictions using in vivo or in vitro recordings.

Mechanistic Insights:<br /> Provide a more detailed mechanistic explanation of how specific ionic currents (e.g., HCN, sodium, leak) interact during gap junctional vs. chemical synaptic inputs.

Implications for Neural Coding:<br /> Discuss how the observed differences in EFP signatures might influence neural coding, especially in circuits with heavy gap junctional connectivity.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #1 (Public review):

Summary:

This study identifies NK2R as an intestinal GPCR that tunes enterocyte lipid uptake, lipid droplet storage, and chylomicron output, with loss or antagonism enhancing post‑prandial triglyceridemia and epithelial lipid stores, and agonism reducing adiposity and improving glycemia in DIO mice. Through bulk RNA‑seq, deconvolution, DSS colitis, and 16S profiling, the authors link Tacr2 deletion to coordinated induction of epithelial lipid‑metabolic programs, dampened immune gene expression, sex‑specific remodeling of secretory lineages, and male‑biased protection from experimental colitis despite dysbiotic microbiota. This is an overall important and thorough paper on an emerging obesity drug target, but it should temper some interpretations, and the following points would be needed to strengthen the claims in the manuscript.

Strengths:


The study uses an impressive combination of genetic loss‑of‑function, pharmacological agonism/antagonism, transcriptomics, and in vivo physiology to establish NK2R as a bidirectional regulator of epithelial lipid handling. The integration of RNA‑seq, epithelial cell‑type deconvolution, DSS colitis, and microbiome profiling provides a rich, systems‑level view of how Tacr2 deletion reshapes epithelial metabolism, lineage allocation, and inflammatory responsiveness in a sex‑specific manner. The gain- and loss‑of‑function data particularly support a model in which NK2R acts as an epithelial metabolic rheostat that restrains lipid absorption and chylomicron export, with downstream consequences for barrier fitness and immune tone.

Weaknesses:

Major points

While the data convincingly establish NK2R's role in epithelial lipid handling, the manuscript arguably overstates a primary "pro‑inflammatory" function for NK2R, given that Tacr2‑/‑ mice show enhanced enterocyte lipid uptake and storage, higher post‑prandial triglycerides, and a dysbiotic microbiota yet reduced mucosal immune gene expression and, in males, protection from DSS colitis. It remains equally plausible that the apparent "protection" reflects a mucosa that is less reactive to unfavorable microbiota rather than genuinely protected, and that NK2R's main function is metabolic, with immune changes emerging secondarily. Such a model would actually help reconcile the long-standing question as to why NK2R antagonism has not translated into clear benefit in clinical trials for GI inflammation over the past several decades.

Without temporal resolution, it is equally plausible that antagonists primarily perturb epithelial lipid homeostasis rather than directly and beneficially modulating immune tone. To discriminate between these possibilities and strengthen the potential direct inflammatory claims, the authors should:

(1) generate epithelial‑specific, immune‑cell-specific, and nociceptor‑specific Tacr2 deletions in the DSS model

(2) test gut‑restricted NK2R agonism versus antagonism under controlled dietary fat conditions for effects on LD load, barrier integrity, and colitis severity

(3) perform ex vivo tachykinin/NK2R stimulation of isolated epithelial versus immune compartments with functional readouts

(4) assess whether microbiota transfer from Tacr2‑/‑ versus WT donors into germ‑free or antibiotic‑treated recipients can recapitulate protection or susceptibility independently of epithelial NK2R status.

Minor points

Additional clarifications on Tac1 and tachykinin receptor expression in male/female colitis models, and validation of the NK2R antibody in KO tissue (or in situ hybridization), would also be needed to strengthen key mechanistic and localization claims.

Reviewer #1 (Public review):

Summary:

The authors have attempted to establish a role for XAP5, a transcriptional regulator they have previously identified for flagellar biogenesis in Chlamydomonas and mice, in primary cilia differentiation.

Strengths:

Genetic and biochemical analysis using a cultured mouse cell line, NIH3T3.

Weaknesses:

(1) The authors have ignored established data that, like in C. elegans and Drosophila, there is in vivo genetic evidence that primary cilia formation is regulated by the RFX transcriptional module (for example, PMID 19887680, PMID 29510665).

(2) The analysis with one mammalian cell line, NIH3T3, while done quite rigorously, is not sufficient. Also, the effect on cilia differentiation is very modest - a shortening of cilia length on XAP5, NONO and SOX5 knockout - which can happen for a variety of reasons, especially in culture conditions. In my view, this relatively mild phenotype does not establish that the XAP5/NONO and SOX5 axis is an important regulator of primary cilia differentiation.

(3) The lack of any data that validates the findings in the model vertebrate is a major weakness of this paper. Validation using clean genetics (whole body knockouts or tissue-specific conditional knockouts) is absolutely essential for these data to be acceptable.

Reviewer #1 (Public review):

Summary:

Heller et al use a murine model of AIRE deficiency, a disease that leads to systemic autoimmune disease, to demonstrate differential effects of selective JAK inhibitors. This group and others have previously demonstrated the efficacy of the JAK1/2 inhibitor ruxolitinib in patients with AIRE deficiency. Here, they focus on the ability of ruxolitinib versus drugs inhibiting either JAK1, JAK2, or JAK3 to alter organ pathology and accumulation of interferon-gamma producing immune cells in the lungs, which are important mediators of inflammation in patients with this disease. The current study provides evidence that selective JAK2 or JAK1 both reduce disease in this mouse model. There is potentially a more beneficial effect of selective JAK2 inhibition, although these differences are minor, and it is uncertain whether this is clinically relevant for patients. They demonstrate that inhibition of JAK3 alone in the mouse was clearly not beneficial for disease. Overall, this study provides evidence for consideration of more selective JAK inhibition in patients with AIRE deficiency.

Strengths:

(1) Robust model for investigating AIRE deficiency.

(2) They combine cellular studies (immune cell production of IFN-g) and robust organ pathology scoring to evaluate the effects of the drugs tested here.

(3) Data clearly demonstrates that JAK3 inhibition, at least as used here, may increase IFN-g production and does not reduce organ pathology.

Weaknesses:

(1) There is no direct comparison of the effects of JAK2 vs. JAK1 inhibition to support that JAK2 inhibition is clearly superior.

(2) They were not able to perform pharmacokinetic studies or measure the efficacy of JAK inhibition in their model, and it is uncertain how the doses of drug used here will translate to the treatment of patients.

(3) It is uncertain whether this study, performed in a murine model, will correspond to tissue/cell specificity of JAK inhibition in patients.

Reviewer #1 (Public review):

Summary:

This study examines the role of the long non-coding RNA Dreg1 in regulating Gata3 expression and ILC2 development. Using Dreg1 deficient mice, the authors show a selective loss of ILC2s but not T or NK cells, suggesting a lineage-specific requirement for Dreg1. By integrating public chromatin and TF-binding datasets, they propose a Tcf1-Dreg1-Gata3 regulatory axis. The topic is relevant for understanding epigenetic regulation of ILC differentiation.

Strengths:

(1) Clear in vivo evidence for a lineage-specific role of Dreg1.

(2) Comprehensive integration of genomic datasets.

(3) Cross-species comparison linking mouse and human regulatory regions.

Weaknesses:

(1) Mechanistic conclusions remain correlative, relying on public data.

(2) Lack of direct chromatin or transcriptional validation of Tcf1-mediated regulation.

(3) Human enhancer function is not experimentally confirmed.

(4) Insufficient methodological detail and limited mechanistic discussion.

Comments on revisions:

The authors have provided clear evidence that Dreg1 is necessary for ILC2 development, but their refusal to perform any mechanistic experiment remains a significant weakness. While their appeal to the 3Rs and the use of public datasets is noted, re-analyzing external data from heterogeneous sources cannot substitute for direct, internal validation of the Tcf1-Dreg1-Gata3 axis in their specific knockout model. This is particularly problematic because ILC2 progenitors, though rare, can be isolated from bone marrow, especially since assays like CUT&Tag and others are specifically designed for low cell numbers. By relying on public T-cell CRISPR screens to justify human ILC2 functions, the authors are substituting cross-cell-type correlation for definitive functional proof. Consequently, the manuscript currently describes a discovery of necessity without providing a verified molecular mechanism, which should be more explicitly reflected in the title and conclusions.

Reviewer #1 (Public review):

In this manuscript, the authors combine single-nucleus RNA sequencing with spatial transcriptomics to generate a spatiotemporal atlas of mouse placental development and explore the role of glycogen trophoblast cells in fetal viability. The study integrates several computational approaches, including trajectory analysis, regulatory network inference, and spatial mapping, together with histology and glycogen measurements. Based on these analyses, the authors propose that glycogen trophoblast cells provide metabolic support that is important for maintaining placental function and fetal survival.

One of the main strengths of the study is the quality and scope of the dataset. The integration of snRNA-seq with Stereo-seq spatial transcriptomics provides a detailed view of placental organization across regions and developmental stages. This type of combined spatial and transcriptional analysis is still relatively rare in placental biology and represents an important contribution to the field. The atlas itself will likely be a valuable resource for future studies.

Another strength is the effort to connect transcriptional findings with tissue-level validation. The glycogen staining and biochemical measurements support the interpretation that glycogen trophoblast cells contribute to placental metabolic function. The spatial analyses identifying macrophage accumulation in the labyrinth region of mutant placentas are also interesting and illustrate how spatial approaches can reveal microenvironmental changes that are difficult to detect otherwise.

The main limitation of the study is that the conclusion that glycogen cells are essential mediators of metabolic support for fetal viability remains partly indirect. The transcriptomic and spatial data strongly suggest a role for these cells, but it is still difficult to determine whether glycogen cell dysfunction is the primary cause of fetal lethality or a consequence of broader placental abnormalities. Clarifying this point would strengthen the central message of the paper.

Similarly, the macrophage accumulation observed in the labyrinth appears consistent with a response to tissue stress or injury, but its relationship to glycogen cell function is not fully explained. A clearer discussion of whether this represents a primary mechanism or a secondary effect would improve the interpretation.

Overall, this is a strong dataset and a useful spatial atlas of placental development. The study provides convincing descriptive insight into glycogen trophoblast biology, and with some clarification of the mechanistic conclusions, the manuscript will be even stronger.

Annotation #1 (5/3/2026): In this segment of the text, we learn that William Henry Singleton and other black men petitioning to enlist in the Union Army to assist in gaining their freedom. I feel that this segment is quite significant because we see how despite their willingness to put themselves at risk for the ideals they stood for, they were not accepted right away nor treated equally as citizens. This should remind all of us how the African Americans had to fight not only against their slavery but also for recognition and basic rights.

Reviewer #1 (Public review):

Summary:

The authors present a new autofocusing method, LUNA (Locking Under Nanoscale Accuracy), designed to overcome severe focus drift, a major challenge in long-term time-lapse microscopy. Using this method, they address a fundamental question in bacterial cold shock response: whether cells halt growth and division following an abrupt temperature downshift. Through single-cell analysis, the authors uncover a multi-phase adaptation process with distinct growth deceleration dynamics, and show that bacterial cells adapt to cold shock in a largely uniform manner across the population. Overall, this work provides new insights into the bacterial cold shock response at the single-cell level, extending beyond what can be inferred from population-level measurements.

Strengths:

(1) The LUNA method shows improved performance compared to existing autofocusing systems, achieving nanoscale precision over a large focusing range. Its focusing speed is sufficient for the experiments presented, with potential for further improvement through faster motors and optimized control algorithms, suggesting broad applicability. Theoretical simulations and experimental validation together provide strong support for the method's robustness.

(2) Using LUNA, the authors address a long-standing question in bacterial physiology: whether cells arrest growth and division during the acclimation phase following cold shock. Single-cell analyses across the full course of cold adaptation reveal features that are obscured in bulk-culture studies. Cells continue to grow and divide at reduced rates while maintaining cell size regulation, and exhibit a three-phase adaptation program with distinct growth dynamics. This response appears uniform across the population, with no evidence for bet-hedging. Overall, the experiments are well designed, and the analyses are solid and support the authors' conclusions.

(3) The authors further propose a model describing how population-level optical density (OD) depends on cell dry mass density, volume, and concentration. Following cold shock, cells grow more slowly and exhibit smaller sizes, explaining the apparently unchanged OD. This model provides a valuable conceptual framework for interpreting OD-based growth measurements, a widely used method in microbiology, and will be of broad interest to the field.

Weaknesses:

No major weaknesses identified.

Comments on revisions:

The authors have thoroughly addressed all of my questions. I thank them for their clear clarifications and thoughtful revisions, and I greatly appreciate their efforts in improving the manuscript.

Reviewer #1 (Public review):

Summary:

The manuscript, titled Hippocampal Single-Cell RNA Atlas of Chronic Methamphetamine Abuse-Induced Cognitive Decline in Mice, focuses on single-cell RNA sequencing (scRNA-seq) analysis following chronic methamphetamine (METH) treatment in mice. The authors propose two hypotheses: (1) METH induces neuroinflammation involving T and NKT cells, and (2) METH alters neuronal stem cell differentiation.

Strengths:

The authors provide a substantial dataset with numerous replicates, offering valuable resources to the research community.

Weaknesses:

Concerns remain regarding the interpretation of the data and the appropriateness of the statistical analyses.

Although the authors provided detailed responses to the reviewer's concerns, I am still concerned that several key issues have not yet been fully addressed in the revised manuscript.

First, in Figure 5, the authors state that neural stem cells (NSCs) preferentially differentiate into astrocytes rather than neuroblasts following METH treatment. However, based on the presented trajectories, it is difficult to visually confirm differences in the relative proportions of astrocyte versus neuroblast differentiation between the control and METH-treated conditions. The current figures do not provide a quantitative or clearly interpretable comparison of lineage allocation that would support this conclusion.

Moreover, in Figures 5C and 5F, the inferred pseudotime trajectories differ both the starting cell populations and the intermediate and terminal cell identities. As a result, the trajectories are not directly comparable between the control and METH conditions. Under these circumstances, it is inappropriate to interpret gene expression changes as occurring along equivalent differentiation paths, and the current analysis does not convincingly support the stated conclusions regarding altered NSC differentiation.

If the authors intend to claim differential gene expression associated with altered differentiation trajectories, the analysis should at minimum present the expression of the same set of genes (e.g., Bsg, Ccl4, Fos, Sox11, Flt1, Hspb1, Igfbp7, and Tmsb10) plotted along a matched trajectory (for example, NSC-to-astrocyte or NSC-to-neuroblast lineages) in both control and METH-treated samples, so that readers can directly compare expression dynamics across conditions.

In addition, several statements throughout the manuscript describing changes in cell-type proportions are not supported by corresponding statistical analyses. For example, in Figure 2C (around line 430), the authors report changes in cell proportions of ~0.1% or 2-3%. Without appropriate statistical testing, it is unclear whether such marginal differences are biologically meaningful or reproducible. The authors should either provide statistical testing (e.g., sample-level proportion analysis with p-values or confidence intervals) or revise the text to describe these findings as descriptive rather than significant changes.

Finally, the reported decrease in astrocyte proportion following METH exposure (from 6.6% to 5.5%), together with the lack of reported changes in neuroblast proportions, appears inconsistent with the trajectory-based conclusion that NSCs preferentially differentiate into astrocytes in METH-treated mice. This apparent discrepancy should be clarified or the conclusions appropriately tempered.

Reviewer #1 (Public review):

Summary:

This study combined high-field fMRI with computational modelling (including a Bayesian population receptive field [pRF] model and functional gradient analysis) in humans to demonstrate that the architecture of the corpus callosum (CC) and its interhemispheric connections is organized into parallel ipsilateral and contralateral streams, rather than functioning as a mixed integration of inputs from both hemispheres. The human findings were validated through preclinical experiments in mice using viral axonal tracing, which revealed a non-overlapping laminar arrangement of axons carrying left and right visual field information.

These results suggest that the CC operates as a set of parallel, segregated pathways, with each stream independently conveying information from one side of the visual field. This organization preserves the spatial origin of visual signals within the white matter. Although the overall concept of interhemispheric parallel pathways is not entirely unexpected, this refined understanding of callosal organization provides important scientific and clinical insights in relation to pathway-specific perturbations and in neurological disorders.

Strengths:

The manuscript is well written, the methodology is sound, and the analyses are carefully conducted. I particularly appreciate the effort to integrate functional and structural approaches and to validate the human neuroimaging findings with more sensitive preclinical techniques, such as viral tracing.

Weaknesses:

Several points require clarification to allow a more complete interpretation of the results. In addition, some further analyses are necessary to fully substantiate the claims made in the manuscript. These are detailed below

Comment 1:

BOLD signals in white matter remain a matter of debate, although this is not the central focus of the present study. Nevertheless, it is important to establish whether the underlying data have sufficient tSNR to support robust pRF estimation in white matter. In Figure 1, the EV appears relatively robust; however, it seems that only the best-fitting examples are shown. In contrast, the group-average EV reported in Figure 2, and the individual maps in the Supplementary Information indicate very low EV values, typically below 5%. In conventional fMRI analyses, thresholds of approximately 15-20% EV are often applied to exclude poor fits that may bias pRF parameter estimates. It appears that no such threshold was applied here. Interestingly, in Figure S6, the average EV for dual pRF models appears to be approximately 17%. Do dual and triple pRF models systematically produce higher EV compared to single pRF models? Additionally, Figure 2 suggests the presence of baseline activation that is captured by the model. Could this be related to a delayed or altered hemodynamic response function (HRF) in white matter? Clarification would be helpful. To better assess the robustness of the reported findings, the authors should provide quantitative measures of tSNR within the white matter tracts where the pRF model was fitted. Furthermore, a plot showing the average BOLD signal during visual stimulation versus baseline in those tracts would greatly strengthen confidence in the signal quality.

Although the reported linear relationship between pRF size and eccentricity, as well as the test-retest reliability analyses, suggest the presence of consistent receptive field estimates, these analyses are based on distributions and may lack the sensitivity required to differentiate single, dual, and triple pRF models. Moreover, the pRF estimates within the FMA appear noisy, particularly at the individual level (Figure S4), making it difficult to clearly dissociate information originating from the left and right hemifields.

Comment 2.1:

The Bayesian modelling approach is interesting and robust. However, as I understand it, the authors must specify a priori the number of pRFs to be estimated. This introduces a strong assumption about the expected underlying receptive field structure. An alternative Bayesian approach, such as micro-probing (Carvalho et al., 2020), does not require prior assumptions regarding the number or shape of pRFs. Instead, it estimates receptive field profiles in a more data-driven manner and provides a direct visualization of the pRF structure. Implementing such an approach, or at least comparing it with the current modelling strategy, could yield more reliable and potentially less biased estimates of multiple pRFs, particularly in white matter where signal quality is limited.

Comment 2.2:

Some clarifications regarding the pRF model are needed: in the Methods section, the authors mention the use of a Difference-of-Gaussians (DoG) model. However, it appears from the Results that the analyses were performed using a single-Gaussian model. Additionally, in Section 5.6, the authors state that six different pRF models were tested. Which specific models were included in this comparison? A clear description of each model, along with justification for the final model selection criteria, would help better understand the study

Comment 3:

Throughout the manuscript, the authors repeatedly refer to laminar-specific findings. However, the reported functional resolution of 1.6 mm isotropic is insufficient to reliably resolve cortical layers. Given this limitation, the laminar interpretations appear overstated. For example, in the Discussion section titled "Integrating White Matter with Laminar-Resolved Function", the authors state: "The combination of anatomically segregated white matter pathways with functionally specific cortical laminae presents a powerful synergy for human brain circuit research." Given the spatial resolution of the functional data, how are laminar-specific functional claims justified?

Similarly, the authors suggest that: "It becomes possible to assess not just if the CC is damaged, but precisely which directional pathways are compromised-either the pathways projecting from the lesioned hemisphere, or those projecting to the other, or both." It is unclear to me how the current methodology uniquely enables this level of directional specificity, and whether this was not already feasible using existing structural and diffusion-based approaches. The authors should clarify what is genuinely novel in this study.

Comment 4:

In the Discussion, the authors state: "These findings fundamentally reframe our understanding of interhemispheric communication, moving beyond static connectivity to reveal a dynamic, directionally specific highway where spatial location encodes the origin of information. This framework provides a novel blueprint for decoding directional information flow in the living human brain." Based on the analyses presented, it is unclear how the findings of this study demonstrate dynamic connectivity or true directional specificity. The reported results appear to characterize spatial organization and segregation of callosal pathways, but they do not measure the directionality of information flow, temporal dynamics, or causal directionality between hemispheres. To substantiate claims regarding dynamic or directional communication, additional analyses, such as connective field model (Haak et al.2013), effective connectivity modelling, time-resolved approaches, or perturbation-based methods (neuromodulation) would be required. As currently presented, the findings seem to support structural and functional segregation rather than dynamic or directionally resolved interhemispheric information transfer. The authors should either provide stronger evidence for these claims or moderate them.

Comment 5:

I agree with the authors that pooling of information across hemispheres represents a plausible explanation for the presence of dual pRFs. As discussed in the manuscript, such an effect would be expected to predominantly affect pRFs located near the vertical meridian. However, Figures S6C and S6D do not appear to demonstrate that bilateral pRFs are preferentially located along the vertical meridian.

Reviewer #1 (Public review):

Summary:

The manuscript entitled "Autonomic reflex plasticity associates with time-dependent SUDEP susceptibility in a murine model with hyperactive stress circuits" by Dr. Saunders and colleagues combined a traditional mouse model of SUDEP, ventral intrahippocampal kainite (vIHKA) injection, with a genetic model of chronic hyperactivity of central corticotropin-releasing hormone (CRH) neurons (Kcc2/Crh) that further increases the risk of SUDEP in the weeks following seizure.

Strengths:

Their results show during spontaneous seizures Kcc2/Crh mice had more pronounced reflex-like ictal bradycardias compared to WT controls that notably occurred prior (~10 sec) to seizure termination and had greater autonomic disturbances compared to WT controls, including a pronounced serotonin-mediated Bezold Jarisch reflex. These results show chronic hyperactivity of central corticotropin-releasing hormone (CRH) neurons (Kcc2/Crh) increased autonomic disturbances and risk of SUDEP in a kainic acid model of epilepsy.

Weaknesses:

This study could be improved with a more thorough assessment of heart rate, blood pressure and breathing during and following the seizures, and in particular the fatal event. It is unclear if the bradycardias were spontaneous or a result of preceding central or obstructive apneas, oxygen desaturations, hypercapnia, arrhythmias, or other possible triggers.

Considerable prior work in the literature suggests SUDEP could be mediated, in some patients, by a burst of parasympathetic activity to the heart. Were the heart rate changes in these animals during seizures inhibited or blocked by atropine or atenolol?<br /> The injection of the 5HT agonist phenylbiguanide into the right jugular is not a selective approach for activating the Bezold Jarisch Reflex (BJR), which is caused by increased activity of intracardiac sensory neurons (generally activated with ischemia or a combination of low preload with high contractility). The results should be interpreted more cautiously, as a response to systemic administration of phenylbiguanide only.

Reviewer #1 (Public review):

In this study, Szinte et al. measured the spatial selectivity of fMRI BOLD responses while subjects viewed dynamic noise stimuli vignetted by a moving bar aperture. Subjects viewed these moving bar stimuli as they fixated at one of three screen locations. This design enabled the authors to test whether fMRI responses are better explained by a model in which stimulus location is encoded relative to the retina or relative to the screen (in other words, 'retintopic' vs. 'spatiotopic' encoding). In retinotopic encoding, the pRFs should move with the eyes. In spatiotopic encoding, the pRFs should be locked to particular screen locations, regardless of eye position. The results are unambiguous: the retinotopic model wins.

A number of prior human fMRI studies have addressed this issue, and there is an overwhelming consensus in the field that spatial encoding throughout human visual cortex (and high-level cortex) is retinotopic (during fixation). All of the results shown in the present manuscript are consistent with these earlier observations. Szinte et al. also find that the degree of retinotopic selectivity is not affected by the task or locus of spatial attention. This too has been observed in multiple prior studies.

So, while this manuscript is primarily confirmatory, the study does nonetheless provide valuable measurements at 7T with a higher signal-to-noise ratio and high spatial resolution than previous studies. The authors also apply an innovative Bayesian decoding analysis (which is beautifully documented on their webpage, with a step-by-step tutorial and ample examples). So, a major strength of this paper is the methods; this study does set a high standard and is an ideal example for a rigorous, replicable analysis pipeline and cutting-edge statistical inference.

The results focus on the spatial profile of pRFs with different eye positions. However, the main idea behind eye-position gain fields is that the amplitude of the visual responses changes with eye position. I could not find any analysis testing response amplitude as a function of eye position. In the Discussion, the authors assert: "We did not find an influence of gaze position at the level of individual voxels nor at the level of visual areas." The authors speculate that this might be because gain fields have a salt-and-pepper organization in the cortex that cancel out when pooled across a voxel. While the salt-and-pepper explanation seems like perfectly fine speculation, here they are discussing a result that isn't shown in the Results!

Several prior human fMRI studies have reported eye position gain fields in humans, suggesting that the salt-and-pepper explanation is not correct. Rather, it is likely the case that the authors did not test a sufficiently wide range of eye positions to detect a gain modulation. For example, a study from Merriam et al. (J. Neurosci, 2013), which is mysteriously not cited here, measured both the spatial selectivity of visual receptive fields AND the response amplitude at 8 different eye positions that were spaced by as much as 24 degrees of visual angle (including both vertical and horizontal changes in eye position). Under these conditions, Merriam et al. did find reliable modulation in response amplitude with changes in eye position, even though the spatial selectivity of the responses did not change. Importantly, Merriam et al. found that visual response selectivity was consistent with a retinotopic reference frame (not a spatiotopic reference frame) and that this selectivity was invariant to the attention task. Consideration of these issues suggests that the experimental design used in the current experiment may have precluded the detection of eye position gain fields. The current manuscript would be much improved by a careful consideration of this prior literature, which is so closely related to what the authors report here.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

A previous concern was that the recognition effect appeared restricted to a subgroup of participants. The authors clarify that the bilateral STG and left IFG effects were present in both groups - it was only the right IFG modulation that was group-dependent. This is an important distinction and is now clearer in the revised manuscript. The timing of the effect emerging in a specific testing window also appears less arbitrary given the authors' explanation that prior work guided the analytical approach, and that task difficulty was expected to determine whether recognition would appear in earlier or later test blocks.

The sample size question is handled honestly. A power analysis based on a related ANOVA study produced an implausibly small estimate of N=5-7, which the authors rightly set aside. Aligning with fNIRS neonate studies - where mean sample sizes around N=24 are standard - is defensible, and the within-subject design with mixed-model analysis does improve sensitivity relative to simpler approaches. This is now explained in the manuscript.

The episodic memory framing has been scaled back appropriately. The revised discussion is clear that the study demonstrates what-who binding - an early component of episodic-like processing - rather than mature episodic memory in the Tulvingian sense. This is a more honest characterization of what the paradigm can show, and it opens a reasonable developmental question about how the remaining components (where, when) come online over the first months and years of life.

Weaknesses

The weaknesses are largely interpretive rather than fatal to the core findings. The absence of a same-speaker interference control within the current paradigm means the causal role of speaker change cannot be established entirely from internal evidence alone - the inference relies partly on comparison with Benavides-Varela et al. (2011), which used a somewhat different design. This is a reasonable approach given the ethical and practical constraints of testing newborns, and the authors are transparent about it, but readers should keep in mind that the conclusion about speaker change as the critical variable is supported by converging evidence across studies rather than a direct within-study manipulation.

Overall, the study contributes new and meaningful data on an underexplored aspect of early speech processing: the role of the speaker as a contextual dimension in word memory. The findings, taken together with the prior literature, tell a coherent story and have real implications for theories of early language acquisition and the developmental origins of episodic-like memory. The paradigm is sound and the results are worth pursuing in larger and more controlled follow-up studies.

Reviewer #1 (Public review):

Summary

The manuscript by K.H. Lee et al. presents Spyglass, a new open-source framework for building reproducible pipelines in systems neuroscience. The framework integrates the NWB (Neurodata Without Borders) data standard with the DataJoint relational database system to organize and manage analysis workflows. It enables the construction of complete pipelines, from raw data acquisition to final figures. The authors demonstrate their capabilities through examples, including spike sorting, LFP filtering, and sharp-wave ripple (SWR) detection. Additionally, the framework supports interactive visualizations via integration with Figurl, a platform for sharing neuroscience figures online.

Strengths:

Reproducibility in data analysis remains a significant challenge within the neuroscience community, posing a barrier to scientific progress. While many journals now require authors to share their data and code upon publication, this alone does not ensure that the code will execute properly or reproduce the original results. Recognizing this gap, the authors aim to address the community's need for a robust tool to build reproducible pipelines in systems neuroscience.

Comments on revisions:

In this revised version, the authors have addressed the majority of the concerns raised in the initial review. The manuscript is clearer, the documentation and explanations have been strengthened, and several important practical issues-particularly regarding usability, terminology, and deployment-have been meaningfully improved. While the framework continues to position itself both as a flexible analysis environment and as a mechanism for freezing and preserving reproducible pipelines, the authors have clarified their rationale for maintaining this dual role. I have no additional comments at this stage.

Reviewer #1 (Public review):

Summary:

The authors wanted to determine whether the set-19 gene, one of 38 SET-domain containing genes in C elegans, has a clear function in vivo with respect to lysine methylation. The question is not only whether it can modify this histone tail residue, but also what the impact of a loss of this locus is on the inheritance of repressive chromatin states.

Strengths:

The authors clearly achieved their goal, and it is convincingly shown that SET_19 is indeed a somatic cell histone methyltransferase with a striking specificity for H3K23. There is both recombinant protein work, quantitative mapping in vivo, of histone marks and transcriptional changes, and the authors rule out some other hypotheses that have been in the literature. Overall, this provides a compelling argument that SET-19 is indeed the major somatic cell HMT for this residue. Interestingly, the phenotypes are rather minimal, consistent with redundancy in the physiological roles of histone methylation, and redundancy as well in HMT function. For the most part, the data are not over-interpreted. The genetic alleles used, assuming they are confirmed, were revealing and well-documented.

Weaknesses:

The major weaknesses are easily fixed. The major weaknesses mainly reflect a slight overstatement of certain data (claiming insignificance, when it is not clear how that was determined) and claiming a bit too much about SET-32, which was independently claimed to be an H3K23 HMT. Clearly, the two SET domain enzymes are not redundant, nor is the claim that SET-32 has no role in H3K23 methylation completely convincing. Especially in germline or embryonic conditions. Finally, the imaging is not of very high quality, nor are the images fully quantitated. These points can be easily remedied.

Reviewer #2 (Public review):

Summary:

This study aims to examine the effects of the subcellular localization of the mammalian clock protein PER2 and its dedicated binding partners CRY1 and the kinase CK1. Using a combination of transient transfection and a Dox-inducible expression system, they show that CRY1 promotes nuclear retention of PER2, and that phosphorylation of PER2 by CK1 promotes cytoplasmic localization and release of CRY1. Changes in complex assembly and subcellular localization could impact the transcriptional repressive function of the CK1-PER2-CRY1 complex in the molecular clock.

Strengths:

The study establishes a system of transient transfection and Dox-inducible expression that allows for strict temporal control of the presence of fluorescently-tagged clock proteins. This is essential to conduct time-lapse microscopy studies that determine changes in the apparent subcellular localization and stability of associated clock proteins. With the potential caveats of overexpression set aside, the authors make use of good controls and supplement cell-based work with in vitro experiments where possible. The discovery that phosphorylation of PER2 by CK1 in the nucleus leads to cytoplasmic localization of PER2 and PER2-CRY1 complexes is a new finding. Moreover, the apparent dissociation of CRY1 from PER2 after CK1 phosphorylation provides a potentially new mechanism by which the repressive activity of this complex could be regulated.

Weaknesses:

Overexpression of circadian clock components, normally expressed at low levels, could disrupt the stoichiometry of native interactions, Although the authors provide a reasonable rationale for the Dox-inducible approach and use appropriate controls throughout the experiments, there is still concern that overexpression of the components of this transcriptional repressive complex far exceed the concentration of the transcription factor they regulate, and this has not been taken into consideration here. In addition, the interesting discovery that CK1 phosphorylation of PER2 leads to dissociation of CRY1 has not identified the phosphorylation site(s) responsible for this, so the mechanism by which this occurs is still unknown. Still, this study provides some interesting hypotheses regarding CK1 regulation of PER2 and CRY1 that could drive future work in the field.

Comments on latest version:

This manuscript has already undergone two rounds of review at a reputable journal, and we have been provided with the previous reviewers' comments and the authors' responses. I am satisfied with the responses and changes to the manuscript made in these previous rounds of review and don't have any further experiments to suggest that wouldn't represent significant additional work.

Reviewer #2 (Public review):

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

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

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

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

Comments on revisions:

None remaining.

Reviewer #1 (Public review):

The manuscript presents a compelling new in vitro system based on isogenic co-cultures of human iPSC-derived hepatocytes and macrophages, enabling the modelling of hepatic immune responses with unprecedented physiological relevance. The authors show that co-culture leads to enhanced maturation of hepatocytes and tissue-resident macrophage identity, which cannot be achieved through conditioned media alone. Using this system, they functionally validate immune-driven hepatotoxic responses to a panel of drugs and compare the system's predictive power to that of monocyte-derived macrophages. The results underscore the necessity of macrophage-hepatocyte crosstalk for accurate modelling of liver inflammation and drug toxicity in vitro. The manuscript is clearly written and addresses a key limitation in liver organoid systems: the lack of immune complexity and tissue-specific macrophage imprinting.

Strengths:

• Novelty and Relevance: The study presents a highly innovative co-culture system based on isogenic human iPSCs, addressing an unmet need in modelling immune-mediated hepatotoxicity.

• Mechanistic Insight: The reciprocal reprogramming between iHeps and iMacs, including induction of KC-specific pathways and hepatocyte maturation markers, is convincingly demonstrated.

• Functional Readouts: The application of the model to detect IL-6 responses to hepatotoxic compounds enhances its translational relevance.

Weaknesses:

The co-culture model with monocyte-derived macrophages is not fully characterised, making comparisons less informative.

Reviewer #1 (Public review):

The authors previously reported that Heliconius, one genus of the Heliconiini butterflies, evolved to be efficient foragers to feed pollen of specific plants and have massively expanded mushroom bodies. Using the same image dataset, the authors segmented the central complex and associated brain regions and found that the volume of the central complex relative to the rest of brain are largely conserved across the Heliconiini butterflies. By performing immunostaining to label specific subset of neurons, the authors found several potential sites of evolutional divergence in the central complex neural circuits, including the numbers of GABAergic ellipsoid body ring neurons and the innervation patterns of Allatostatin A expressing neurons in the noduli. These neuroanatomical data will be helpful to guide the future studies to understand the evolution of the neural circuits for vector-based navigations.

Strength

The authors used sufficiently large scale of dataset from 307 individuals of 41 specifies of Heliconiini butterflies to solidify the quantitative conclusions, and present new microscopy data for fine neuroanatomical comparison of the central complex.

Weakness

(1) Although the figures display a concise summary of anatomical findings, it would be difficult for non-experts to learn from this manuscript to identify the same neuronal processes in the raw confocal stacks. It would be helpful to have instructive movies to show step by step guide for identifications of neurons of interests, segmentations and 3D visualizations (rotation) for several examples including ER neurons (to supplement texts in line 347-353) and Allatostatin A neurons.

(2) Related to (1), it was difficult for me to access if the data in Fig 7 support the author's conclusions that ER neuron number increased in Heliconius Melpomene. By my understanding, the resolution of this dataset isn't high enough to trace individual axons and therefore authors do not rule out that the portion of "ER ring neurons" in Heliconius may not innervate the ER, as stated in Line 635 "Importantly, we also found that some ER neurons bypass the ellipsoid body and give rise to dense branches within distinct layers in the fan-shaped body (ER-FB)". If they don't innervate the ellipsoid body, why are they named as "ER neurons"?

(3) Discussions around the line 577-584 requires the assumption that each ellipsoid body (EB) ring neuron typically arborise in a single microglomerulus to form largely one-to-one connection with TuBu neurons within the bulb (BU), and therefore the number of BU microglomeruli should provide an estimation of the number of ER neurons. Explain this key assumption or provide an alternative explanation.

(4) The details of antibody information are missing in the Key resource table. Instead of citing papers, list the catalogue numbers and identifier for commercially available antibodies, and describe the antigen and if they are monoclonal or polyclonal. Are antigens conserved across species?

(5) I did not understand why authors assume that foraging to feed on pollens is more difficult cognitive task than foraging to feed on nectars. Would it be possible that they are equality demanding tasks but pollen feeding allows Heliconius to pass more proteins and nucleic acids to their offsprings and therefore they can develop larger mushroom bodies?

Comments on revisions:

The authors fully addressed my concerns and significantly improved the accessibility of the manuscript.

Reviewer #1 (Public review):

The author presents a new method for microRNA target prediction based on (1) a publicly available pretrained Sentence-BERT language model that the author fine-tunes using MeSH information and (2) downstream classification analysis for microRNA target prediction. In particular, the author's approach, named "miRTarDS", attempts to solve the microRNA target prediction problem by utilizing disease information (i.e., semantic similarity scores) from their language model. The author then compares the prediction performance with other sequence- and disease-based methods and attempts to show that miRTarDS is superior or at least comparable to existing methods. The author's general approach to this microRNA target prediction problem seems promising, but fails to demonstrate concrete computational evidence that miRTarDS outperforms other existing methods. The author's claim that disease information-based language models are sufficient is unfounded. The manuscript requires substantial rewriting and reorganization for readers with a strong background in biomedical research.

A major issue related to the author's claim of computational advance of miRTarDS: The author does not introduce existing biomedical-specific language models, and does not compare them against miRTarDS's fine-tuned model. The performance of miRTarDS is largely dependent on the semantic embedding of disease terms. The author shows in Figure 5 that MeSH-based fine-tuning leads to a substantial improvement in MeSH-based correlation compared to the publicly available pretrained SBERT model "multi-qa-MiniLM-L6-cos-v1" without sacrificing a large amount of BIOSSES-based correlation. However, the author does not compare the performance of MeSH- and BIOSSES-based correlation with existing language models such as ChatGPT, BioBERT, PubMedBERT, and more. Also, the substantial improvement in MeSH-based correlation is a mere indication that the MeSH-based fine-tuning strategy was reasonable and not that it's superior to the publicly available pretrained SBERT model "multi-qa-MiniLM-L6-cos-v1".

Another major issue is in the author's claim that disease-information from miRTarDS's language model is "sufficient" for accurate microRNA target prediction. Available microRNA targets with experimental evidence are largely biased for those with disease implications that have been reported in the biomedical literature. It's possible that their language model is biased by existing literature that has also been used to build microRNA target databases. Therefore, it is important that the author provides strong evidence that excludes the possibility of data leakage circularity. Similar concerns are prevalent across the manuscript, and so I highly recommend that the author reassess the evaluation frameworks and account for inflated performance, biased conclusions, and self-confirming results.

Last but not least, the manuscript requires a deeper and careful description and computational encoding of microRNA biology. I'd advise the author to include an expert in microRNA biology to improve the quality of this manuscript. For example, the author uses the pre-miRNA notation and replaces the mature miRNA notation to maintain computational encoding consistency across databases. However, the mature microRNA notation "the '-3p' or '-5p' is critical as the 3p and 5p mature microRNAs have different seed sequences and thus different mRNA targets. The 3p mature microRNA would most likely not target an mRNA targeted by the 5p mature microRNA.

Reviewer #1 (Public review):

Summary:

This study examines how traumatic brain injury (TBI) alters hippocampal network dynamics and single-unit activity in awake, behaving rats. Using laminar recordings, the authors report reductions in theta power, theta-gamma phase-amplitude coupling, and spike-field entrainment, alongside impairments in spatial memory performance.

Strengths of the study include the use of high-density laminar electrodes to localize activity across hippocampal layers and the integration of electrophysiological and behavioral measures. Analyses that consider behavioral state and account for broadband power changes improve confidence in the interpretation of oscillatory effects. Additional controls suggest that the observed differences are unlikely to be explained by gross motor or motivational deficits. The reported relationships between theta amplitude, phase-amplitude coupling, and spike entrainment provide useful insight into how network coordination is disrupted following injury.

There are a few minor weaknesses. The analyses of single-unit activity across environments are relatively limited, and more comprehensive approaches to characterizing spatial coding would strengthen conclusions about how TBI impacts hippocampal representations. The behavioral assessment relies primarily on a single task, which constrains the interpretation of the cognitive deficits. In addition, the relatively small number of animals is a limitation, although this is partially mitigated by the number of recorded units and the consistency of effects across measures.

Overall, this work provides a careful characterization of hippocampal circuit dysfunction following TBI and contributes to understanding how disruptions in oscillatory coordination and spike timing may relate to cognitive impairment.

Comments on revisions:

The authors have adequately addressed all of my concerns.

Reviewer #1 (Public review):

Summary:

This study utilizes fNIRS to investigate the effects of undernutrition on functional connectivity patterns in infants from a rural population in Gambia. fNIRS resting-state data recording spanned ages 5 to 24 months, while growth measures were collected from birth to 24 months. Additionally, executive functioning tasks were administered at 3 or 5 years of age. The results show an increase in left and right frontal-middle and right frontal-posterior connections with age and, contrary to previous findings in high-income countries, a decrease in frontal interhemispheric connectivity. Restricted growth during the first months of life was associated with stronger frontal interhemispheric connectivity and weaker right frontal-posterior connectivity at 24 months of age. Additionally, the study describes some connectivity patterns, including stronger frontal interhemispheric connectivity, which is associated with better cognitive flexibility at preschool age.

Strengths:

- The study analyses longitudinal data from a large cohort (n = 204) of infants living in a rural area of Gambia. This already represents a large sample for most infant studies, and it is impressive, considering it was collected outside the lab in a population that is underrepresented in the literature. The research question regarding the effect of early nutritional deficiency on brain development is highly relevant and may highlight the importance of early interventions. The study may also encourage further research on different underrepresented infant populations (i.e., infants not residing in Western high-income countries) or in settings where fMRI is not feasible.

- The preprocessing and analysis steps are carefully described, which is very welcome in the fNIRS field, where well-defined standards for preprocessing and analysis are still lacking.

Weaknesses:

- The study provides a solid description of the functional connectivity changes in the first two years of life at the group level and investigates how restricted growth influences connectivity patterns at 24 months. However, it does not explore the links between adverse situations and developmental trajectories for functional connectivity. Given the longitudinal nature of the dataset, future work should expand the analysis using more sophisticated tools to link undernutrition to specific developmental trajectories in functional connectivity, and eventually incorporate additional data to increase statistical power.

- Connectivity was assessed in 6 big ROIs to reduce variability due to head size and optode placement. Nevertheless, this also implies a significant reduction in spatial resolution. Individual digitalisation and co-registration of the optodes to a head model, followed by image reconstruction, could provide better spatial resolution. This is not a weakness specific to this study but rather a limitation common to most fNIRS studies, which typically analyse data at the channel level since digitalisation and co-registration can be challenging, especially in complex setups like this. The authors made an important effort to identify subjects with major optode displacement; however, future work might use tools to digitally record the positions of optodes and head markers.

Reviewer #1 (Public review):

Summary:

Hoverflies are renowned for their striking sexual dimorphism in eye morphology and early visual system physiology, as well as in sexually dimorphic behaviors. Surprisingly, male and female flight behaviors in response to optic flow exhibit only subtle differences. Nicholas et al. investigate the sensorimotor transformation of sexually dimorphic visual information into flight steering commands via descending neurons. Using a combination of intracellular and extracellular recordings, neuroanatomical analysis, and behavioral assays, the authors convincingly demonstrate that descending neurons-particularly at high optic flow velocities-exhibit pronounced sexual dimorphisms, while wing steering responses remain largely monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1 and OFS DN2. Their likely counterparts in Drosophila connect to neck, wing and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - is complex and less straightforward than one might expect.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known in hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

Revision comment:

I thank the authors for their detailed replies to my questions and the additional clarifications and analysis included in the paper. All my concerns have been addressed.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The editors have determined that the authors adequately addressed the prior reviewer comments.]

Summary:

The author's goal was to arrest PsV capsids on the extracellular matrix using cytochalasin D. The cohort was then released and interaction with the cell surface, specifically with CD151 was assessed.

Note on previous revisions:

The authors did an excellent job in their revision to include data from the effect of proteolytic priming on their observed virion transfer to the cell body. All other minor issues were addressed adequately.

The work could be especially critical to understanding the process of in vivo infection.

Reviewer #1 (Public review):

Summary:

Using comprehensive profiling of normal and cancerous tissue via bulk and single-cell RNA sequencing, the authors identified that high-grade serous ovarian cancer is likely to originate from the epithelial progenitor cells from the distal fimbrial region of the fallopian tube, where it has been previously shown to be most prone to ovulatory stress and other microenvironmental influences. The authors also included a CNV analysis to identify hotspots in HGSOCs.

The findings are preliminary, but the resource on its own has great potential and can be used for developing methods for early detection, stratification and treatment.

The main limitation of this study is that the lineage is purely inferred from bioinformatics analysis. More validation work is required, perhaps using cell models / other model organisms.

Strengths and weaknesses:

The authors investigated the origin of high-grade serous ovarian cancer, which is one of the deadliest. They performed comparative analysis using both bulk and single-nucleus RNA sequencing between cancerous and normal tissues (fallopian tube and ovaries) and identified a population of epithelial progenitor cells from the distal fimbrial region that are exposed to ovulatory stress, as the most plausible cells of origin. The extensive profiling of the molecular signatures can also be used for early detection and stratification for treating the disease.

Previous studies have shown that HGSOCs likely originated from the epithelial lining of the fallopian tubes (PMID 32349388). The bulk RNAseq data is confusing in that neither the overall correlation of the transcriptome nor the sample clustering (Figure 1) supports the idea that the HGSOCs are close to the fallopian tube. The authors could perform a more comprehensive marker gene-based analysis to demonstrate their relationship.

The authors also performed a comprehensive analysis of single-cell datasets on both normal and cancerous tissue in humans. From there, they performed a combination of RNA velocity, PAGA and pseudotime, etc, to try and delineate the relationship amongst related cell populations. It would be helpful if the authors could clarify why they applied this particular suite of tools (explaining the differences between tools and bioinformatic approaches) to assist the broader readership who may not be familiar with this type of single-cell bioinformatic analysis.

It also seems to me that the authors did not account for patient effect when they performed the data integration (this point is discussed in the text). This may explain at least partially why the clusters are segregated by patient samples. Another explanation is that it could be due to uneven sampling, as only very few cells (1000s) were captured from each of the tumour samples, and this is clear when a dramatic difference can be seen in their cellular composition.

The trajectory analysis of normal and cancer single-cell data should also include other cells to prevent confirmation bias, as these analyses would only consider relationships amongst the cells available in the model.

As the authors indicated in the limitations, the cell lineage in the studies is largely inferred from the bioinformatics analysis. Experimental lineage tracing via other experimental models (organoids/animal models) would be required.

Despite these limitations, this study will serve as an important resource for the scientific community. I would also suggest that the authors should share this resource via additional portals in addition to the GEO data deposit (e.g. the HCA, or single-cell portals such as at the Broad Institute or CellXGene Discover).

Reviewer #1 (Public review):

This work demonstrates that MORC2 undergoes phase separation (PS) in cells to form nuclear condensates, and the authors demonstrate convincingly the interactions responsible for this phase separation. Specifically, the authors make good use of crystallography and NMR to identify multiple protein:protein interactions and use EMSA to confirm protein:DNA interactions. These interactions work together to promote in vitro and in cell phase separation and boosted ATPase activity by the catalytic domain of MORC2.

Moreover, the authors show solid evidence supporting their important claim that MORC2 PS is important for MORC2-mediated gene regulation. Exploring causal links between PS and function is an important need in the phase separation field, particularly as regards the role of condensates in gene regulation, and is a non-trivial matter. It is crucial and challenging to properly explore the alternative possibility that soluble complexes, existing in the same conditions as phase-separated condensates, are the functional species. The authors have attempted to address this concern by manipulating the physical nature of the MORC2 condensates using a killswitch (KS) peptide (MORC2 +KS), finding that reducing condensates dynamics results in a cellular phenotype very similar to that of the phase separation-deficient MORC2 condensates. While not fully ruling out the alternative, soluble-complex hypothesis, this experiment suggests that function is indeed localized inside the MORC2 condensates, and that perturbing the condensate can be functionally equivalent to removing condensate formation.

The authors also look at several disease related mutants of MORC2. While most of these do not seem to have an obvious connection to the phase separation data, it is quite interesting that one mutant, E236G, displays similar intra-condensate dynamics compared to MORC2 +KS, strengthening the claim that MORC2 phase separation is important for function and suggesting that the observations in this paper may indeed have some disease relevance.

Strengths

Static light scattering and crystallography are nicely used to demonstrate the dimerization of MORC2FL and to discover the structure of the CC3 domain dimer, presumably responsible for the dimerization of MORC2FL (Figure 1).

Extensive use of deletion mutants in multiple cell lines is used to identify regions of MORC2 that are important for forming condensates in the nucleus: the IBD, IDR, and CC3 domains are found to both be essential for condensate formation, while the CW domain plays an unknown role in condensate morphology (Figure 3). The authors use NMR to further identify that the IBD domain seems to interact with the first third of the centrally located IDR, termed IDRa, but not with the latter two thirds of the IDR domain (Figure 4). This leads them to propose that phase separation is the product of IDB:IDRa interaction, CC3 dimerization, and an unknown but important role for the CW domain.

Based on the observation that removal of the NLS resulted in diffuse cytoplasmic localization, they hypothesized that DNA may play an important role in MORC2 PS. EMSA was used to demonstrate interaction between DNA and several MORC2 domains: CC1, CC2, IDR, and TCD-CC3-IBD. Further in vitro microscopy with purified MORC2 showed that DNA addition significantly reduces MORC2 saturation concentration (Figure 5).

These assays convincingly demonstrate that MORC2 phase separates in cells and identifies the protein domains and interactions responsible for this phenomenon.

Weaknesses

The connection between condensates and function, while improved from the original manuscript, still has some weak points.

The central experiment demonstrating that MORC2 condensates mediate function takes the form of RNA-Seq in MORC2 KO HeLa cells (Figure 6), rescued with WT, condensate-deficient mutants, and a KS peptide mutant that reduces dynamics by increasing homotypic protein interactions. The observation that rescuing with MORC2 +KS is ineffective, in a manner similar to rescue with condensate-deficient MORC2 mutants, suggests that unperturbed condensates are important for function. An alternative possibility, however, is that condensates are non-functional bystanders, and that the increased homotypic interactions present in MORC2 +KS result in stronger MORC2 +KS recruitment to condensates, reducing the pool of functional, dilute phase MORC2 +KS and squashing function via sequestration. Similar ideas have been explored by others for transcription factors (e.g. Chong et al, Mol Cell, 2022). This possibility is neither discussed nor ruled out. The absence of microscopy data showing similar localization of MORC2 and MORC2 +KS (particularly the amount of diffuse MORC2 outside condensates) amplifies this concern.

The RNA-Seq data presented in Figure 6h also has some concerning qualities. Inter-replicate variability is higher than ideal, particularly for MORC2 deltaCC3. This may be a product of the transient transfection system used for these experiments, which inherently results in stochasticity. Specific sets of genes regulated by MORC2 are consistent with the main conclusion (Figure 6i, individual genes in 6h, showing that all mutants are more similar to one another than to WT MORC2), but global transcription shifts seem quite different between MORC2 condensate-deficient mutants and MORC2 +KS (Figure 6h heatmap), suggesting much more than simple condensate disruption is taking place. Together, this weakens the conclusion that MORC2 condensates are the functional form of MORC2.