Reviewer #2 (Public review):

Summary:

In this manuscript, Li and colleagues study the fate of endothelial cells in a mouse model of ischemic stroke. Using genetic lineage tracing approaches, they find that endothelial cells give rise to non-endothelial cells, which they term "E-pericytes." They further show that depleting these cells exacerbates blood-brain barrier leakage and worsens functional recovery. The authors also provide evidence that endothelial-to-mesenchymal transition, myeloid cell-derived TGFβ1, and endothelial TGFβRII are involved in this process. These are potentially interesting findings, however, the experimental evidence that endothelial cells undergo transdifferentiation to non-endothelial cells is weak, as is the evidence that these cells are pericytes. Addressing this foundational weakness will facilitate interpretation of the other findings.

In this revised manuscript, the authors corrected labeling errors and included negative controls for flow cytometry and immunohistochemistry data. They did not, however, substantively address the major weaknesses below related to rigorously demonstrating the cellular origin and identity of "E-pericytes."

Strengths:

(1) The authors address an important question about blood vessel function and plasticity in the context of stroke.

(2) The authors use a variety of genetic approaches to understand cell fate in the context of stroke. Particularly commendable is the use of several complementary lineage tracing strategies, including an intersectional strategy requiring both endothelial Cre activity and subsequent mural cell NG2 promoter activity.

(3) The authors address upstream cellular and molecular mechanisms, including roles for myeloid-derived TGFβ.

Weaknesses:

(1) The authors use Cdh5-CreERT2; Ai47 mice to permanently label endothelial cells and their progeny with eGFP. They then isolate eGFP+ cells from control and MCAO RP7D and RP34D brains, and use single cell RNA-seq to identify the resulting cell types. Theoretically, all eGFP+ cells should be endothelial cells or their progeny. This is a very powerful and well-conceived experiment. The authors use the presence of a pericyte cluster as evidence that endothelial to pericyte transdifferentiation occurs. However, pericytes are also present in the scRNA-seq data from sham mice, as are several other cell types such as fibroblasts and microglia. This suggests that pericytes and these other cell types might have been co-purified (e.g., as doublets) with eGFP+ endothelial cells during FACS and may not themselves be eGFP+. Pericyte-endothelial doublets are common in scRNA-seq given that these cell types are closely and tightly associated. Additionally, tight association (e.g., via peg-socket junctions) can cause fragments of endothelial cells to be retained on pericytes (and vice-versa) during dissociation. Finally, it is possible that after stroke or during the dissociation process, endothelial cells lyse and release eGFP that could be taken up by other cell types. All of these scenarios could lead to purification of cells that were not derived (transdifferentiated) from endothelial cells. Authors note that the proportion of pericytes increased in the stroke groups, but it does not appear this experiment was replicated and thus this conclusion is not supported by statistical analysis. The results of pseudotime and trajectory analyses rely on the foundation that the pericytes in this dataset are endothelial-derived, which, as discussed above, has not been rigorously demonstrated.

(2) I have the same concern regarding inadvertent purification of cells that were not derived from endothelial cells in the context of the bulk RNA-seq experiment (Fig. S4), especially given the sample-to-sample variability in gene expression in the RP34D, eGFP+ non-ECs group (e.g., only 2/5 samples are enriched for mesenchymal transcription factor Tbx18, only 1/5 samples are enriched for mural cell TF Heyl). If the sorted eGFP+ non-ECs were pericytes, I would expect a strong and consistent pericyte-like gene expression profile.

(3) Authors use immunohistochemistry to understand localization, morphology, and marker expression of eGFP+ cells in situ. The representative "E-pericytes" shown in Fig. 3A-D are not associated with blood vessels, and the authors' quantification also shows that the majority of such cells are not vessel-associated ("avascular"). By definition, pericytes are a component of blood vessels and are embedded within the vascular basement membrane. Thus, concluding that these cells are pericytes ("E-pericytes") may be erroneous.

(4) CD13 flow cytometry and immunohistochemistry are used extensively to identify pericytes. In the context of several complementary lineage tracing strategies noted in Strength #2, CD13 immunohistochemistry is the only marker used to identify putative pericytes (Fig. S3J-M). In stroke, CD13 is not specific to pericytes; dendritic cells and other monocyte-derived cells express CD13 (Anpep) in mouse brain after stroke (PMID: 38177281, https://anratherlab.shinyapps.io/strokevis/).

(5) Authors conclude that "EC-specific overexpression of the Tgfbr2 protein by a virus (Tgfbr2) decreases Evans blue leakage, promotes CBF recovery, alleviates neurological deficits and facilitates spontaneous behavioral recovery after stroke by increasing the number of E-pericytes." All data in Fig. 10, however, compare endothelial Tgfbr2 overexpression to a DsRed overexpression control. There is no group in which Tgfbr2 is overexpressed but "E-pericytes" are eliminated with DTA (this is done in Fig. 9B, but this experiment lacks the Tgfbr2 overexpression-only control). Thus, the observed functional outcomes cannot be ascribed to "E-pericytes"; it remains possible that endothelial Tgfbr2 overexpression affects EB leakage, CBF, and behavior through alternative mechanisms.

In response to this comment, authors wrote: "in Figures 9A-B, we observed no significant difference in Evans blue leakage between the Tgfbr2 overexpression group and the Tgfbr2 overexpression + DTA group (P=0.8153), this suggests that the impact of Tgfbr2 overexpression on the blood-brain barrier (BBB) is primarily attributed from the E-pericytes generated by Tgfbr2 expression."

I do not see data from a Tgfbr2 overexpression-only group in Fig. 9B. Further, I do not understand authors' logic: If the mechanism by which EC Tgfbr2 overexpression acts to reduce BBB leakage is by increasing the number of "E-pericytes," depleting "E-pericytes" with DTA in this context should increase BBB leakage.

(6) Single-cell and bulk RNA-seq data are not available in a public repository (such as GEO). Depositing these data would facilitate their independent reevaluation and reuse.

In response to this comment, authors indicated they submitted data to GEO, but did not provide an accession number.

Reviewer #2 (Public review):

Summary:

In this manuscript, Tolossa et al. analyze Inter-spike intervals from various freely available datasets from the Allen Institute and from a dataset from Steinmetz et al.. They show that they can modestly decode between gross brain regions (Visual vs. Hippocampus vs. Thalamus), and modestly separate sub areas within brain regions (DG vs. CA1 or various visual brain areas). The core result is that a multi-layer perceptron trained on the ISI distributions can modestly classify different brain areas and perhaps in a reasonably compelling way generalize across animals. The result is interesting but the exact problem formulation still feels a tad murky to me because I am worried the null is a strawman and I'm unsure if anyone has ever argued for this null hypothesis ("the impact of anatomy on a neuron's activity is either nonexistent or unremarkable"). Given the patterns of inputs to different brain areas and the existence of different developmental origin and different cell types within these areas, I am unclear why this would be a good null hypothesis. Nevertheless, the machine learning is reasonable, and the authors demonstrate that a nonlinear population based classifier can pull out reasonable information about the brain area and layer.

Strengths:

The paper is reasonably well written, and the definitions are quite well done. For example, the authors clearly explained transductive vs. inductive inference in their decoders. E.g., transductive learning allows the decoder to learn features from each animal, whereas inductive inference focuses on withheld animals and prioritizes the learning of generalizable features. The authors walk the reader through various analyses starting as simply as PCA, then finally showing a MLP trained on ISI distributions and PSTHs performs modestly well in decoding brain area. The key is ISI distributions work well in inductive settings for generalizing from one mouse to the other.

Weaknesses:

As articulated in my overall summary, I still found the null hypothesis a tad underwhelming. I am not sure this is really a valid null hypothesis ("the impact of anatomy on a neuron's activity is either nonexistent or unremarkable"), although in the statistical sense it is fine. The authors took on board some of the advice from the first review and clarified the paper but there are portions that are unnecessarily verbose (e.g., "Beyond fundamental scientific insight, our findings may be of benefit in various practical applications, such as the continued development of brain-machine interfaces and neuroprosthetics"). Also, given that ISIs cannot separate between visual areas, why is the statement that these are conserved. I still find it somewhat underwhelming that the thalamus, hippocampus , and visual cortex have different ISI distributions. Multiple researchers have reported similar things in cortex perhaps without the focus on decoding area from these ISI distributions.

All in all, it is an interesting paper with the notion that ISI distributions can modestly predict brain area and layer. It could have some potential for a tool for neuropixels, although this needs to be developed further for this use case.

Reviewer #2 (Public review):

This paper tests the hypothesis that perceptual switches during the presentation of ambiguous stimuli are accompanied by changes in neuromodulation that alter neural gain and trigger abrupt changes in brain activity. To test this hypothesis, the study combines pupillometry, artificial recurrent network (RNN) analysis and fMRI recording. In particular, the study uses methods of energy landscape analysis inspired by physics, which is particularly interesting.

Strengths<br /> - The authors should be commended for combining different methods (pupillometry, RNNs, fMRI) to test their hypothesis. This combination provides a mechanistic insight into perceptual switches in the brain and artificial neural networks.<br /> - The study combines different viewpoints and fields of scientific literature, including neuroscience, psychology, physics, dynamical systems. In order to make this combination more accessible to the reader, the different aspects are presented in a pedagogical way to be accessible to all fields.<br /> - This combination of methods and viewpoints is rarely done, so it is very useful.<br /> - The authors introduce dynamic gain modulation in their recurrent neural network, which is novel. They devote a section of the paper to studying the dynamics, fixed points and convergence of this type of network.

Weaknesses<br /> - The study may not be specific to perceptual switches. This is because the study relies on a paradigm in which participants report when they identify a switch in the item category. Therefore, it is unclear whether the effects reported in the paper are related to the perceptual switch itself, to attention, or to the detection of behaviourally relevant events. The authors are cautious and explicitly acknowledge this point in their study.<br /> - The demonstration of the causal role of gain modulation in perceptual switches is partial. This causality is clearly demonstrated in the simulation work with the RNN. However, it is not fully demonstrated in the pupil analysis and the fMRI analysis. One reason is that this work is correlative (which is already very informative).<br /> - Some effects may reflect the expectation of a perceptual switch rather than the perceptual switch itself. To mitigate this risk, the design of the fMRI task included catch trials, in which no switch occurs, to reduce the expectation of a switch. The pupil study, however, did not include such catch trials.<br /> - The paper uses RNN-based modelling to provide mechanistic insight into the role of gain modulation in perceptual switches. However, the RNN solves a task that differs from that performed by human participants, which may limit the explanatory value of the model. The RNN is provided with two inputs characterising the sensory evidence supporting the first and last image category in the sequence (e.g. plane and shark). In contrast, observers in the task don't know in advance the identity of the last image at the beginning of the sequence. The brain first receives sensory evidence about the image category (e.g. plane) with which the sequence begins, which is very easy to recognise, then it sees a sequence of morphed images and has to discover what the final image category will be. To discover the final image category, the brain considers several possibilities for the second images (it is a shark?, a frog?, a bird?, etc.), rather than comparing the likelihood of just two categories. This search process among many alternatives and the perceptual switch in the task is therefore different from the competition between only two inputs in the RNN.<br /> - Another aspect of the motivation for the RNN model remains unclear. The authors introduce dynamic gain modulation in the RNN, but it is not clear what the added value of dynamic gain modulation is. Both static (Fig. S1) and dynamic (Fig. 2F) gain modulation lead to the predicted effect: faster switching when the gain is larger.<br /> - The authors are to be commended for addressing their research questions with multiple tools and approaches. There are links between the different parts of the study. The RNN and the pupil are linked by the notion of gain modulation, the RNN and the fMRI analysis are linked by the study of the energy landscape, the fMRI study and the pupil study are indirectly linked by previous work for this group showing that the peak in LC fMRI activity precedes a flattening of the energy landscape. These links are very interesting but could have been stronger and more complete.

Comments on revisions:

I thank the authors for their responses.<br /> My review presents points that the authors themselves present as weaknesses or limitations. It also includes points that cannot be addressed in a revision (e.g. causality).<br /> Regarding the fact that the RNN only considers two categories, whereas subjects consider more categories (because they don't know the final image), I have toned down my remark (removing "markedly" different, removing the fact that the hypothesis space is vast given that participants have some priors). I also removed the qualifier "mechanistically" different, because it can be understood in different ways. The point remains that the proposed model has 2 inputs, the corresponding network in the brain has >2 inputs (because it considers more categories than the RNN), which is different, and which is the point of my remark. I think it may limit the value of the model, but I don't think it is not "sensible".

Reviewer #2 (Public review):

Summary:

Baniulyte and Wade describe how translation of an 8-codon uORF denoted toiL upstream of the topAI-yjhQP operon is responsive to different ribosome-targeting antibiotics, consequently controlling translation of the TopAI toxin as well as Rho-dependent termination with the gene.

Strengths:

The authors used multiple different approaches such as a genetic screen to identify factors such as 23S rRNA mutations that affect topA1 expression and ribosome profiling to examine the consequences of various antibiotics on toiL-mediated regulation.

Weaknesses: Future experiments will be needed to better understand the physiological role of the toiL-mediated regulation and elucidate the mechanism of specific antibiotic sensing.

The results are clearly described, and the revisions have helped to improve the presentation of the data.

Reviewer #3 (Public review):

Summary:

The authors were trying to show that a novel neuronal metallothionein of poorly defined function, GIF/MT3, is actually heavily persulfidated in both the Zn-bound and apo (metal-free) forms of the molecule as purified from a heterologous (bacterial) or native host. Evidence in support of this conclusion is strong, with both spectroscopic and mass spectrometry evidence strongly consistent with this general conclusion. The authors would appear to have achieved their aims.

Strengths:

The analytical data in support of the author's primary conclusions are strong. The authors also provide some modeling evidence that supports the contention that MT3 (and other MTs) can readily accommodate a sulfane sulfur on each of the 20 cysteines in the Zn-bound structure, with little perturbation of the overall structure. This is not the case with Cys trisulfides, which suggests that the persulfide-metallated state is clearly positioned at lower energy relative to the immediately adjacent thiolate- or trisulfidated metal coordination complexes.

Weaknesses:

The biological significance of the findings is not entirely clear. On the one hand, the analytical data are solid (albeit using a protein derived from a bacterial over-expression experiment), and yes, it's true that sulfane S can protect Cys from overoxidation, but everything shown in the summary figure (Fig. 9D) can be done with Zn release from a thiol by ROS, and subsequent reduction by the Trx/TR system. In addition, it's long been known that Zn itself can protect Cys from oxidation. I view this as a minor shortcoming that will motivate follow-up studies.

Impact:

The impact will be high since the finding is potentially disruptive to the MT field for sure. The sulfane sulfur counting experiment (the HPE-IAM electrophile trapping experiment) may well be widely adopted by the field. Those in the metals field always knew that this was a possibility, and it will interesting to see the extent to which metal binding thiolates broadly incorporate sulfane sulfur into their first coordination shells.

Comments on revisions:

The revised manuscript is only slightly changed from the original, with the inclusion of a supplementary figure (Fig. S2) and minor changes in the text. The authors did not choose to carry out the quantitative Zn binding experiment (which I really wanted to see), but given the complexities of the experiment, I'll let it go.

Reviewer #2 (Public review):

Summary:

In 2021 (PMID: 33503405) and 2024 (PMID: 38578830) Constantinou and colleagues published two elegant papers in which they demonstrated that the Topbp1 checkpoint adaptor protein could assemble into mesoscale phase-separated condensates that were essential to amplify activation of the PIKK, ATR, and its downstream effector kinase, Chk1, during DNA damage signalling. A key tool that made these studies possible was the use of a chimeric Topbp1 protein bearing a cryptochrome domain, Cry2, which triggered condensation of the chimeric Topbp1 protein, and thus activation of ATR and Chk1, in response to irradiation with blue light without the myriad complications associated with actually exposing cells to DNA damage.

In this current report Morano and co-workers utilise the same optogenetic Topbp1 system to investigate a different question, namely whether Topbp1 phase-condensation can be inhibited pharmacologically to manipulate downstream ATR-Chk1 signalling. This is of interest, as the therapeutic potential of the ATR-Chk1 pathway is an area of active investigation, albeit generally using more conventional kinase inhibitor approaches.

The starting point is a high throughput screen of 4730 existing or candidate small molecule anti-cancer drugs for compounds capable of inhibiting the condensation of the Topbp1-Cry2-mCherry reporter molecule in vivo. A surprisingly large number of putative hits (>300) were recorded, from which 131 of the most potent were selected for secondary screening using activation of Chk1 in response to DNA damage induced by SN-38, a topoisomerase inhibitor, as a surrogate marker for Topbp1 condensation. From this the 10 most potent compounds were tested for interactions with a clinically used combination of SN-38 and 5-FU (FOLFIRI) in terms of cytotoxicity in HCT116 cells. The compound that synergised most potently with FOLFIRI, the GSK3-beta inhibitor drug AZD2858, was selected for all subsequent experiments.

AZD2858 is shown to suppress the formation of Topbp1 (endogenous) condensates in cells exposed to SN-38, and to inhibit activation of Chk1 without interfering with activation of ATM or other endpoints of damage signalling such as formation of gamma-H2AX or activation of Chk2 (generally considered to be downstream of ATM). AZD2858 therefore seems to selectively inhibit the Topbp1-ATR-Chk1 pathway without interfering with parallel branches of the DNA damage signalling system, consistent with Topbp1 condensation being the primary target. Importantly, neither siRNA depletion of GSK3-beta, or other GSK3-beta inhibitors were able to recapitulate this effect, suggesting it was a specific non-canonical effect of AZD2858 and not a consequence of GSK3-beta inhibition per se.

To understand the basis for synergism between AZD2858 and SN-38 in terms of cell killing, the effect of AZD2858 on the replication checkpoint was assessed. This is a response, mediated via ATR-Chk1, that modulates replication origin firing and fork progression in S-phase cell under conditions of DNA damage or when replication is impeded. SN-38 treatment of HCT116 cells markedly suppresses DNA replication, however this was partially reversed by co-treatment with AZD2858, consistent with the failure to activate ATR-Chk1 conferring a defect in replication checkpoint function.

Figures 4 and 5 demonstrate that AZD2858 can markedly enhance the cytotoxic and cytostatic effects of SN-38 and FOLFIRI through a combination of increased apoptosis and growth arrest according to dosage and treatment conditions. Figure 6 extends this analysis to cells cultured as spheroids, sometimes considered to better represent tumor responses compared to single cell cultures.

Significance:

Liquid phase separation of protein complexes is increasingly recognised as a fundamental mechanism in signal transduction and other cellular processes. One recent and important example was that of Topbp1, whose condensation in response to DNA damage is required for efficient activation of the ATR-Chk1 pathway. The current study asks a related but distinct question; can protein condensation be targeted by drugs to manipulate signalling pathways which in the main rely on protein kinase cascades?

Here, the authors identify an inhibitor of GSK3-beta as a novel inhibitor of DNA damage-induced Topbp1 condensation and thus of ATR-Chk1 signalling.

This work will be of interest to researchers in the fields of DNA damage signalling, biophysics of protein condensation, and cancer chemotherapy.

Comments on latest version:

Morano et al. have revised their manuscript in response to the points raised by reviewer #3 as follows.

1) Fig. 2E: Correcting the previously erroneous labelling of this Fig. makes it match the textual description.

2) Figs 3A and B: The revised textual description of the flow cytometry BrdU incorporation is now precise.

3) Fig. 3E: Removing the suspect WB images is a pragmatic decision that does not significantly affect the overall conclusions of the paper.

4) Fig. 3D: Despite its puzzling appearance this data is now described accurately in the text as "DSBs remained elevated after the combined treatment" rather than "increased after the combined treatment. A more convincing increase in the presumed damaged DNA band is evident in Fig. 4D when AZD2858 is combined with a much lower concentration of SN38 (1.5nM) which could mean that the concentration used in Fig. 3D (300nM) induced maximal damage that could not be further enhanced.

Reviewer #2 (Public review):

Despite over 50 years of investigation, our understanding of how the ubiquitous heat shock response, governed by the transcription factor HSF1, was regulated was minimal. In recent years, a coordinated yet simple negative feedback circuit has been elucidated in high detail that centers on the chaperone Hsp70 as a direct-binding inhibitor of HSF1 transcriptional activation. However, roles for the obligatory Hsp70 J-domain partner co-chaperones are currently poorly understood. The present study applies several orthogonal techniques to the question and uncovers an unexpected role for the nuclear JDP Apj1 in attenuation of the heat shock response (HSR) via removal of Hsf1 from HSEs in heat shock gene promoter regions. Interestingly, Apj1 appears to play no role in initiating repression of Hsf1, as null mutants do not exhibit constitutive derepression of the HSR. This role is likely filled by the general nucleo/cytoplasmic JDP Ydj1, as previously reported. These results enhance understanding of HSR regulation and underscore the pivotal role that chaperones play in controlling pro-survival gene expression.

Overall, the work is exceptionally well done and controlled, and the results are properly and appropriately interpreted. Several of the approaches, while powerful, are somewhat indirect (i.e., following gene expression via ribosomal profiling) but ultimately provide a compelling answer to the main question being asked. However, at the end of the day, there is really only one major finding here: Apj1 regulates Hsf1 attenuation via Hsp70. That finding is strongly supported by the experimental data but lacks the one piece of mechanistic evidence found in other recent papers - differential binding of Ssa1/2 to Hsf1 at either the N- or C-terminal binding sites.

Reviewer #2 (Public review):

I would like to thank the authors for the revision and the input they invested in this study.

With the revised text of the study, my earlier criticism holds, and your arguments about the counterfactual approach are irrelevant to that. The recent rise of the counterfactual approach might likely mirror the fact that there are too many scientists behind their computers, and few go into the field to collect in situ data. Studies like the one presented here are a good intellectual exercise but the real impact is questionable. All your main conclusions are inferred from published studies on 7! bird species. In addition, spatial sampling in those seven species was not ideal in relation to your target questions. Thus, no matter how fancy your findings look, the basic fact remains that your input data were for 7 bird species only! Your conclusion, „our study provides a novel understanding of how QTP shapes migration patterns of birds, " is simply overstretching.

The way you respond to my criticism on L 81-93 is something different than what you admit in the rebuttal letter. The text of the ms is silent about the drawbacks and instead highlights your perspective. I understand you; you are trying to sell the story in a nice wrapper. In the rebuttal you state: „we assume species' responses to environments are conservative and their evolution should not discount our findings." But I do not see that clearly stated in the main text.

In your rebuttal, you respond to my criticism of "No matter how good the data eBird provides is, you do not know population-specific connections between wintering and breeding sites" when you responded: ... "we can track the movement of species every week, and capture the breeding and wintering areas for specific populations" I am having a feeling that you either play with words with me or do not understand that from eBird data nobody will be ever able to estimate population-specific teleconnections between breeding and wintering areas. It is simply impossible as you do not track individuals. eBird gives you a global picture per species but not for particular populations. You cannot resolve this critical drawback of your study. I am sorry that you invested so much energy into this study, but I see it as a very limited contribution to understanding the role of a major barrier in shaping migration.

My modest suggestion for you is: go into the field. Ideally use bird radars along the plateau to document whether the birds shift the directions when facing the barrier.

Reviewer #2 (Public review):

Summary :

The paper proposes a model to explain the learning that occurs in brain-computer interface (BCI) tasks when animals need to adapt to novel BCI decoders. The model consists of a network formulation of the "re-aiming" learning strategy, which assumes that BCI learning does not modify the underlying neural circuitry, but instead occurs through a reorganization of existing neural activity patterns.

The authors formalize this in a recurrent neural network (RNN) model, driven by upstream inputs that live in a low-dimensional space.

They show that modelling BCI learning as reorganization of these upstream inputs can explain several experimental findings, such as the difference in the ability of animals to adapt to within vs outside-manifold perturbations, biases in the decoded behaviour after within-manifold perturbations, or qualitative changes in the neural responses observed during credit assignment rotation perturbations or operant conditioning of individual neurons.

Overall, while the idea of re-aiming as a learning strategy has previously been proposed in the literature, the authors show how it can be formalized in a network model, which allows for more direct comparisons to experimental data.

Strengths:

The paper is very well written. The presentation of the model is clear, and the use of vanilla RNN dynamics driven by upstream inputs that are constant in time is consistent with the broader RNN modeling literature.

The main value of the paper lies in the fact that it proposes a network implementation for a learning strategy that had been proposed previously. The network model has a simple form, but the optimization problem is performed in the space of inputs, which requires the authors to solve a nonlinear optimization problem in that space.

While some of the results (eg the fact that the model can adapt to within but not outside-manifold perturbations) are to be expected based on the model assumptions, having a network model allows to make more direct and quantitative comparisons to experiments, to investigate analytically how much the dimension of the output is constrained by the input, and to make predictions that can be tested in data.

The authors perform such comparisons across three different experiments. The results are clearly presented, and the authors show that they hold for various RNN connectivities.

Weaknesses :

The authors mention alternative models (eg, based on synaptic plasticity in the RNN and/or input weights) that can explain the same experimental data that they do, they do not provide any direct comparisons to those models.

Thus, the main argument that the authors have in favor of their model is the fact that it is more plausible because it relies on performing the optimization in a low-dimensional space. It would be nice to see more quantitative arguments for why the re-aiming strategy may be more plausible than synaptic plasticity (either by showing that it explains data better, or explaining why it may be more optimal in the context of fast learning).

In particular, the authors model the adaptation to outside-manifold perturbations (OMPs) through a "generalized re-aiming strategy". This assumes the existence of additional command variables, which are not used in the original decoding task, but can then be exploited to adapt to these OMPs. While this model is meant to capture the fact that optimization is occurring in a low-dimensional subspace, the fact that animals take longer to adapt to OMPs suggests that WMPs and OMPs may rely on different learning mechanisms, and that synaptic plasticity may actually be a better model of adaptation to OMPs. It would be important to discuss how exactly generalized re-aiming would differ from allowing plasticity in the input weights, or in all weights in the network. Do those models make different predictions, and could they be differentiated in future experiments?

Reviewer #2 (Public review):

Summary:

I am not familiar with mathematical modeling of gene expression, so I will evaluate this manuscript solely from a biological point of view.

Kerlin et al. combined single-molecule RNA FISH and mathematical modeling approaches to quantitatively characterize changes in the transcriptional dynamics of three neighboring genes at the FOS locus in response to estradiol (E2) stimulation. They showed that the neighboring JDP2 and BATF genes, located on the same side of the TAD boundary, exhibit highly coordinated bursting dynamics. While FOS and JDP2/BATF are strongly insulated (~7:1 intra-to-inter-domain contact ratio) by the TAD boundary, correlated bursting dynamics were still observed between these gene pairs, suggesting that enhancers can bypass strong insulation sites. The authors proposed that burst co-occurrence arises from the activity of ERα-bound enhancers at the locus. They also proposed that the burst size correlation between two neighboring genes located on the same side of the TAD boundary results from local spreading of histone marks.

Strengths:

The direct visualization of coordinated transcriptional bursting across a strong insulation site is novel. This finding was carefully analyzed using the mathematical framework developed by the authors.

Weaknesses:

Several models were proposed based on single-molecule RNA FISH analysis of the FOS locus, but the generality of these findings remains uncertain. The proposed models were not directly tested through follow-up experiments, leaving the authors' conclusions largely speculative.

Reviewer #2 (Public review):

Summary:

While immune cell distribution in tuberculosis (TB) is well documented, research on its disruption in diabetes-tuberculosis (DM-TB) comorbidity remains limited. In this study, Chaudhary et al. explore immune cell perturbations in DM-TB using single-cell RNA sequencing (scRNA-seq), providing key insights into the impaired host immune response. By elucidating the molecular mechanisms underlying immune dysfunction in DM-TB, this study addresses an important knowledge gap. The study demonstrates that diabetes impairs lung immune cell infiltration and contributes to a dampened immune response against Mycobacterium tuberculosis. Reduced Th1 and M1 macrophage populations indicate a compromised ability to mount an effective pro-inflammatory response, which is essential for TB control. The observed increase in IL-16 signaling and reduction in TNF and IFN-II responses suggest a shift toward a more immunosuppressive or dysregulated inflammatory state. The interplay between chronic inflammation, hyperglycemia, and dyslipidemia in diabetes further exacerbates immune dysfunction, reinforcing the idea that metabolic disorders significantly impact TB pathogenesis.

Strengths:

This well-designed study employs robust methodology, well-executed experiments, and a well-written manuscript. The use of scRNA-seq is a notable strength, offering high-resolution analysis of immune cell heterogeneity in the lung environment. Additionally, the study corroborates its findings in a long-term infection model, demonstrating that chronic M. tuberculosis (H37Rv) infection in diabetic mice leads to increased bacterial burden and worsened tissue pathology.

Weaknesses:

(1) The study focuses on CD3⁺ and CD11c⁺ cells but does not extensively examine other key immune players that may contribute to DM-TB pathogenesis. Given that diabetes affects multiple immune compartments, a broader immune profiling approach would provide a more comprehensive understanding.

(2) While the study identifies increased IL-16 signaling and reduced TNF/IFN-II responses, the precise molecular mechanisms driving these changes remain unclear. Further investigation into metabolic-immune crosstalk (e.g., how hyperglycemia affects immune cell differentiation and cytokine secretion) would strengthen the mechanistic depth of the findings.

(3) The study suggests targeting IL-16 and Th17 cells as potential therapeutic strategies; however, no experimental validation (e.g., testing IL-16 inhibitors in DM-TB models) is provided. Validating these interventions would enhance their translational relevance.

(4) Incorporating clinical samples (e.g., PBMCs from DM-TB patients) could help bridge the gap between murine and human studies, offering more translational insights into disease mechanisms.

Overall, this study provides valuable findings, but addressing these concerns would further strengthen its impact on understanding DM-TB immunopathogenesis.

Reviewer #2 (Public review):

Summary:

Garibova et al. investigated the conservation of sperm recognition and interaction with the egg envelope in two groups of distantly related animals: mammals (mouse) and fish (zebrafish). Previous work and key physiological differences between these two animal groups strongly suggest that mouse sperm would be incapable of interaction with the zebrafish egg envelope (chorion) and its constituent proteins, though homologous to the mammalian zona pellucida (ZP). Indeed, the authors showed that mouse sperm do not bind recombinant zebrafish ZP proteins nor the intact chorion. Surprisingly, however, mouse sperm are able to locate and bind to the zebrafish micropyle, a specialized canal within the chorion that serves as the egg's entry point for sperm. This study suggests that sperm attraction to the egg might be highly conserved from fish to mammals and depends on the presence of a still unknown glycosylated protein within the micropyle. The authors further demonstrate that mouse sperm are able to enter the micropyle and accumulate within the intrachorionic space, potentially through a CatSper-dependent mechanism.

Strengths:

The authors convincingly demonstrate that mouse sperm do not bind zebrafish ZP proteins or the chorion. Furthermore, they make the interesting observation that mouse sperm are able to locate and enter the zebrafish micropyle in an MP-dependent manner, which is quite unexpected given the large evolutionary distance between these species, the many physiological differences between mouse and zebrafish gametes, and the largely different modes of both fertilization and reproduction in these species. This may indicate that the sperm chemoattractant in the egg is conserved between mammals and fish; however, whether zebrafish sperm are attracted to mouse eggs was not tested.

Weaknesses:

The key weakness of this study lies in the rationale behind the overall investigation. In mammals, the zona pellucida (ZP) has been implicated in binding sperm in a taxon-specific manner, such that human sperm are incapable of binding the mouse ZP. Indeed, work by the corresponding author showed that this specificity is mediated by the N-terminal region of the ZP protein ZP2 (Avella et al., 2014). The N-termini of human and mouse ZP2 share 48% identity, which is higher than the overall identity between mouse and zebrafish ZP2, with the latter ortholog entirely lacking the N-terminal domain that is essential for sperm binding to the ZP. Given this known specificity for mouse vs. human sperm-ZP binding, it does not follow that mouse sperm would bind ZP proteins from not only a species that is much more distantly related, but also one that is not even a mammal, the zebrafish. Furthermore, the fish chorion does not play a role in sperm binding at all, while the mammalian ZP can bind sperm at any location. On the contrary, the zebrafish chorion prevents polyspermy by limiting sperm entry to the single micropyle.

In addition, though able to provide some information regarding the broad conservation of sperm-egg interaction mechanisms, the biological relevance of these findings is difficult to describe. Fish and mammals are not only two very distinct and distantly related animal groups, but also employ opposite modes of fertilization and reproduction (external vs. internal, oviparous vs viviparous). Fish gametes interact in a very different environment compared to mammals and lack many typically mammalian features of fertilization (e.g., sperm capacitation, presence of an acrosome, interaction with the female reproductive tract), making it difficult to make any physiologically relevant claims from this study. While this study may indicate conserved mechanisms of sperm attraction to the egg, the identity of the molecular players involved is not investigated. With this knowledge, the reader is forced to question the motivation behind much of the study.

During fertilization in fish, the sperm enters the micropyle and subsequently, the egg, as it is simultaneously activated by exposure to water. During egg activation, the chorion lifts as it separates from the egg and fills with water. This mechanism prevents supernumerary sperm from entering the egg after the successfully fertilizing sperm has bound and fused. In this study, the authors show that mouse sperm enter the micropyle and accumulate in the intrachorionic space. Whether any sperm successfully entered the egg is not addressed, and the status of egg activation is not reported. In Supplementary Videos 3-4, the egg shown has been activated for some time, as evident by the separation of yolk and cytoplasm, yet the chorion is only partially expanded (likely due to mouse IVF conditions). How multiple sperm were able to enter the micropyle but presumably not the egg is not addressed, yet this suggests that the zebrafish mechanism of blocking polyspermy (fertilization by multiple sperm) is not effective for mouse sperm or is rendered ineffective due to mouse IVF conditions. The authors do not discuss these observations in the context of either species' physiological process of fertilization, highlighting the lack of biological context in interpreting the results.

The authors further show that the zebrafish micropyle does not trigger the acrosome reaction in mouse sperm. Whether the acrosome reacts is not correlated with a sperm's ability to cross the micropyle opening, as both acrosome-intact and acrosome-reacted sperm were observed within the intrachorionic space. While the acrosome reaction is a key event during mammalian fertilization and is required for sperm to fertilize the egg, zebrafish sperm do not contain an acrosome. Thus, these results are particularly difficult to interpret biologically, bringing into question whether this observation has biological relevance or is a byproduct of egg activation/chorion lifting that indirectly draws sperm into the chorion.

The final experiments regarding CatSper1's role in mediating mouse sperm entry into the micropyle/chorion are not convincing. As no molecular interactions are described or perturbed, the reader cannot be sure whether the sperm's failure to enter is due to signaling via CatSper1 or whether the overall failure to undergo hyperactivation limits sperm motility such that the mutant sperm can no longer find and enter the zebrafish micropyle. Indeed, in Figure 5E, no CatSper1 mutant sperm are visible near any part of the egg, suggesting that overall motility is impaired, and this is not a phenotype specific to interactions with the micropyle.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the mechanism by which chronic stress induces locus coeruleus (LC) neuron degeneration. The authors demonstrate that chronic stress leads to internalization of α2A-adrenergic receptors (α2A-ARs) on LC-neurons, causing increased cytosolic noradrenaline (NA) accumulation and subsequent production of the neurotoxic metabolite DOPEGAL via monoamine oxidase A (MAO-A). The study suggests a mechanistic link between stress-induced α2A-AR internalization, disrupted autoinhibition, elevated NA metabolism, asparagine endopeptidase (AEP) activation, and Tau pathology relevant to Alzheimer's disease (AD). The conclusions of this paper are mostly well supported by data, but some aspects of image acquisition need to be extended.

Strengths:

This study clearly demonstrates the effects of chronic stimulation on the excitability of LC neurons using electrophysiological techniques. It also elucidates the role of α2-adrenergic receptor (α2-AR) internalization and the associated upstream and downstream signaling pathways of GIRK1 using a range of pharmacological agents, highlighting the innovative nature of the work.

Additionally, the study identifies the involvement of the MAO-A-DOPEGAL-AEP pathway in this process. The topic is timely, the proposed mechanistic pathway is compelling, and the findings have translational relevance, particularly regarding therapeutic strategies targeting α2A-AR internalization in neurodegenerative diseases.

Weaknesses:

(1) The manuscript reports that chronic stress for 5 days increases MAO-A levels in LC neurons, leading to the production of DOPEGAL, activation of AEP, and subsequent tau cleavage into the tau N368 fragment, ultimately contributing to neuronal damage. However, the authors used wild-type C57BL/6 mice, and previous literature has indicated that AEP-mediated tau cleavage in wild-type mice is minimal and generally insufficient to cause significant behavioral alterations. Please clarify and discuss this apparent discrepancy.

(2) It is recommended that the authors include additional experiments to examine the effects of different durations and intensities of stress on MAO-A expression and AEP activity. This would strengthen the understanding of stress-induced biochemical changes and their thresholds.

(3) Please clarify the rationale for the inconsistent stress durations used across Figures 3, 4, and 5. In some cases, a 3-day stress protocol is used, while in others, a 5-day protocol is applied. This discrepancy should be addressed to ensure clarity and experimental consistency.

(4) The abbreviation "vMAT2" is incorrectly formatted. It should be "VMAT2," and the full name (vesicular monoamine transporter 2) should be provided at first mention.

Reviewer #2 (Public review):

In K. Kostanjevec et.al, the authors study a possible mechanism for the formation of spiral patterns in the cornea. First the authors analyze an inferred velocity field, which is deduced from images of fixed corneas, and then determine the position-dependent spiral angle of this velocity fields. Next, the authors analysed two possible markers of cell polarity: the direction of the centrosome-nuclei and the axis of mitosis. Then the authors introduce a stochastic agent-based model of self-propelled particles with over-damped dynamics and with aligning interactions to the orientation of the nearest neighbors and to the particle's velocity. The authors claim to be able to reproduce the equal-time autocorrelation function and the velocity Fourier spectrum. Then the authors introduce the geometry of the cornea by constraining the dynamics on a spherical cap and show that their model can reproduce a typical trajectory in experiments. Finally, the authors produce a phase diagram of the states at a fixed time point as a function of the spherical cap radius and the strength of the coupling aligning constant. Finally, the authors propose an interpretation of the cell fluxes based on the equation of mass conservation.

Reviewer #2 (Public review):

This is a short yet very clear manuscript demonstrating that two methods (END-seq and S1-END-seq), previously developed in the Nussenzweig laboratory to study DSBs in the genome, can also be applied to the 5' ends of mammalian telomeres and the accumulation of telomeric single-stranded DNA.

The authors first validate the applicability of END-seq using different approaches and confirm that mammalian telomeres preferentially end with an ATC 5' end through a mechanism that requires intact POT1 (POT1a in mice). They then extend their analysis to cells that maintain telomeres through the ALT mechanism and demonstrate that, in these cells as well, telomeres frequently end in an ATC 5' sequence via a POT1-dependent mechanism. Using S1-END-seq, the authors further show that ALT telomeres contain single-stranded DNA and estimate that each telomere in ALT cells harbors at least five regions of ssDNA.

I find this work very interesting and incisive. It clearly demonstrates that END-seq can be applied with unprecedented depth and precision to the study of telomeric features such as the 5' end and ssDNA. The data are very clear and thoroughly interpreted, and the manuscript is well written. The results are carefully analyzed and effectively presented. Overall, I find this manuscript worthy of publication, as the optimized END-seq methods described here will likely be widely utilized in the telomere field.

I only have a few minor suggestions:

How can we be sure that all telomeres are equally represented? The authors seem to assume that END-seq captures all chromosome ends equally, but can we be certain of this? While I do not see an obvious way to resolve this experimentally, I recommend discussing this potential bias more extensively in the manuscript.

I believe Figures 1 and 2 should be merged.

Scale bars should be added to all microscopy figures.

Reviewer #2 (Public review):

Summary:

In this paper, the authors describe the results of a longitudinal study of pertussis infection in mother/infant dyads in Lusaka, Zambia. Unlike many past studies, the authors assessed the infection status of individuals independently of whether they were symptomatic for a respiratory infection. As a result, this work represents one of the first studies specifically designed to assess asymptomatic transmission of pertussis. Using qPCR, the authors find strong evidence for the role of asymptomatic transmission from mothers to infants and also evidence for long-term bacterial carriage. This work represents an important contribution to our understanding of the global burden of pertussis. Also, it highlights the still under-appreciated role of asymptomatic transmission across many infectious diseases (including vaccine-preventable ones).

Strengths:

Unlike many past studies, the authors assessed the infection status of individuals independently of whether they were symptomatic for a respiratory infection. As a result, this work represents one of the first studies specifically designed to assess asymptomatic transmission of pertussis. Using qPCR, the authors find strong evidence for the role of asymptomatic transmission from mothers to infants and also evidence for long-term bacterial carriage.

Weaknesses:

While I am quite enthusiastic about the work, I am concerned that a number of likely relevant confounders were not discussed and that the broader implications of their findings were not well grounded in the existing literature. For example, I could not find information on the vaccination status of the mothers in the study. Given the conclusions about asymptomatic transmission and the durability of immunity, it is important to know the vaccination status of the mothers. Moreover, did the authors have other metadata on the mother/infant dyads, e.g., household size, vaccination status of household members, etc.? Given the potential implications of more widespread asymptomatic transmission associated with pertussis infection, I believe the authors should better couch their results in the context of the broader debate around asymptomatic transmission.

Reviewer #3 (Public review):

NCXs are key Ca2+ transporters located on the plasma membrane, essential for maintaining cellular Ca2+ homeostasis and signaling. The activities of NCX are tightly regulated in response to cellular conditions, ensuring precise control of intracellular Ca2+ levels, with profound physiological implications. Building upon their recent breakthrough in determining the structure of human NCX1, the authors obtained cryo-EM structures of NCX1 in complex with its modulators, including the cellular activator PIP2 and the small molecule inhibitor SEA0400. Structural analyses revealed mechanistically informative conformational changes induced by PIP2 and elucidated the molecular basis of inhibition by SEA0400. These findings underscore the critical role of the interface between the transmembrane and cytosolic domains in NCX regulation and small molecule modulation. Overall, the results provide key insights into NCX regulation, with important implications for cellular Ca2+ homeostasis.

Comments on revisions:

The authors have adequately addressed my previous comments.

Reviewer #3 (Public review):

Summary:

The authors aim to establish that cipargamin can be used for the treatment of infection caused by Babesia organisms.

Strengths:

The study provides strong evidence that cipargamin is effective against various Babesia species. In vitro growth assays were used to establish that cipargamin is effective against Babesia bovis and Babesia gibsoni. Infection of mice with Babesia microti demonstrated that cipargamin is as effective as the combination of atovaquone plus azithromycin. Cipargamin protected mice from lethal infection with Babesia rodhaini. Mutations that confer resistance to cipargamin were identified in the gene encoding ATP4, a P-type Na ATPase that is found in other apicomplexan parasites, thereby validating ATP4 as the target of cipargamin. A 7-day treatment of cipagarmin, when combined with a single dose of tafenoquine, was sufficient to eradicate Babesia microti in a mouse model of severe babesiosis caused by a lack of adaptive immunity.

Weaknesses:

Cipargamin was tested in vivo at a single dose administered daily for 7 days. Despite the prospect of using cipargamin for the treatment of human babesiosis, there was no attempt to identify the lowest dose of cipagarmin that protects mice from Babesia microti infection.

Comments on revisions:

The authors have edited the manuscript and, in doing so, have addressed all queries pertaining to experimental design. The authors have decided to keep the discussion unchanged, but have replied to this reviewer regarding comments on interpretation of some data. The reader could have benefited from the authors' explanation. Nonetheless, the manuscript in its present form describes a valuable and significant body of work.

Reviewer #2 (Public review):

Summary:

While technical advances have enabled large-scale, multi-site neural recordings, characterizing inter-regional communication and its behavioral relevance remains challenging due to intrinsic properties of the brain such as shared inputs, network complexity, and external noise. This work by Saiki-Ishkawa et al. examines the functional hierarchy between premotor (PM) and primary motor (M1) cortices in mice during a directional reaching task. The authors find some evidence consistent with an asymmetric reciprocal influence between the regions, but overall, activity patterns were highly similar and equally predictive of one another. These results suggest that motor cortical hierarchy, though present, is not fully reflected in firing patterns alone.

Strengths:

Inferring functional hierarchies between brain regions, given the complexity of reciprocal and local connectivity, dynamic interactions, and the influence of both shared and independent external inputs, is a challenging task. It requires careful analysis of simultaneous recording data, combined with cross-validation across multiple metrics, to accurately assess the functional relationships between regions. The authors have generated a valuable dataset simultaneously recording from both regions at scale from mice performing a cortex-dependent directional reaching task.

Using electrophysiological and silencing data, the authors found evidence supporting the traditionally assumed asymmetric influence from PM to M1. While earlier studies inferred a functional hierarchy based on partial temporal relationships in firing patterns, the authors applied a series of complementary analyses to rigorously test this hierarchy at both individual neuron and population levels, with robust statistical validation of significance.

In addition, recording combined with brief optogenetic silencing of the other region allowed authors to infer the asymmetric functional influence in a more causal manner. This experiment is well designed to focus on the effect of inactivation manifesting through oligosynaptic connections to support the existence of a premotor to primary motor functional hierarchy.

Subsequent analyses revealed a more complex picture. CCA, PLS, and three measures of predictivity (Granger causality, transfer entropy, and convergent cross mapping) emphasized similarities in firing patterns and cross-region predictability. However, DLAG suggested an imbalance, with RFA capturing CFA variance at a negative time lag, indicating that RFA 'leads' CFA. Taken together these results provide useful insights for current studies of functional hierarchy about potential limitations in inferring hierarchy solely based on firing rates.

While I would detail some questions and issues on specifics of data analyses and modeling below, I appreciate the authors' effort in training RNNs that match some behavioral and recorded neural activity patterns including the inactivation result. The authors point out two components that can determine the across-region influence - 1) the amount of inputs received and 2) the dependence on across-region input, i.e., relative importance of local dynamics, providing useful insights in inferring functional relationships across regions.

Weaknesses:

(1) Trial-averaging was applied in CCA and PLS analyses. While trial-averaging can be appropriate in certain cases, it leads to the loss of trial-to-trial variance, potentially inflating the perceived similarities between the activity in the two regions (Figure 4). Do authors observe comparable degrees of similarity, e.g., variance explained by canonical variables? Also, the authors report conflicting findings regarding the temporal relationship between RFA and CFA when using CCA/PLS versus DLAG. Could this discrepancy be due to the use of trial-averaging in former analyses but not in the latter?

(2) A key strength of the current study is the precise tracking of forelimb muscle activity during a complex motor task involving reaching for four different targets. This rich behavioral data is rarely collected in mice and offers a valuable opportunity to investigate the behavioral relevance of the PM-M1 functional interaction, yet little has been done to explore this aspect in depth. For example, single-trial time courses of inter-regional latent variables acquired from DLAG analysis can be correlated with single-trial muscle activity and/or reach trajectories to examine the behavioral relevance of inter-regional dynamics. Namely, can trial-by-trial change in inter-regional dynamics explain behavioral variability across trials and/or targets? Does the inter-areal interaction change in error trials? Furthermore, the authors could quantify the relative contribution of across-area versus within area dynamics to behavioral variability. It would also be interesting to assess the degree to which across-area and within-area dynamics are correlated. Specifically, can across-area dynamics vary independently from within-area dynamics across trials, potentially operating through a distinct communication subspace?

(3) While network modeling of RFA and CFA activity captured some aspects of behavioral and neural data, I wonder if certain findings such as the connection weight distribution (Figure 7C), across-region input (Figure 7F), and the within-region weights (Figure 7G), primarily resulted from fitting the different overall firing rates between the two regions with CFA exhibiting higher average firing rates. Did the authors account for this firing rate disparity when training the RNNs?

(4) Another way to assess the functional hierarchy is by comparing the time courses of movement representation between the two regions. For example, a linear decoder could be used to compare the amount of information about muscle activity and/or target location as well as time courses thereof between the two regions. This approach is advantageous because it incorporates behavior rather than focusing solely on neural activity. Since one of the main claims of this study is the limitation of inferring functional hierarchy from firing rate data alone, the authors should use the behavior as a lens for examining inter-areal interactions.

Comments on revisions:

I appreciate the authors' thoughtful revisions in response to prior reviews, which I believe have substantially improved the manuscript. In particular, I found the addition of the new section "Manifestations of hierarchy in firing patterns" to be valuable, as it begins to address some of the more complex and potentially conflicting observations

Reviewer #2 (Public review):

Summary:

This study explores the potential of inhibiting the p38-MK2 signaling pathway to enhance the efficacy of microtubule-targeting agents (MTAs) in breast cancer treatment using a dual-target inhibitor.

Strengths:

The study identifies the p38-MK2 pathway as a promising target to enhance the efficacy of microtubule-targeting agents (MTAs), offering a novel therapeutic strategy for breast cancer treatment. The study also employs a wide range of techniques, especially live-cell imaging, to assess the microtubule dynamics in TNBC cells. The revised manuscript added new in vitro and in vivo evidence that furtherly supported the conclusions.

Comments on revisions:

The authors have appropriately addressed all of my comments and concerns. Specifically, they performed additional in vitro experiments using MCF10A cells and p53 knockout cells to determine the IC50 of CMPD1. They also repeated the in vivo treatment experiment and evaluated the toxicity of the drug treatment in the CAL-51 model. Furthermore, they provided genetic evidence for the combination treatment. I'm satisfied with the revision and have no further major comments. Minor comment: make sure the name of the chemo drug shown in Fig. 3 is consistent.

Reviewer #2 (Public review):

Summary:

The author of this manuscript aimed to uncover the mechanisms behind miRNA retention within cells. They identified PCBP2 as a crucial factor in this process, revealing a novel role for RNA-binding proteins. Additionally, the study discovered that SYNCRIP is essential for PCBP2's function, demonstrating the cooperative interaction between these two proteins. This research not only sheds light on the intricate dynamics of miRNA retention but also emphasizes the importance of protein interactions in regulating miRNA behavior within cells.

Strengths:

This paper makes important progress in understanding how miRNAs are kept inside cells. It identifies PCBP2 as a key player in this process, showing a new role for proteins that bind RNA. The study also finds that SYNCRIP is needed for PCBP2 to work, highlighting how these proteins work together. These discoveries not only improve our knowledge of miRNA behavior but also suggest new ways to develop treatments by controlling miRNA locations to influence cell communication in diseases. The use of liver cell models and thorough experiments ensures the results are reliable and show their potential for RNA-based therapies

Weaknesses:

The manuscript is well-structured and presents compelling data, but I noticed a few minor corrections that could further enhance its clarity:

Figure References: In the response to Reviewer 1, the comment states, "It's not Panel C, it's Panel A of Figure 1"-this should be cross-checked for consistency.<br /> Supplementary Figure 2 is labeled as "Panel A"-please verify if additional panels (B, C, etc.) are intended.

Western Blot Quality: The Alix WB shows some background noise. A repeat with optimized conditions (or inclusion of a cleaner replicate) would strengthen the data. Adding statistical analysis for all WBs would also reinforce robustness.

These are relatively small refinements, and the manuscript is already in excellent shape. With these adjustments, it will be even stronger.

Reviewer #2 (Public review):

Hawes et al. investigated the role of striatal neurons in the patch compartment of the dorsal striatum. Using Sepw1-Cre line, the authors combined a modified version of the light/dark transition box test that allows them to examine locomotor activity in different environmental valence with a variety of approaches, including cell-type-specific ablation, miniscope calcium imaging, fiber photometry, and opto-/chemogenetics. First, they found ablation of patchy striatal neurons resulted in an increase in movement vigor when mice stayed in a safe area or when they moved back from more anxiogenic to safe environments. The following miniscope imaging experiment revealed that a larger fraction of striatal patchy neurons was negatively correlated with movement speed, particularly in an anxiogenic area. Next, the authors investigated differential activity patterns of patchy neurons' axon terminals, focusing on those in GPe, GPi, and SNr, showing that the patchy axons in SNr reflect movement speed/vigor. Chemogenetic and optogenetic activation of these patchy striatal neurons suppressed the locomotor vigor, thus demonstrating their causal role in the modulation of locomotor vigor when exposed to valence differentials. Unlike the activation of striatal patches, such a suppressive effect on locomotion was absent when optogenetically activating matrix neurons by using the Calb1-Cre line, indicating distinctive roles in the control of locomotor vigor by striatal patch and matrix neurons. Together, they have concluded that nigrostriatal neurons within striatal patches negatively regulate movement vigor, dependent on behavioral contexts where motivational valence differs.

In my view, this study will add to the important literature by demonstrating how patch (striosomal) neurons in the striatum control movement vigor. This study has applied multiple approaches to investigate their functionality in locomotor behavior, and the obtained data largely support their conclusions. Nevertheless I have some suggestions for improvements in the manuscript and figures regarding their data interpretation, accuracy, and efficacy of data presentation.

(1) The authors found that the activation of the striatonigral pathway in the patch compartment suppresses locomotor speed, which contradicts with canonical roles of the direct pathway. It would be great if the authors could provide mechanistic explanations in the Discussion section. One possibility is that striatal D1R patch neurons directly inhibit dopaminergic cells that regulate movement vigor (Nadal et al., Sci. Rep., 2021; Okunomiya et al., J Neurosci., 2025). Providing plausible explanations will help readers infer possible physiological processes and give them ideas for future follow-up studies.

(2) On page 14, Line 301, the authors stated that "Cre-dependent mCheery signals were colocalized with the patch marker (MOR1) in the dorsal striatum (Fig. 1B)". But I could not find any mCherry on that panel, so please modify it.

(3) From data shown in Figure 1, I've got the impression that mice ablated with striatal patch neurons were generally hyperactive, but this is probably not the case, as two separate experiments using LLbox and DDbox showed no difference in locomotor vigor between control and ablated mice. For the sake of better interpretation, it may be good to add a statement in Lines 365-366 that these experiments suggest the absence of hyperactive locomotion in general by ablating these specific neurons.

(4) In Line 536, where Figure 5A was cited, the author mentioned that they used inhibitory DREADDs (AAV-DIO-hM4Di-mCherrry), but I could not find associated data on Figure 5. Please cite Figure S3, accordingly.

(5) Personally, the Figure panel labels of "Hi" and "ii" were confusing at first glance. It would be better to have alternatives.

(6) There is a typo on Figure 4A: tdTomata → tdTomato

Reviewer #2 (Public review):

Summary:

The authors investigated the mechanisms behind breeding season-dependent feeding behavior using medaka, a well-known photoperiodic species, as a model. Through a combination of molecular, cellular, and behavioral analyses, including tests with mutants, they concluded that AgRP1 plays a central role in feeding behavior, mediated by ovarian estrogenic signals.

Strengths:

This study offers valuable insights into the neuroendocrine mechanisms that govern breeding season-dependent feeding behavior in medaka. The multidisciplinary approach, which includes molecular and physiological analyses, enhances the scientific contribution of the research.

Comments on revised version:

My concerns from the first review have been addressed. The manuscript's key points are clearly presented, and the conclusions are readily comprehensible

Reviewer #2 (Public review):

Summary:

This paper formulates an individual-based model to understand the evolution of division of labor in vertebrates. A main conclusion of the paper is that direct fitness benefits are the primary factor causing the evolution of vertebrate division of labor, rather than indirect fitness benefits.

Strengths:

The paper formulates an individual-based model that is inspired by vertebrate life history. The model incorporates numerous biologically realistic details, including the possibility to evolve age polytheism where individuals switch from work to defence tasks as they age or vice versa, as well as the possibility of comparing the action of group augmentation alone with that of kin selection alone.

Weaknesses:

The model makes assumptions that restrict the possibility that kin selection leads to the evolution of helping. In particular, the model assumes that in the absence of group augmentation, subordinates can only help breeders but cannot help non-breeders or increase the survival of breeders, whereas with group augmentation, subordinates can help both breeders and non-breeders and increase the survival of breeders. This is unrealistic as subordinates in real organisms can help other subordinates and increase the survival of non-breeders, even in the absence of group augmentation, for instance, with targeted helping to dominants or allies. This restriction artificially limits the ability of kin selection alone to lead to the evolution of helping, and potentially to division of labor. Hence, the conclusion that group augmentation is the primary driving factor driving vertebrate division of labor appears forced by the imposed restrictions on kin selection. The model used is also quite particular, and so the claimed generality across vertebrates is not warranted.

I describe some suggestions for improving the paper below, more or less in the paper's order.

First, the introduction goes to great lengths trying to convince the reader that this model is the first in this or another way, particularly in being only for vertebrates, as illustrated in the abstract where it is stated that "we lack a theoretical framework to explore the conditions under which division of labor is likely to evolve" (line 13). However, this is a risky and unnecessary motivation. There are many models of division of labor and some of them are likely to be abstract enough to apply to vertebrates even if they are not tailored to vertebrates, so the claims for being first are not only likely to be wrong but will put many readers in an antagonistic position right from the start, which will make it harder to communicate the results. Instead of claiming to be the first or that there is a lack of theoretical frameworks for vertebrate division of labor, I think it is enough and sufficiently interesting to say that the paper formulates an individual-based model motivated by the life history of vertebrates to understand the evolution of vertebrate division of labor. You could then describe the life history properties that the model incorporates (subordinates can become reproductive, low relatedness, age polyethism, etc.) without saying this has never been done or that it is exclusive to vertebrates; indeed, the paper states that these features do not occur in eusocial insects, which is surprising as some "primitively" eusocial insects show them. So, in short, I think the introduction should be extensively revised to avoid claims of being the first and to make it focused on the question being addressed and how it is addressed. I think this could be done in 2-3 paragraphs without the rather extensive review of the literature in the current introduction.

Second, the description of the model and results should be clarified substantially. I will give specific suggestions later, but for now, I will just say that it is unclear what the figures show. First, it is unclear what the axes in Figure 2 show, particularly for the vertical one. According to the text in the figure axis, it presumably refers to T, but T is a function of age t, so it is unclear what is being plotted. The legend explaining the triangle and circle symbols is unintelligible (lines 227-230), so again it is unclear what is being plotted; part of the reason for this unintelligibility is that the procedure that presumably underlies it (section starting on line 493) is poorly explained and not understandable (I detail why below). Second, the axes in Figure 3 are similarly unclear. The text in the vertical axis in panel A suggests this is T, however, T is a function of t and gamma_t, so something else must be being done to plot this. Similarly, in panel B, the horizontal axis is presumably R, but R is a function of t and of the helping genotype, so again some explanation is lacking. In all figures, the symbol of what is being plotted should be included.

Third, the conclusions sound stronger than the results are. A main conclusion of the paper is that "kin selection alone is unlikely to select for the evolution of defensive tasks and division of labor in vertebrates" (lines 194-195). This conclusion is drawn from the left column in Figure 2, where only kin selection is at play, and the helping that evolves only involves work rather than defense tasks. This conclusion follows because the model assumes that without group augmentation (i.e., xn=0, the kin selection scenario), subordinates can only help breeders to reproduce but cannot help breeders or other subordinates to survive, so the only form of help that evolves is the least costly, not the most beneficial as there is no difference in the benefits given among forms of helping. This assumption is unrealistic, particularly for vertebrates where subordinates can help other group members survive even in the absence of group augmentation (e.g., with targeted help to certain group members, because of dominance hierarchies where the helping would go to the breeder, or because of alliances where the helping would go to other subordinates). I go into further details below, but in short, the model forces a narrow scope for the kin selection scenario, and then the paper concludes that kin selection alone is unlikely to be of relevance for the evolution of vertebrate division of labor. This conclusion is particular to the model used, and it is misleading to suggest that this is a general feature of such a particular model.

Overall, I think the paper should be revised extensively to clarify its aims, model, results, and scope of its conclusions.

Reviewer #2 (Public review):

In this study, Cai and colleagues investigate how one component of the m6A methyltransferase complex, the WTAP protein, responds to IFNb stimulation. They find that viral infection or IFNb stimulation induces the transition of WTAP from aggregates to liquid droplets through dephosphorylation by PPP4. This process affects the m6A modification levels of ISG mRNAs and modulates their stability. In addition, the WTAP droplets interact with the transcription factor STAT1 to recruit the methyltransferase complex to ISG promoters and enhance m6A modification during transcription. The investigation dives into a previously unexplored area of how viral infection or IFNb stimulation affects m6A modification on ISGs. The observation that WTAP undergoes a phase transition is significant in our understanding of the mechanisms underlying m6A's function in immunity. However, there are still key gaps that should be addressed to fully accept the model presented.

Major points:<br /> (1) More detailed analyses on the effects of WTAPsgRNA on the m6A modification of ISGs:<br /> a. A comprehensive summary of the ISGs, including the percentage of ISGs that are m6A-modified,<br /> b. The distribution of m6A modification across the ISGs, and<br /> c. A comparison of the m6A modification distribution in ISGs with non-ISGs.<br /> In addition, since the authors propose a novel mechanism where the interaction between phosphorylated STAT1 and WTAP direct the MTC to the promoter regions of ISGs to facilitate co-transcriptional m6A modification, it is critical to analyze whether the m6A modification distribution holds true in the data.

(2) Since a key part of the model includes the cytosol-localized STAT1 protein undergoing phosphorylation to translocate to the nucleus to mediate gene expression, the authors should focus on the interaction between phosphorylated STAT1 and WTAP in Figure 4, rather than the unphosphorylated STAT1. Only phosphorylated STAT1 localizes to the nucleus, so the presence of pSTAT1 in the immunoprecipitate is critical for establishing a functional link between STAT1 activation and its interaction with WTAP.

(3) The authors should include pSTAT1 ChIP-seq and WTAP ChIP-seq on IFNb-treated samples in Figure 5 to allow for a comprehensive and unbiased genomic analysis for comparing the overlaps of peaks from both ChIP-seq datasets. These results should further support for their hypothesis that WTAP interacts with pSTAT1 to enhance m6A modifications on ISGs.

Minor points:<br /> (1) Since IFNb is primarily known for modulating biological processes through gene transcription, it would be informative if the authors discussed the mechanism of how IFNb would induce the interaction between WTAP and PPP4.

(2) The authors should include mCherry alone controls in Figure 1D to demonstrate that mCherry does not contribute to the phase separation of WTAP. Does mCherry have or lack a PLD?

(3) The authors should clarify the immunoprecipitation assays in the methods. For example, the labeling in Fig. 2A suggests that antibodies against WTAP and pan-p were used for two immunoprecipitations. Is that accurate?

(4) The authors should include overall m6A modification levels quantified of GFPsgRNA and WTAPsgRNA cells, either by mass spectrometry (preferably) or dot blot.

Comments on revisions:

The authors thoroughly addressed the aforementioned points during the review process.

Reviewer #2 (Public review):

Summary:

This paper investigates the neural underpinnings of an interactive speech task requiring verbal coordination with another speaker. To achieve this, the authors recorded intracranial brain activity from the left (and to a lesser extent, the right) hemisphere in a group of drug-resistant epilepsy patients while they synchronised their speech with a 'virtual partner'. Crucially, the authors were able to manipulate the degree of success of this synchronisation by programming the virtual partner to either actively synchronise or desynchronise their speech with the participant, or else to not vary its speech in response to the participant (making the synchronisation task purely one-way). Using such a paradigm, the authors identified different brain regions that were either more sensitive to the speech of the virtual partner (primary auditory cortex), or more sensitive to the degree of verbal coordination (i.e. synchronisation success) with the virtual partner (left secondary auditory cortex and bilateral IFG). Such sensitivity was measured by (1) calculating the correlation between the index of verbal coordination and mean power within a range of frequency bands across trials, and (2) calculating the phase-amplitude coupling between the behavioural and brain signals within single trials (using the power of high-frequency neural activity only). Overall, the findings help to elucidate some of the brain areas involved in interactive speaking behaviours, particularly highlighting high-frequency activity of the bilateral IFG as a potential candidate supporting verbal coordination.

Strengths:

This study provides the field with a convincing demonstration of how to investigate speaking behaviours in more complex situations that share many features with real-world speaking contexts e.g. simultaneous engagement of speech perception and production processes, the presence of an interlocutor and the need for inter-speaker coordination. The findings thus go beyond previous work that has typically studied solo speech production in isolation, and represent a significant advance in our understanding of speech as a social and communicative behaviour. It is further an impressive feat to develop a paradigm in which the degree of cooperativity of the synchronisation partner can be so tightly controlled; in this way, this study combines the benefits of using pre-recorded stimuli (namely, the high degree of experimental control) with the benefits of using a live synchronisation partner (allowing the task to be truly two-way interactive, an important criticism of other work using pre-recorded stimuli). A further key strength of the study lies in its employment of stereotactic EEG to measure brain responses with both high temporal and spatial resolution, an ideal method for studying the unfolding relationship between neural processing and this dynamic coordination behaviour.

Weaknesses:

One limitation of the current study is the relatively sparse coverage of the right hemisphere by the implanted electrodes (91 electrodes in the right compared to 145 in the left). Of course, electrode location is solely clinically motivated, and so the authors did not have control over this. In a previous version of this article, the authors therefore chose not to include data from the right hemisphere in reported analyses. However, after highlighting previous literature suggesting that the right hemisphere likely has high relevance to verbal coordination behaviours such as those under investigation here, the authors have now added analyses of the right hemisphere data to the results. These confirm an involvement of the right hemisphere in this task, largely replicating left hemisphere results. Some hemispheric differences were found in responses within the STG; however, interpretation should be tempered by an awareness of the relatively sparse coverage of the right hemisphere meaning that some regions have very few electrodes, resulting in reduced statistical power.

Reviewer #2 (Public review):

uORFs, short open reading frames located in the 5' UTR, are pervasive in genomes. However, their roles in maintaining protein abundance are not clear. In this study, the authors propose that uORFs act as "molecular dam", limiting the fluctuation of the translation of downstream coding sequences. First, they performed in silico simulations using an improved ICIER model, and demonstrated that uORF translation reduces CDS translational variability, with buffering capacity increasing in proportion to uORF efficiency, length, and number. Next, they analysed the translatome between two related Drosophila species, revealing that genes with uORFs exhibit smaller fluctuations in translation between the two species and across different developmental stages within the same species. Moreover, they identified that bicoid, a critical gene for Drosophila development, contains a uORF with substantial changes in translation efficiency. Deleting this uORF in Drosophila melanogaster significantly affected its gene expression, hatching rates, and survival under stress conditions. Lastly, by leveraging public Ribo-seq data, the authors showed that the buffering effect of uORFs is also evident between primates and within human populations. Collectively, the study significantly advances our understanding of how uORFs regulate the translation of downstream coding sequences at the genome-wide scale, as well as during development and evolution. It would be particularly interesting to explore whether similar buffering functions are conserved in other organisms, and whether their regulatory effects could be harnessed for practical applications, such as improving crop traits or benefiting human health.

Comments on revisions:

The authors have fully addressed all of my concerns, and the revisions have substantially improved the manuscript. I have no further comments.

Reviewer #2 (Public review):

Summary

Schubert et al. recorded MEG and eye tracking activity while participants were listening to stories in single-speaker or multi-speaker speech. In a separate task, MEG was recorded while the same participants were listening to four types of pure tones in either structured (75% predictable) or random (25%) sequences. The MEG data from this task was used to quantify individual 'prediction tendency': the amount by which the neural signal is modulated by whether or not a repeated tone was (un)predictable, given the context. In a replication of earlier work, this prediction tendency was found to correlate with 'neural speech tracking' during the main task. Neural speech tracking is quantified as the multivariate relationship between MEG activity and speech amplitude envelope. Prediction tendency did not correlate with 'ocular speech tracking' during the main task. Neural speech tracking was further modulated by local semantic violations in the speech material and by whether or not a distracting speaker was present. The authors suggest that part of the neural speech tracking is mediated by ocular speech tracking. Story comprehension was negatively related with ocular speech tracking.

Strengths

This is an ambitious study, and the authors' attempt to integrate the many reported findings related to prediction and attention in one framework is laudable. The data acquisition and analyses appear to be done with great attention to methodological detail. Furthermore, the experimental paradigm used is more naturalistic than was previously done in similar setups (i.e.: stories instead of sentences).

Weaknesses

While the analysis pipeline is outlined in much detail, some analysis choices appear ad-hoc and could have been more uniform and/or better motivated (other than: this is what was done before).

Reviewer #2 (Public review):

Summary:

This work concerns the evolution of ZDBF2 imprinting in mammalian species via initiation of GPR1 antisense (AS) transcription from a lineage-specific long-terminal repeat (LTR) retrotransposon. It extends previous work describing the mechanism of ZDBF2 imprinting in mice and humans by demonstrating conservation of GPR1-AS transcripts in rabbits and non-human primates. By identifying the origin of GPR1-AS transcription as the LTR MER21C, the authors claim to account for how imprinting evolved in these species but not in those lacking the MER21C insertion. This illustrates the principle of LTR co-option as a means of evolving new gene regulatory mechanisms, specifically to achieve parent-of-origin allele specific expression (imprinting). Examples of this phenomenon have been described previously, but usually involve initiation of transcription during gametogenesis rather than post-fertilization, as in this work. The findings of this paper are therefore relevant to biologists studying imprinted genes or interested more generally in the evolution of gene regulatory mechanisms.

Strengths:

(1) The authors convincingly demonstrate the existence of GPR1-AS orthologs in specific mammalian lineages using high quality RNA-seq libraries collected from diverse mammalian species.

(2) The authors demonstrate imprinting of the ZDBF2 locus in rabbits and Rhesus macaques using allele-specific expression analysis. The transcription of GPR1-AS orthologs therefore correlates with imprinting of the ZDBF2 locus.

Weaknesses:

(1) Experimental evidence directly linking GPR1-AS transcription to ZDBF2 imprinting in rabbits and non-human primates is lacking. Consideration should be given to the challenges associated with studying non-model species and manipulating repeat sequences. Further, this mechanism is established in humans and mice, so the authors' model is arguably sufficiently supported merely by the existence of GPR1-AS orthologs in other mammalian lineages.

Reviewer #2 (Public Review):

In this manuscript, Glaser et al. describe a new selective plane illumination microscope designed to image a large field of view that is optimized for expanded and cleared tissue samples. For the most part, the microscope design follows a standard formula that is common among many systems (e.g. Keller PJ et al Science 2008, Pitrone PG et al. Nature Methods 2013, Dean KM et al. Biophys J 2015, and Voigt FF et al. Nature Methods 2019). The primary conceptual and technical novelty is to use a detection objective from the metrology industry that has a large field of view and a large area camera. The authors characterize the system resolution, field curvature, and chromatic focal shift by measuring fluorescent beads in a hydrogel and then show example images of expanded samples from mouse, macaque, and human brain tissue.

Glaser et al. have responded to the reviewer comments by removing some of the overstated claims from the prior manuscript and editing portions of the manuscript text to enhance the clarity. Although the manuscript would be stronger if the authors had been able to provide data that justified the original high-impact claims from the initial publication (e.g. that the images could be used for robust and automated neuronal tracing across large volumes), the amended manuscript text now more closely matches the supporting data. As with the initial submission, I believe that the microscope design and characterization is a useful contribution to the field and the data are quite stunning.

Reviewer #2 (Public review):

Summary:

In this work the authors show that dopaminergic neurons (DANs) from the DL1 cluster in Drosophila larvae are required for the formation of aversive memories. DL1 DANs complement pPAM cluster neurons which are required for the formation of attractive memories. This shows the compartmentalized network organization of how an insect learning center (the mushroom body) encodes memory by integrating olfactory stimuli with aversive or attractive teaching signals. Interestingly, the authors found that the 4 main dopaminergic DL1 neurons act partially redundant, and that single cell ablation did not result in aversive memory defects. However, ablation or silencing of a specific DL1 subset (DAN-f1,g1) resulted in reduced salt aversion learning, which was specific to salt but no other aversive teaching stimuli tested. Importantly, activation of these DANs using an optogenetic approach was also sufficient to induce aversive learning in the presence of high salt. Together with the functional imaging of salt and fructose responses of the individual DANs and the implemented connectome analysis of sensory (and other) inputs to DL1/pPAM DANs this represents a very comprehensive study linking the structural, functional and behavioral role of DL1 DANs. This provides fundamental insight into the function of a simple yet efficiently organized learning center which displays highly conserved features of integrating teaching signals with other sensory cues via dopaminergic signaling.

Strengths:

This is a very careful, precise and meticulous study identifying the main larval DANs involved in aversive learning using high salt as a teaching signal. This is highly interesting because it allows to define the cellular substrates and pathways of aversive learning down to the single cell level in a system without much redundancy. It therefore sets the basis to conduct even more sophisticated experiments and together with the neat connectome analysis opens the possibility to unravel different sensory processing pathways within the DL1 cluster and integration with the higher order circuit elements (Kenyon cells and MBONs). The authors' claims are well substantiated by the data and balanced, putting their data in the appropriate context. The authors also implemented neat pathway analyses using the larval connectome data to its full advantage, thus providing network pathways that contribute towards explaining the obtained results.

Weaknesses:

Previous comments were fully addressed by the authors.

Reviewer #2 (Public review):

The study by Setogawa et al. aims to understand the role that different striatal subregions belonging to parallel brain circuits have in associative learning and discrimination learning (S-O-R and S-R tasks). Strengths of the study are the use of multiple methodologies to measure and manipulate brain activity in rats, from microPET imaging to excitotoxic lesions and multielectrode recordings across anterior dorsolateral (aDLS), posterior ventral lateral (pVLS)and dorsomedial (DMS) striatum.

The main conclusions are that the aDLS promotes stimulus-response association and suppresses response-outcome associations. The pVLS is engaged in the formation and maintenance of the stimulus-response association. There is a lot of work done and some interesting findings however, the manuscript can be improved by clarifying the presentation and reasoning. The inclusion of important controls will enhance the rigor of the data interpretation and conclusions.

Comments on revisions:

The authors have made important revisions to the manuscript and it has improved in clarity. They also added several figures in the rebuttal letter to answer questions by the reviewers. I would ask that these figures are also made public as part of the authors' response or if not, included in the manuscript.

Reviewer #2 (Public review):

This is potentially interesting work, but the analyses are attempted in a rather scattergun way, with little evident critical thought. The structure of the work (Results before Methods) can work in some manuscripts, but it is not ideal here. The authors discuss results before we know anything about the underlying data that the results come from. It gives the impression that the authors regard data as a resource to be exploited, without really caring where the data comes from. The methods can provide meaningful insights if correctly used, but while I don't have reasons to doubt that the analyses were conducted correctly, findings are presented with little discussion or interpretation. No follow-up analyses are performed.

In summary, there are likely some gems here, but the whole manuscript is essentially the output from an analytic pipeline.

Taking the researchers aims in turn:

(1) Meta-GWAS - while combining two datasets together can provide additional insights, the contribution of this analysis above existing GWAS is not clear. The PRACTICAL consortium has already reported the GWAS of 70% of these data. What additional value does this analysis provide? (Likely some, but it's not clear from the text.) Also, the presentation of results is unclear - authors state that only 5 gene regions contained variants at p<5x10-8, but Figure 1 shows dozens of hits above 5x10-8. Also, the red line in Figure 1 (supposedly at 5x10-8) is misplaced.

(2) Cross-phenotype analysis. It is not really clear what this analysis is, or why it is done. What is the iCPAGdb? A database? A statistical method? Why would we want to know cross-phenotype associations? What even are these? It seems that the authors have taken data from an online resource and have written a paragraph based on this existing data with little added value.

(3) PW-MR. I can see the value of this work, but many details are unclear. Was this a two-sample MR using PRACTICAL + FinnGen data for the outcome? How many variants were used in key analyses? Again, the description of results is sparse and gives little added value.

(4) Colocalization - seems clear to me.

(5) Additional post-GWAS analyses (pathway + druggability) - again, the analyses seem to be performed appropriately, although little additional insight other than the reporting of output from the methods.

Minor points:

(6) The stated motivation for this work is "early detection". But causality isn't necessary for early detection. If the authors are interested in early detection, other analysis approaches are more appropriate.

(7) The authors state "193 proteins were associated with PCa risk", but they are looking at MR results - these analyses test for disease associations of genetically-predicted levels of proteins, not proteins themselves.

Strengths:

The data and methods used are state-of-the-art.

Weaknesses:

The reader will have to provide their own translational insight.

Reviewer #2 (Public review):

Summary:

The study provides an updated literature review and meta-analysis for the 5-year survival estimates in breast cancer patients across continental Africa. The findings reveal substantial disparities between regions and other factors, highlighting the disadvantaged areas in Africa and the urgent need to address these inequities across the continent.

Strengths:

The main strengths of this study include:<br /> (1) the thorough literature search with an increasing number of included studies that enhances result reliability;<br /> (2) standard and appropriate statistical methods with clear reporting;<br /> (3) a comprehensive discussion.

Overall, the paper is well-structured, clearly presented, and provides useful insights.

Weaknesses:

However, I have a few concerns that I would like the authors to address.

(1) The conclusion "A country-wise comparison with 2018 estimates suggests a declining survival tendency, with WHO AFRO countries reporting the poorest estimates among other WHO regions." appears to have been drawn from the comparisons across both different regions and different time periods, which is incorrect! As shown in Figure 8, survival in Africa has increased from below 30% (WHO AFRO 2017) to around 50% (AFRICA 2024, presumably the current study). Section 3.5 is confusing and headed in the wrong direction. The key message in Figure 8 is that the current survival estimate in Africa is still lower than that of other WHO regions from a few years ago, highlighting the urgent need to improve survival in Africa.

(2) The previous review by Ssentongo et al. classified countries into North Africa and sub-Saharan Africa (SSA), regions divided by the Sahara Desert. This classification is not only geographical-based, but also accounts for the significant differences in ethnicity, health system, and socioeconomic factors. North Africa (especially Egypt, Tunisia, Morocco) has better cancer registries, earlier detection, more treatment access, and therefore better survival outcomes (as shown in Figure 2). SSA tends to have worse outcomes, due to later-stage diagnosis, limited pathology, and access barriers. Given that the survival in women with breast cancer is among the lowest for several SSA countries, the study would benefit from an additional comparison between pooled estimates of North African and SSA, and comparisons with previous pooled estimates.

(3) The authors classified studies under the female group. Females constituted at least 80% of the sample population, and subgroup analysis revealed only a marginal discrepancy in survival rates between the two sexes. However, most of the breast cancer patients and related studies consist predominantly of females. Given the non-negligible differences in various aspects between females and males, sensitivity analyses restricted to studies among females (as in Figure 2-3) would be informative for future research in breast cancer patients.

(4) Stage at diagnosis and treatment are the strongest prognostic factors for breast cancer survival. Though data regarding these variables are not available for all studies, and it's complicated to compare or pool the results from different studies (as mentioned in the limitation), could the authors perform the regression analyses regarding early vs. late stages, and the percentage of treatment received? These two factors are too significant to overlook in studies on breast cancer survival.

(5) The authors reported that studies published before 2019 had a higher survival than those conducted from 2019 onwards, which could be misleading and requires further explanation. As the authors noted ─"the year of publication may not be a reliable measure of the effect in question"─ a better approach would be to use the year of inclusion, i.e., the year the studies were conducted.

(6) Northern and Western Africa both have the highest incidence of breast cancer in Africa, yet their 5-year survival estimates differ substantially. However, the authors have discussed the survival disparities without considering their similarly higher incidence rates. Could this disparity reflect different contributing factors, with the higher incidence rate in Northern Africa resulting from better screening programs (leading to more detections), while that in Western Africa stems from other epidemiological factors despite lower screening participation? Though the incidence rate is not the primary focus of this study, briefly exploring this dichotomy would enhance the discussion and provide valuable insights for readers.

Reviewer #2 (Public review):

This is a timely and insightful study aiming to explore the general physical principles for the sub-compartmentalization--or lack thereof--in the phase separation processes underlying the assembly of postsynaptic densities (PSDs), especially the markedly different organizations in three-dimensional (3D) droplets on one hand and the two-dimensional (2D) condensates associated with a cellular membrane on the other. Simulation of a highly simplified model (one bead per protein domain) is carefully executed. Based on a thorough consideration of various control cases, the main conclusion regarding the trade-off between repulsive excluded volume interactions and attractive interactions among protein domains in determining the structures of 3D vs 2D model PSD condensates is quite convincing. The results in this manuscript are novel; however, as it stands, there is substantial room for improvement in the presentation of the background and the findings of this work. In particular, (i) conceptual connections with prior works should be better discussed, (ii) essential details of the model should be clarified, and (iii) the generality and limitations of the authors' approach should be better delineated. Specifically, the following items should be addressed (with the additional references mentioned below cited and discussed):

(1) Excluded volume effects are referred to throughout the text by various terms and descriptions such as "repulsive force according to the volume" (e.g., in the Introduction), "nonspecific volume interaction", and "volume effects" in this manuscript. This is somewhat curious and not conducive to clarity, because these terms have alternate or connotations of alternate meanings (e.g., in biomolecular modeling, repulsive interactions usually refer to those with longer spatial ranges, such as that between like charges). It will be much clearer if the authors simply refer to excluded volume interactions as excluded volume interactions (or effects).

(2) Inasmuch as the impact of excluded volume effects on subcompartmentalization of condensates ("multiple phases" in the authors' terminology), it has been demonstrated by both coarse-grained molecular dynamics and field-theoretic simulations that excluded volume is conducive to demixing of molecular species in condensates [Pal et al., Phys Rev E 103:042406 (2021); see especially Figures 4-5 of this reference]. This prior work bears directly on the authors' observation. Its relationship with the present work should be discussed.

(3) In the present model setup, activation of the CaMKII kinase affects only its binding to GluN2Bc. This approach is reasonable and leads to model predictions that are essentially consistent with the experiment. More broadly, however, do the authors expect activation of the CaMKII kinase to lead to phosphorylation of some of the molecular species involved with PSDs? This may be of interest since biomolecular condensates are known to be modulated by phosphorylation [Kim et al., Science 365:825-829 (2019); Lin et al, eLife 13:RP100284 (2025)].

(4) The forcefield for confinement of AMPAR/TARP and NMDAR/GluN2Bc to 2D should be specified in the main text. Have the authors explored the sensitivity of their 2D findings on the strength of this confinement?

(5) Some of the labels in Figure 1 are confusing. In Figure 1A, the structure labeled as AMPAR has the same shape as the structure labeled as TARP in Figure 1B, but TARP is labeled as one of the smaller structures (like small legs) in the lower part of AMPAR in Figure 1A. Does the TARP in Figure 1B correspond to the small structures in the lower part of AMPAR? If so, this should be specified (and better indicated graphically), and in that case, it would be better not to use the same structural drawing for the overall structure and a substructure. The same issue is seen for NMDAR in Figure 1A and GluN2Bc in Figure 1B.

(6) In addition to clarifying Figure 1, the authors should clarify the usage of AMPAR vs TARP and NMDAR vs GluN2Bc in other parts of the text as well.

(7) The physics of the authors' model will be much clearer if they provide an easily accessible graphical description of the relative interaction strengths between different domain-representing spheres (beads) in their model. For this purpose, a representation similar to that given by Feric et al., Cell 165:1686-1697 (2016) (especially Figure 6B in this reference) of the pairwise interactions among the beads in the authors' model should be provided as an additional main-text figure. Different interaction schemes corresponding to inactive and activated CAMKII should be given. In this way, the general principles (beyond the PSD system) governing 3D vs 2D multiple-component condensate organization can be made much more apparent.

(8) Can the authors' rationalization of the observed difference between 3D and 2D model PSD condensates be captured by an intuitive appreciation of the restriction on favorable interactions by steric hindrance and the reduction in interaction cooperativity in 2D vs 3D?

(9) In the authors' model, the propensity to form 2D condensates is quite weak. Is this prediction consistent with the experiment? Real PSDs do form 2D condensates around synapses.

(10) More theoretical context should be provided in the Introduction and/or Discussion by drawing connections to pertinent prior works on physical determinants of co-mixing and de-mixing in multiple-component condensates (e.g., amino acid sequence), such as Lin et al., New J Phys 19:115003 (2017) and Lin et al., Biochemistry 57:2499-2508 (2018).

(11) In the discussion of the physiological/neurological significance of PSD in the Introduction and/or Discussion, for general interest it is useful to point to a recently studied possible connection between the hydrostatic pressure-induced dissolution of model PSD and high-pressure neurological syndrome [Lin et al., Chem Eur J 26:11024-11031 (2020)].

(12) It is more accurate to use "perpendicular to the membrane" rather than "vertical" in the caption for Figure 3E and other such descriptions of the orientation of the CaMKII hexagonal plane in the text.

Reviewer #2 (Public review):

Summary:

This article presents Morphonet 2.0, a software designed to visualise and curate segmentations of 3D and 3D+t data. The authors demonstrate their capabilities on five published datasets, showcasing how even small segmentation errors can be automatically detected, easily assessed, and corrected by the user. This allows for more reliable ground truths, which will in turn be very much valuable for analysis and training deep learning models. Morphonet 2.0 offers intuitive 3D inspection and functionalities accessible to a non-coding audience, thereby broadening its impact.

Strengths:

The work proposed in this article is expected to be of great interest to the community by enabling easy visualisation and correction of complex 3D(+t) datasets. Moreover, the article is clear and well written, making MorphoNet more likely to be used. The goals are clearly defined, addressing an undeniable need in the bioimage analysis community. The authors use a diverse range of datasets, successfully demonstrating the versatility of the software.

We would also like to highlight the great effort that was made to clearly explain which type of computer configurations are necessary to run the different datasets and how to find the appropriate documentation according to your needs. The authors clearly carefully thought about these two important problems and came up with very satisfactory solutions.

Weaknesses:

There is still one concern: the quantification of the improvement of the segmentations in the use cases and, therefore, the quantification of the potential impact of the software. While it appears hard to quantify the quality of the correction, the proposed work would be significantly improved if such metrics could be provided.

The authors show some distributions of metrics before and after segmentations to highlight the changes. This is a great start, but there seem to be two shortcomings: first, the comparison and interpretation of the different distributions does not appear to be trivial. It is therefore difficult to judge the quality of the improvement from these. Maybe an explanation in the text of how to interpret the differences between the distributions could help. A second shortcoming is that the before/after metrics displayed are the metrics used to guide the correction, so, by design, the scores will improve, but does that accurately represent the improvement of the segmentation? It seems to be the case, but it would be nice to maybe have a better assessment of the improvement of the quality.

Reviewer #2 (Public review):

Summary:

Poge et al. present a workflow for studying plant tissue by combining high-pressure freezing, cryo-fluorescence microscopy, FIB milling, and cryo-electron tomography (cryo-ET). They tested various plant tissues, including Physcomitrium patens, Arabidopsis thaliana, and Limonium bicolor. The authors successfully produce thin lamellae suitable for cryo-ET studies. Using sub-tomogram averaging, they determined the Rubisco structure at subnanometer resolution, demonstrating the potential of this workflow for plant tissue studies.

Strengths:

This manuscript is likely the first to systematically apply FIB milling and cryo-ET to plant tissue samples. It provides a detailed methodological description, which is not only valuable for plant tissue studies but also adaptable to a broader range of biological tissue samples. The study compares the plunge freezing method with a high-pressure freezing method, demonstrating that high-pressure freezing can vitrify thick tissues while preserving their native state. Additionally, the authors explore two methods for plant tissue sample preparation, the "waffle" method and in-carrier high-pressure freezing combined with the "lift-out" approach. The "waffle" method is suitable for samples less than 25um, while the in-carrier high-pressure freezing method can process samples up to 100um.

Weaknesses:

The described workflow is very complicated and requires special expertise. The success rate of this workflow is not very high, particularly for high-pressure freezing and life-out technology. Further improvements are needed for automation and increasing throughput.

Reviewer #2 (Public review):

This valuable paper studies the problem of learning from feedback given by sources of varying credibility. The solid combination of experiment and computational modeling helps to pin down properties of learning, although some ambiguity remains in the interpretation of results.

Summary:

This paper studies the problem of learning from feedback given by sources of varying credibility. Two bandit-style experiments are conducted in which feedback is provided with uncertainty, but from known sources. Bayesian benchmarks are provided to assess normative facets of learning, and alternative credit assignment models are fit for comparison. Some aspects of normativity appear, in addition to deviations such as asymmetric updating from positive and negative outcomes.

Strengths:

The paper tackles an important topic, with a relatively clean cognitive perspective. The construction of the experiment enables the use of computational modeling. This helps to pinpoint quantitatively the properties of learning and formally evaluate their impact and importance. The analyses are generally sensible, and parameter recovery analyses help to provide some confidence in the model estimation and comparison.

Weaknesses:

(1) The approach in the paper overlaps somewhat with various papers, such as Diaconescu et al. (2014) and Schulz et al. (forthcoming), which also consider the Bayesian problem of learning and applying source credibility, in terms of theory and experiment. The authors should discuss how these papers are complementary, to better provide an integrative picture for readers.

Diaconescu, A. O., Mathys, C., Weber, L. A., Daunizeau, J., Kasper, L., Lomakina, E. I., ... & Stephan, K. E. (2014). Inferring the intentions of others by hierarchical Bayesian learning. PLoS computational biology, 10(9), e1003810.<br /> Schulz, L., Schulz, E., Bhui, R., & Dayan, P. Mechanisms of Mistrust: A Bayesian Account of Misinformation Learning. https://doi.org/10.31234/osf.io/8egxh

(2) It isn't completely clear what the "cross-fitting" procedure accomplishes. Can this be discussed further?

(3) The Credibility-CA model seems to fit the same as the free-credibility Bayesian model in the first experiment and barely better in the second experiment. Why not use a more standard model comparison metric like the Bayesian Information Criterion (BIC)? Even if there are advantages to the bootstrap method (which should be described if so), the BIC would help for comparability between papers.

(4) As suggested in the discussion, the updating based on random feedback could be due to the interleaving of trials. If one is used to learning from the source on most trials, the occasional random trial may be hard to resist updating from. The exact interleaving structure should also be clarified (I assume different sources were shown for each bandit pair). This would also relate to work on RL and working memory: Collins, A. G., & Frank, M. J. (2012). How much of reinforcement learning is working memory, not reinforcement learning? A behavioral, computational, and neurogenetic analysis. European Journal of Neuroscience, 35(7), 1024-1035.

(5) Why does the choice-repetition regression include "only trials for which the last same-pair trial featured the 3-star agent and in which the context trial featured a different bandit pair"? This could be stated more plainly.

(6) Why apply the "Truth-CA" model and not the Bayesian variant that it was motivated by?

(7) "Overall, the results from this study support the exact same conclusions (See SI section 1.2) but with one difference. In the discovery study, we found no evidence for learning based on 50%-credibility feedback when examining either the feedback effect on choice repetition or CA in the credibility-CA model (SI 1.2.3)" - this seems like a very salient difference, when the paper reports the feedback effect as a primary finding of interest, though I understand there remains a valence-based difference.

(8) "Participants were instructed that this feedback would be "a lie 50% of the time but were not explicitly told that this meant it was random and should therefore be disregarded." - I agree that this is a possible explanation for updating from the random source. It is a meaningful caveat.

(9) "Future studies should investigate conditions that enhance an ability to discard disinformation, such as providing explicit instructions to ignore misleading feedback, manipulations that increase the time available for evaluating information, or interventions that strengthen source memory." - there is work on some of this in the misinformation literature that should be cited, such as the "continued influence effect". For example: Johnson, H. M., & Seifert, C. M. (1994). Sources of the continued influence effect: When misinformation in memory affects later inferences. Journal of experimental psychology: Learning, memory, and cognition, 20(6), 1420.

(10) Are the authors arguing that choice-confirmation bias may be at play? Work on choice-confirmation bias generally includes counterfactual feedback, which is not present here.

Reviewer #2 (Public review):

Summary:

The paper examines the diversity of complex amacrine neurons in the ventral lobe of the adult octopus brain, a structure involved in learning and memory. The work builds on a recent paper by the authors that described the connectivity of the much larger population of simple amacrine (SAM) interneurons from the same pioneering EM volume.

Strengths:

While the EM volume only provides a snapshot of a tiny fraction of an adult octopus' brain, the authors can make specific conclusions and formulate precise hypotheses about neuron function, synaptic pathways, and developmental trajectories. One example is the reconstruction of a putative maturation sequence for the SAM neuronal lineage, based on the correlation of soma position and the number of synapses, uncovering a plausible developmental sequence of cell morphologies, with interesting parallels to vertebrate neurogenesis.

Weaknesses:

The weakness of the study is that it is examining a relatively small volume (260 × 390 × 27 µm), and several neurons are only incompletely reconstructed. It also remains unclear approximately how many neurons remain to be reconstructed from this volume.

To improve the presentation, the authors should consider showing videos with the volumetric reconstructions of the different types with their partners/synapses and their relation to the SFL track and SAMs. Such videos would help the reader to appreciate the morphological differences between the cell types. The authors could also consider carrying out further morphological analyses to strengthen their cell-type classification, including Sholl value, radial density of input and output synapses, the number of branch nodes, and similar measures.

Reviewer #2 (Public review):

Summary:

Building on previous models of multisensory integration (including their earlier correlation-detection framework used for non-spatial signals), the author introduces a population-level Multisensory Correlation Detector (MCD) that processes raw auditory and visual data. Crucially, it does not rely on abstracted parameters, as is common in normative Bayesian models," but rather works directly on the stimulus itself (i.e., individual pixels and audio samples). By systematically testing the model against a range of experiments spanning human, monkey, and rat data, the authors show that their MCD population approach robustly predicts perception and behavior across species with a relatively small (0-4) number of free parameters.

Strengths:

(1) Unlike prior Bayesian models that used simplified or parameterized inputs, the model here is explicitly computable from full natural stimuli. This resolves a key gap in understanding how the brain might extract "time offsets" or "disparities" from continuously changing audio-visual streams.

(2) The same population MCD architecture captures a remarkable range of multisensory phenomena, from classical illusions (McGurk, ventriloquism) and synchrony judgments, to attentional/gaze behavior driven by audio-visual salience. This generality strongly supports the idea that a single low-level computation (correlation detection) can underlie many distinct multisensory effects.

(3) By tuning model parameters to different temporal rhythms (e.g., faster in rodents, slower in humans), the MCD explains cross-species perceptual data without reconfiguring the underlying architecture.

Weaknesses:

(1) The authors show how a correlation-based model can account for the various multisensory integration effects observed in previous studies. However, a comparison of how the two accounts differ would shed light on the correlation model being an implementation of the Bayesian computations (different levels in Marr's hierarchy) or making testable predictions that can distinguish between the two frameworks. For example, how uncertainty in the cue combined estimate is also the harmonic mean of the unimodal uncertainties is a prediction from the Bayesian model. So, how the MCD framework predicts this reduced uncertainty could be one potential difference (or similarity) to the Bayesian model.

2) The authors show a good match for cue combination involving 2 cues. While Bayesian accounts provide a direction extension to more cues (also seen empirically, for eg, in Hecht et al. 2008), discussion on how the MCD model extends to more cues would benefit the readers.

Likely Impact and Usefulness:

The work offers a compelling unification of multiple multisensory tasks- temporal order judgments, illusions, Bayesian causal inference, and overt visual attention - under a single, fully stimulus-driven framework. Its success with natural stimuli should interest computational neuroscientists, systems neuroscientists, and machine learning scientists. This paper thus makes an important contribution to the field by moving beyond minimalistic lab stimuli, illustrating how raw audio and video can be integrated using elementary correlation analyses.

Reviewer #2 (Public review):

Summary:

In their study, Osorio and colleagues present 'retriever,' an innovative computational tool designed to extract disease-specific transcriptional drug response profiles from the LINCS-L1000 project. This tool has been effectively applied to TNBC, leveraging single-cell RNA sequencing data to predict drug combinations that may effectively target the disease. The public review highlights the significant integration of extensive pharmacological data with high-resolution transcriptomic information, which enhances the potential for personalized therapeutic applications.

Strengths:

A key finding of the study is the prediction and validation of the drug combination QL-XII-47 and GSK-690693 for the treatment of TNBC. The methodology employed is robust, with a clear pathway from data analysis to experimental confirmation.

Comments on revisions:

I commend the authors for their thorough and thoughtful revisions, which have significantly strengthened the manuscript. The expanded discussion on the limitations of the LINCS-L1000 dataset and the inherent challenges of imputation techniques provides critical context for interpreting the tool's predictive accuracy. The addition of clinical implications, including strategies for integrating retriever into clinical trial design and its broader applicability to other diseases, enhances the translational relevance of the work. Addressing drug resistance mechanisms in the context of combination therapy further underscores the biological rationale for the approach.

The transparency regarding computational requirements and ethical considerations-particularly data privacy, bias mitigation, and model validation-demonstrates a responsible and forward-thinking approach to computational biology. These additions not only improve the manuscript's rigor but also set a precedent for ethical practices in personalized medicine research.

With these revisions, the authors have effectively addressed prior concerns and elevated the impact of their work. The manuscript now presents a compelling case for the retriever as a valuable tool in precision oncology.

Reviewer #3 (Public review):

The authors use a generic model framework to study the emergence of habituation and its functional role from information-theoretic and energetic perspectives. Their model features a receptor, readout molecules, and a storage unit, and as such, can be applied to a wide range of biological systems. Through theoretical studies, the authors find that habituation (reduction in average activity) upon exposure to repeated stimuli should occur at intermediate degrees to achieve maximal information gain. Parameter regimes that enable these properties also result in low dissipation, suggesting that intermediate habituation is advantageous both energetically and for the purpose of retaining information about the environment.

A major strength of the work is the generality of the studied model. The presence of three units (receptor, readout, storage) operating at different time scales and executing negative feedback can be found in many domains of biology, with representative examples well discussed by the authors (e.g. Figure 1b). A key takeaway demonstrated by the authors that has wide relevance is that large information gain and large habituation cannot be attained simultaneously. When energetic considerations are accounted for, large information gain and intermediate habituation appear to be the favorable combination.

Comments on the revision:

The authors have adequately addressed the points I raised during the initial review. The text has been clarified at multiple instances, and the treatment of energy expenditure is now more rigorous. The manuscript is much improved both in terms of readability and scientific content.

Reviewer #2 (Public review):

This work presents new genetic tools for enhanced Cre-mediated gene deletion and genetic lineage tracing. The authors optimise and generate mouse models that convert temporally controlled CreER or DreER activity to constitutive Cre expression, coupled with the expression of tdT reporter for the visualizing and tracing of gene-deleted cells. This was achieved by inserting a stop cassette into the coding region of Cre, splitting it into N- and C-terminal segments. Removal of the stop cassette by Cre-lox or Dre-rox recombination results in the generation of modified Cre that is shown to exhibit similar activity to native Cre. The authors further demonstrate efficient gene knockout in cells marked by the reporter using these tools, including intersectional genetic targeting of pericentral hepatocytes.

The new models offer several important advantages. They enable tightly controlled and highly effective genetic deletion of even alleles that are difficult to recombine. By coupling Cre expression to reporter expression, these models reliably report Cre-expressing i.e. gene-targeted cells and circumvent false positives that can complicate analyses in genetic mutants relying on separate reporter alleles. Moreover, the combinatorial use of Dre/Cre permits intersectional genetic targeting, allowing for more precise fate mapping.

The study and the new models have also limitations. The demonstration of efficient deletion of multiple floxed alleles in a mosaic fashion, a scenario where the lines would demonstrate their full potential compared to already existing models, has not been tested in the current study. Mosaic genetics is increasingly recognized as a key methodology for assessing cell-autonomous gene functions. The challenge lies in performing such experiments, as low doses of tamoxifen needed for inducing mosaic gene deletion may not be sufficient to efficiently recombine multiple alleles in individual cells while at the same time accurately reporting gene deletion. In addition, as discussed by the authors, a limitation of this line is the constitutive expression of Cre, which is associated with toxicity in some cases.

Comments on revisions: I have no further comments.

Reviewer #2 (Public review):

Summary:

Canonical Wnt signaling has previously been shown to be responsible for correct patterning of the oral-aboral axis as well as germ layer formation in several cnidarians. The post-gastrula stage, the planula larvae is not only elongated, it has a specific swimming direction due to the decentralized cellular positioning and slanted anchoring of the cilia. This, in turn, is in most other animals the result of a Wnt-Planar-cell polarity pathway. This paper by Uveira et al investigates the role of Wnt3 signaling in serving as a local cue for the PCP pathway which then is responsible for the orientation of the cilia and elongation of the planula larva of the hydrozoan Clytia hemisphaerica. Wnt3 was shown before to activate the canonical pathway via ß-catenin and to act as an axial organizer. The authors provide compelling evidence for this somewhat unusual direct link between the pathways through the same signaling molecule, Wnt3. In conclusion, they propose a two-step model: 1) local orientation by Wnt3 secretion 2) global propagation by the PCP pathway over the whole embryo.

Strengths:

In a series of elegant and also seemingly sophisticated experiments, they show that Wnt3 activates the PCP pathway directly, as it happens in the absence of canonical Wnt signaling (e.g. through co-expression of dnTCF). Conversely, constitutive active ß-catenin was not able to rescue PCP coordination upon Wnt3 depletion, yet restored gastrulation. This uncouples the effect of Wnt3 on axis specification and morphogenetic movements from the elongation via PCP. Through transplantation of single blastomeres providing a local source of Wnt3, they also demonstrate the reorganization of cellular polarity immediately adjacent to the Wnt3 expressing cell patch. These transplantation experiments also uncover that mechanical cues can also trigger the polarization, suggesting a mechanotransduction or direct influence on subcellular structures, e.g. actin fiber orientation.

This is a beautiful and elegant study addressing an important question. The results have significant implications also for our understanding of the evolutionary origin of axis formation and the link of these two ancient pathways, which in most animals are controlled by distinct Wnt ligands and Frizzled receptors. The quality of the data is stunning and the paper is written in a clear and succinct manner. This paper has the potential to become a widely cited milestone paper.

Weaknesses:

I can not detect any major weaknesses. The work only raises a few more follow-up questions, which the authors are invited to comment on.

I acknowledge the revisions made by the authors. Some open questions remain that need to be addressed in future work, and I accept the limitations of this study, as argued by the authors. Besides the elegant and high-quality experiments, I also appreciate the thoughtful and inspiring discussion.

Reviewer #2 (Public review):

This manuscript addresses a fundamental problem of immunology - the persistence mechanisms of tissue-resident memory T cells (TRMs). It introduces a novel quantitative methodology, combining the in vivo tracing of T cell cohorts with rigorous mathematical modeling and inference. Interestingly, the authors show that immigration plays a key role for maintaining CD4+ TRM populations in both skin and lamina propria (LP), with LP TRMs being more dependent on immigration than skin TRMs. This is an original and potentially impactful manuscript.

Comments on revised version: This reviewer is satisfied with the author responses and the changes made in the manuscript.

Reviewer #2 (Public review):

Strengths:

Wang and colleagues successfully uncovered an important function of the Drosophila PRDM16/PRDM3 homolog Hamlet (Ham) - a PR domain containing transcription factor with known roles in the nervous system in Drosophila. To do so, they generated and analyzed new mutants lacking the PR domain, and also employed diverse preexisting tools. In doing so, they made a fascinating discovery: They found that PR-domain containing isoforms of ham are crucial in the intriguing development of the fly genital tract. Wang and colleagues found three distinct roles of Ham: (1) Specifying the position of the testis terminal epithelium within the testis, (2) allowing normal shaping and growth of the anlagen of the seminal vesicles and paragonia and (3) enabling the crucial epithelial fusion between the seminal vesicle and the testis terminal epithelium. The mutant blocks fusion even if the parts are positioned correctly. The last finding is especially important, as there are few models allowing one to dissect the molecular underpinnings of heterotypic epithelial fusion in development. Their data suggest that they found a master regulator of this collective cell behavior. Further, they identified some of the cell biological players downstream of Ham, like for example E-Cadherin and Crumbs. In a holistic approach, they performed RNAseq and intersected them with the CUT&TAG-method, to find a comprehensive list of downstream factors directly regulated by Ham. Their function in the fusion process was validated by a tissue-specific RNAi screen. Meticulously, Wang and colleagues performed multiplexed in situ hybridization and analyzed different mutants, to gain a first understanding of the most important downstream-pathways they characterized - which are Wnt2 and Toll.

This study pioneers a completely new system. It is a model for exploring a process crucial in morphogenesis across animal species, yet not well-understood. Wang and colleagues not only identified a crucial regulator of heterotypic epithelial fusion but took on the considerable effort of meticulously pinning down functionally important downstream effectors by using many state-of-the-art methods. This is especially impressive, as dissection of pupal genital discs before epithelial fusion is a time-consuming and difficult task. This promising work will be the foundation future studies build on, to further elucidate how this epithelial fusion works, for example on a cell biological and biomechanical level.

Weaknesses:

The developing testis-genital disc system has many moving parts. Myotube migration was previously shown to be crucial for testis shape. This means, that there is the potential of non-tissue autonomous defects upon knockdown of genes in the genital disc or the terminal epithelium, affecting myotube behavior which in turn affects epithelial fusion, as myotubes might create the first "bridge" bringing the two epithelia together. Nevertheless, this is outside the scope of this work and could be addressed in the future.

Reviewer #2 (Public review):

This manuscript explores the molecular mechanisms that are involved in substrate recognition by the PP1 phosphatase. The authors previously showed that the PP1 interacting protein (PPI), PhactrI, conferred substrate specificity by remodelling the PP1 hydrophobic substrate groove. In this work, the authors aimed to understand the key determinant of how other PIPs, Neurabin and Spinophilin, mediate substrate recognition.

The authors generated a few PP1-PIP fusion constructs, undertook TMT phosphoproteomics and validated their method using PP1-Phactr1/2/3/4 fusion constructs. Using this method, the authors identified phsophorylation sites controlled by PP1-Neurabin and focussed their work on 4E-BP1, thereby linking PP1-Neurabin to mTORC1 signalling. Upon validating that PP1-Neurabin dephosphorylates 4E-BP1, they determined that 4E-BP1 PBM binds to the PDZ domain of Neurabin with an affinity that was greater than 30 fold as compared to other substrates. PP1-Neurabin dephosphorylated 4E-BP1WT and IRSp53WT with a catalytic efficiency much greater than PP1 alone. However, PP1-Neurabin bound to 4E-BP1 and IRSp53 mutants lacking the Neurabin PDZ domain with a catalytic efficiency lesser than that observed with 4E-BP1WT. These results indicate the involvement of the PDZ domain in facilitating substrate recruitment by PP1-Neurabin. Interestingly, PP1-Phactr1 dephosphorylation of 4E-BP1 phenocopies PP1 alone, while PP1-Phactr1 dephosphorylates IRSp53 to a much higher extent than PP1 alone. These results highlights the importance of the PDZ domain and also shed light on how different PP1-PIP holoenzymes mediate substrate recognition using distinct mechanisms. The authors also show that the remodelling of the hydrophobic PP1 substrate groove which is essential for substrate recognition by PP1-Phactr1, was not required by PP1-Neurabin. Additionally, the authors also resolved the structure of a PP1-4E-BP1 fusion with the PDZ-containing C-terminal of Neurabin and observed that the Neurabin/PP1-4E-BP1 complex structure was oriented at 21{degree sign} to that in the unliganded Spinophilin/PP1 complex (resolved by Ragusa et al., 2010) owing to a slight bend in the C-terminal section that connects it to the RVxF-ΦΦ-R-W string. Since, no interaction was observed with the remodelled PP1-Neurabin hydrophobic groove, the authors utilised AlphaFold3 to further answer this. They observed a high confidence of interaction between the groove and phosphorylated substrate and a low confidence of interaction between the groove and unphosphorylated substrate, thereby suggesting that the hydrophobic groove remodelling is not involved in PP1-Neurabin recognition and dephosphorylation of 4E-BP1.

In this work, the authors provide novel insights into how Neurabin depends on the interaction between its PDZ domain and PBM domains of potential substrates to mediate its recruitment by PP1. Additionally, they uncover a novel PP1-Neurabin substrate, 4E-BP1. They systematically employ phosphoproteomics, biochemical and structural methods to investigate substrate specifity in a robust fashion. Furthermore, the authors also compares the interactions between PP1-Neurabin to 4E-BP1 and IRSp53 (PP1-Phactr1 substrate) with PP1-Phactr1, to showcase the specificity of the mode of action employed by these complexes in mediating substrate specificity. The authors do employ an innovative PP1-PIP fusion strategy previously explored by Oberoi et al., 2016 and the authors themselves in Fedoryshchak et al., 2020. This method, allows for a more controlled investigation of the interactions between PP1-PIPs and its substrates. Furthermore, the authors have substantially characterised the importance of the PDZ domain using their fusion constructs, however, I believe that a further exploration into either structural or AlphaFold3 modelling of PBM domain substrate mutants, or a Neurabin PDZ-domain mutant might further strengthen this claim. Overall, the paper makes a substantial contribution to understanding substrate recognition and specificity in PP1-PIP complexes. The study's innovative methods, biological relevance, and mechanistic insights are strengths, but whether this mechanism occurs in a physiological context is unclear.

Reviewer #2 (Public review):

Summary:

This paper studies the role of hexatic defects in the collective migration of epithelia. The authors emphasize that epithelial migration is driven by cell intercalation events and not just isolated T1 events, and analyze this through the lens of hexatic topological defects. Finally, the authors study the effect of active and passive forces on the dynamics of hexatic defects using analytical results, and numerical results in both continuum and phase-field models.

The results are very interesting and highlight new ways of studying epithelial cell migration through the analysis of the binding and unbinding of hexatic defects.

Strengths:

(1) The authors convincingly argue that intercalation events are responsible for collective cell migration, and that these events are accompanied by the formation and unbinding of hexatic topological defects.

(2) The authors clearly explain the dynamics of hexatic defects during T1 transitions, and demonstrate the importance of active and passive forces during cell migration.

(3) The paper thoroughly studies the T1 transition through the viewpoint of hexatic defects. A continuum model approach to study T1 transitions in cell layers is novel and can lead to valuable new insights.

Weaknesses:

(1) The authors could expand on the dynamics of existing hexatic defects during epithelial cell migration, in addition to how they are created during T1 transitions.

(2) The different terms in the MPF model used to study cell layer dynamics are not fully justified. In particular, it is not clear why the model includes self-propulsion and rotational diffusion in addition to nematic and hexatic stresses, and how these quantities are related to each other.

(3) The authors could provide some physical intuition on what an active extensile or contractile term in the hexatic order parameter means, and how this is related to extensility and contractility in active nematics and/or for cell layers.

Reviewer #2 (Public review):

The authors investigated how experiencing the COVID-19 pandemic affected optimism bias in updating beliefs about the future. They ran a between-subjects design testing participants on cognitive tasks before, during and after the lift of the sanitary state of emergency during the pandemic. The authors show that optimism bias varied depending on the context in which it was tested. Namely, it disappeared during COVID-19 and it re-emerged at the time of lift of sanitary emergency measures. Via advanced computational modelling they are able to thoroughly characterise the nature of such alterations, pinpointing specific mechanisms underlying the lack of optimistic bias during the pandemic.

Strengths pertain to the comprehensive assessment of the results via computational modelling, and from a theoretical point of view, the notion that environmental factors can affect cognition. Power analysis was conducted to ensure that the study was powered to observe the effect of interest despite the relatively small sample size.

As the authors also noted, a major impediment to the interpreting the findings pertains to the lack of additional measures. While information on, for example, risk perception or need for social interaction were collected from participants during the pandemic, the fact that these could not be included in the analysis hindered the interpretation of findings. While the interpretation of the findings remains challenging, this work offers an example of the influence of real-life conditions on the belief-updating process.

Reviewer #3 (Public review):

As a result of a number of rounds of reviews and consultations between reviewers, Jung et al. present important work on the relationship between gamma-aminobutyric acid (GABA) levels within the anterior temporal lobes (ATL) to semantic memory while accounting for inter-individual differences. They provide solid evidence suggesting that inhibitory continuous theta burst transcranial magnetic stimulation (cTBS TMS) increased GABA concentration and decreased the blood-oxygen dependent signal (BOLD) during a semantic task.

The authors fully addressed my comments from the first and second rounds of reviews, and I do not have additional concerns. I have, however, scaled down my short assessment, given the concerns of reviewers 1 and 2.

Reviewer #2 (Public review):

Summary:

The study characterized the dependence of spike-timing-dependent long-term depression (tLTD) on presynaptic NMDA receptors and the intracellular cascade after NMDAR activation possibly involved in the observed decrease in glutamate probability release at L5-L5 synapses of the visual cortex in mouse brain slices.

Strengths:

The genetic and electrophysiological experiments are thorough. The experiments are well-reported and mainly support the conclusions. This study confirms and extends current knowledge by elucidating additional plasticity mechanisms at cortical synapses, complementing existing literature.

Weaknesses:

While one of the main conclusions (preNMDARs mediating presynaptic LTD) is resolved in a very convincing genetic approach, the second main conclusion of the manuscript (non-ionotropic preNMDARs) relies on the use of a high concentration of extracellular blockers (MK801, 2 mM; 7-clorokinurenic acid: 100 microM), but no controls for the specific actions of these compounds are shown. In addition, no direct testing for ions passing through preNMDAR has been performed.

It is not known if the results can be extrapolated to adult brain as the data were obtained from 11-18 days-old mice slices, a period during which synapses are still maturing and the cortex is highly plastic.

Reviewer #2 (Public review):

Summary:

The article builds on the earlier work that both p66Shc and SUMOylation are essential nitric oxide (NO) based development of endothelial vasculature (PMID: 10580504; 28760777 and 35187108). The current manuscript brings forward a finding of how SUMO2ylation of p66Shc mediated ROS production which is essential for endothelial cells. They further identify that lysine 81 of p66Shc is the residue which is conjugated to SUMO2 and is crucial for mitochondrial localization. They further show that K81 SUMO2ylation is essential for S36 phosphorylation.

Strengths:

Convincingly shows that p66Shc is SUMO2ylated on lysine 81 in cells and also shows that the phosphorylation (serine 36) reduces upon loss of this critical SUMOylation site.

Weaknesses:

All the experiments performed here are in overexpression background therefore, it would be crucial to show that p66Shc is SUMO2ylated at physiological levels.

Reviewer #2 (Public review):

This valuable study presents image correlation spectroscopy (ICS) an alternative method to foci counting as a quantitative measurement of recruitment of DNA damage response associated proteins to chromatin following exposure of cells to various genotoxic agents. The evidence presented to demonstrate that this method is more sensitive than traditional foci counting is convincing, although the two methods provide similar results for many of the comparisons. This work will be of interest to scientists using immunostaining to study DNA repair.

Comments on revisions:

The authors adequately addressed the comments raised and improved the manuscript. The authors accurately state that there is subjectivity in foci counting, e.g., different thresholds and/or algorithms produce different absolute counts. In addition, the conditions for pre-extraction also introduce variability, and any pre-extraction may inadvertently remove meaningful signal. Yet it is unclear whether these differences in absolute counts impact the conclusions that can be drawn from these experiments, which do not usually make a claim about the absolute number of foci, but rather a comparison between two different conditions with the same pre-extraction conditions and the same threshold/counting algorithm applied, with appropriate controls. Moreover, when the authors compared ICS to foci counting, the results were largely similar, although ICS was superior in a few instances. Overall, how transitioning from the widely-used foci counting method to ICS will offer a major advantage is unclear.

Reviewer #2 (Public review):

Using a combination of in vivo studies with testosterone-inhibited and aged mice with lower testosterone levels as well as isolated mouse and human seminal vesicle epithelial cells the authors show that testosterone induces an increase in glucose uptake. They find that testosterone induces a difference in gene expression with a focus on metabolic enzymes. Specifically, they identify increased expression of enzymes regulating cholesterol and fatty acid synthesis, leading to increased production of 18:1 oleic acid. The revised version strengthens the role of ACLY as the main regulator of seminal vesicle epithelial cell metabolic programming. The authors propose that fatty acids are secreted by seminal vesicle epithelial cells and are taken up by sperm, positively affecting sperm function. A lipid mixture mimicking the lipids secreted by seminal vesicle epithelial cells, however, only has a small and mostly non-significant effect on sperm motility, suggesting the authors were not apply to pinpoint the seminal vesicle fluid component that positively affects sperm function.

Reviewer #2 (Public review):

Summary:

Antibodies, thanks to their high binding affinity and specificity to cognate protein targets, are increasingly used as research and therapeutic tools. In this work, Zhou et al. have created, curated and made publicly available a new database of antibody-antigen complexes to support research in the field of antibody modelling, development and engineering.

Strengths:

The authors have performed a manual curation of antibody-antigen complexes from the Protein Data Bank, rectifying annotation errors; they have added two methods to estimate paratope-epitope interfaces; they have produced a web interface capable of effective visualisation and of summarising the key useful information in one page. The database is also cross-linked to other databases that contain information relevant to antibody developability and therapeutic applications.

Weaknesses:

The database does not import all the experimental information from PDB and contains only complexes with large protein targets.

Comments on revisions: I thank the authors for having incorporated my feedback and I look forward to the next releases of this database.

Reviewer #2 (Public review):

Summary:

The altered metabolism of tumors enables their growth and survival. Classically, tumor metabolism often involves increased activity of a given pathway in intermediary metabolism to provide energy or substrates needed for growth. Papadopoli et al. investigate the converse - the role of mitochondrial electron transfer flavoprotein dehydrogenase (ETFDH) in cancer metabolism and growth. The authors present compelling evidence that ETFDH insufficiency, which is detrimental in non-malignant tissues, paradoxically enhances bioenergetic capacity and accelerates neoplastic growth in cancer cells in spite of the decreased metabolic fuel flexibility that this affords tumor cells. This is achieved through the retrograde activation of the mTORC1/BCL-6/4E-BP1 axis, leading to metabolic and signaling reprogramming that favors tumor progression.

Strengths:

This review focuses primarily on the cancer metabolism aspects of the manuscript.

The study provides robust evidence linking ETFDH insufficiency to enhanced cancer cell bioenergetics and tumor growth.

The use of multiple cancer cell lines and in vivo models strengthens the generalizability of the findings.

The mechanistic insights into the mTORC1/BCL-6/4E-BP1 axis and its role in metabolic reprogramming are of general interest within and outside the immediate field of tumor metabolism.

Weaknesses:

The ETFDH knockout experiments are well-controlled by the addback of sgRNA-resistant ETFDH, but do not determine if the catalytic activity of this enzyme is required for the phenotypes induced by ETFDH loss.

Although this is not critical, it would be nice to see if the increased labeled aspartate pools result in higher nucleotide pools to support tumor growth.

Conclusion:

This manuscript provides significant insights into the role of ETFDH insufficiency in cancer metabolism and growth. The findings highlight the potential of targeting the mTORC1/BCL-6/4E-BP1 axis in ETFDH-deficient cancers. The compelling data support the conclusions presented in the manuscript, which will be valuable to the cancer metabolism community.

Reviewer #2 (Public review):

Summary:

This paper reports on the discovery of calcarins, a protein family that seems involved in calcification in the sponge Sycon ciliatum, based on specific expression in sclerocytes and detection by mass spectrometry within spicules. Two aspects stand out: (1) the unexpected similarity between Sycon calcarins and the galaxins of stony corals, which are also involved in mineralization, suggesting a surprising, parallel co-option of similar genes for mineralization in these two groups; (2) the impressively cell-type-specific expression of specific calcarins, many of which are restricted to either founder or thickener cells, and to either diactines, triactines, or tetractines. The finding that calcarins likely diversified at least partly by tandem duplications (giving rise to gene clusters) is a nice bonus.

Strengths:

I enjoyed the thoroughness of the paper, with multiple lines of evidence supporting the hypothesized role of calcarins: spatially and temporally resolved RNAseq, mass spectrometry, and whole-mount in situ hybridization using CISH and HCR-FISH (the images are really beautiful and very convincing). The structural predictions and the similarity to galaxins are very surprising and extremely interesting, as they suggest parallel evolution of biomineralization in sponges and cnidarians during the Cambrian explosion by co-option of the same "molecular bricks".

Weaknesses:

I did not detect any major weakness, beyond those inherent to working with sponges (lack of direct functional inhibition of these genes) or with fast-evolving gene families with complex evolutionary histories (lack of a phylogenetic tree that would clarify the history of galaxins/calcarins and related proteins).

Reviewer #2 (Public review):

Summary:

In this paper, the authors study the role of Kruppel in regulating the survival of mushroom body neuroblasts. They first confirm that adult wild-type brains have no proliferation and report that Kruppel mutants and Kruppel RNAi in neuroblasts show a few proliferative clones; they show that these proliferative clones are localized in the mushroom body. They then show that Kruppel is expressed mostly during pupal stages and acts by downregulating the expression of Imp, which has been shown to positively regulate neuroblast proliferation and survival. Expectedly, this also affects neuronal diversity in the mushroom body, which is enriched in gamma neurons that are born during the Imp-expression window. Finally, they show that Kr acts antagonistically to Kr-h1, which is expressed predominantly in larval stages.

Strengths:

The main strength of this paper is that it identified a novel regulator of Imp expression in the mushroom body neuroblasts. Imp is a conserved RNA-binding protein that has been shown to regulate neural stem cell proliferation and survival in different animals.

Weaknesses:

(1) The main weakness of the paper is that the authors want to test adult neurogenesis in a system where no adult neurogenesis exists. To achieve this, they force neuroblasts to survive in adulthood by altering the genetic program that prevents them from terminating their proliferation. If this was reminiscing about "adult neurogenesis", the authors should at least show how adult neurons incorporate into the mushroom body even if they are born much later. On the contrary, this more likely resembles a tumorigenic phenotype, when stem cells divide way past their appropriate timing.

(2) Moreover, the figures are, in many cases, hard to understand, and the interpretation of the figures doesn't always match what one sees. The manuscript would benefit from better figures; for example, in Figure 2C, Miranda expression in insc>GFP in Kr-IF-1 is not visible.

(3) The authors describe a targeted genetic screen, but they don't describe which genes were tested, how they were chosen, and why Kruppel was finally selected.

(4) The authors argue that Kr does not behave as a typical tTF in MBNBs. However, they show no expression in the embryo, limited expression in the larva and early pupa, and a peak around P24-P48. This sounds like a temporally regulated expression of a transcription factor. Importantly, they mentioned that they tested their observations against different datasets (FlyAtlas2, modENCODE, and MBNB-lineage-specific RNA-seq data), but they don't provide the data.

(5) Finally, the contribution of Kr to the neuronal composition of the mushroom body is expected (since Imp is known to regulate neuronal diversity in the MB), but the presentation in the paper is very incomplete.

Unfortunately, based on the above, I am not convinced that the authors can use this framework to infer anything about adult neurogenesis. Therefore, the impact of this work is limited to the role of Kruppel in regulating Imp, which has already been shown to regulate the extent of neuroblast division, as well as the neuronal types that are born at different temporal windows.

Reviewer #2 (Public review):

Summary:

This manuscript presents intriguing findings about the role of alternative oxidase (AOX) from the tunicate Ciona intestinalis in accelerating growth and development when expressed in Drosophila melanogaster.

Strengths:

The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. The present study provides valuable insights into AOX's role in Drosophila development. The paper attempts to explore a unique mechanism by which AOX influences Drosophila development, providing insights into mitochondrial respiration and its physiological effects. This is relevant for understanding mitochondrial dysfunction and potential therapeutic applications. The study employs a variety of approaches, including calorimetry, infrared thermography, and genetic analyses, to investigate AOX's impact on metabolism and development.

Weaknesses:

There are a number of methodological limitations and substantial gaps in the interpretation of the data presented, which reduces the strength of its conclusions. For instance, there is a misunderstanding of the non-proton motive nature of the AOX - it does not uncouple respiration, merely decouple it as it neither contributes to nor dissipates the proton motive force, in contrast to chemical uncouplers or proton uncouplers such as UCPs. The authors need to reassess their data in light of the above.

Reviewer #2 (Public review):

Summary:

The authors of this manuscript performed a fascinating set of zebrafish mutant analyses on hox cluster deletion and pinpointed the cause of the pectoral fin loss in one combinatorial hox cluster mutant of Hoxba and Hoxbb.

Strengths:

The study is based on a variety of existing experimental tools that enabled the authors' past construction of hox cluster mutants, and is well-designed. The manuscript is well written to report the authors' findings on the mechanism that positions the pectoral fin.

Weaknesses:

The study does not focus on the other hox clusters other than ba and bb, and is confined to the use of zebrafish, as well as the comparison with existing reports from mouse experiments.

Reviewer #2 (Public review):

This paper investigates binding epitopes of different anti-Abeta antibodies. Background information on the clinical outcome of some of the antibodies in the paper, which might be important for readers to know, is lacking. There are no references to clinical outcomes from antibodies that have been in clinical trials. This paper would be much more complete if the status of the antibodies were included. The binding characteristics of aducanumab, donanemab, and lecanemab should be compared with data from clinical phase 3 studies.

Aducanumab was identified at Neurimmune in Switzerland and licensed to Biogen and Eisai. Aducanumab was retracted from the market due to a very high frequency of the side-effect amyloid-related imaging abnormalities-edema (ARIA-E). Gantenerumab was developed by Roche and had two failed phase 3 studies, mainly due to a high frequency of ARIA-E and low efficacy of Abeta clearance. Lecanemab was identified at Uppsala University, humanized by BioArctic, and licensed to Eisai, who performed the clinical studies. Eisai and Biogen are now marketing lecanemab as Leqembi on the world market. Donanemab was developed by Ely Lilly and is sold in the US as Kisunla.

Limitations:

(1) Conclusions are based on Abeta antigens that may not be the primary targets for some conformational antibodies like aducanumab and lecanemab. There is an absence of binding data for soluble aggregated species.

(2) Quality controls and characterization of different Abeta species are missing. The authors need to verify if monomers remain monomeric in the blocking studies for Figures 5 and 6.

(3) The authors should discuss the limitations of studying synthetic Abeta species and how aggregation might hide or reveal different epitopes.

(4) The authors should elaborate on the differences between synthetic Abeta and patient-derived Abeta. There is a potential for different epitopes to be available.

Reviewer #2 (Public review):

PROTACs are a class of small molecules that induce an interaction between a target protein and a ubiquitin ligase, thereby leading to the target protein's ubiquitination and subsequent proteasomal degradation. Given that the vast majority of PROTACs rely on the cereblon and VHL ubiquitin ligases, a major goal within this field has been to identify and develop ligands for additional ubiquitin ligases, in particular those whose expression affords tissue or subcellular specificity or those whose structure allows them to degrade targets that are otherwise incompatible with cereblon or VHL.

In this work, Zhou and colleagues from the Bollong group at Scripps utilize a high-throughput fluorescence polarization screen of >350,000 compounds to identify and optimize a novel ligand for KLHDC2, a ubiquitin ligase which had previously been discovered to be capable of proximity-induced degradation of target proteins. Zhou et al go on to show that this ligand can be used as the basis for PROTACs capable of degrading BRD4 in a cell line. Of note, prior to this paper, three other groups had also developed ligands to KLHDC2 and used them to generate active PROTACs. Interestingly, docking studies by Zhou suggest that their compound may bind to a different region of the KLHDC2's kelch domain.

The major strengths of this work are its brevity and the clarity of the writing and figures. Their claim that they have discovered a ligand for KLHDC2, which can be used to develop BRD4-degrading PROTACs, is well-supported by their findings from the screen, SPR, and cellular assays. The weakness of the work then, is not so much relevant to the paper at hand but rather stems from the fact that their story leaves me wanting to know more. Indeed, there are a number of interesting experiments that we need as a field in order to assess 1) how generalizable their findings are across cell lines and targets, and 2) how this new KLHDC2 ligand stacks up against the other recently discovered ligands for KLDHC2 as well as the existing standards, cereblon and VHL.

Nonetheless, Zhou and colleagues provide a valuable addition to the emerging repertoire of KLHDC2 ligands, and I'm certain that with time, we will come to understand what ligands work best for KLHDC2-based PROTACs and how they compare to the growing set of ubiquitin ligases in our armamentarium.

Reviewer #2 (Public review):

The aims of investigating Slit-Robo signaling in cardiac innervation were achieved by the experiments designed. While questions remain regarding signal regulation and interplay between established axon guidance signals and further role of other Slit ligands and Robo expression in endothelium, the results strongly support the conclusions drawn.

Writing and presentation are easy to follow and well structured, Appropriate controls are used, statistical analysis applied appropriately, and experiments directly test aims following a logical story.

The authors demonstrate a novel mechanism for Slit-Robo signaling in cardiac sympathetic innervation. The data establishes a framework for future studies.

Recommendations:

Further assessment of interplay between Slit ligands as well as other signaling pathways (Semaphorin, NGF, etc) could be investigated. Is it possible to rescue the phenotype by modulation of other signaling pathways? Can combined Slit2/Slit3 KO rescue? Additionally, as the authors state, conditional Robo1 knockouts will be important to validate the findings of constitutive knockout.

Reviewer #2 (Public review):

Summary:

Endo et al. investigate the novel role of ubiquitin response upon lysosomal damage in activating cellular signaling for cell survival. The authors provide a comprehensive transcriptome and proteome analysis of aging-related cells experiencing lysosomal damage, identifying transcription factors involved in transcriptome and proteome remodeling with a focus on the NF-κB signaling pathway. They further characterized the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis in controlling gene expression, inflammatory responses, and apoptotic processes.

Strengths:

In the aging-related model, the authors provide a comprehensive transcriptome and characterize the K63-ubiquitin-TAB-TAK1-NF-κB signaling axis. Through compelling experiments and advanced tools, they elucidate its critical role in controlling gene expression, inflammatory responses, and apoptotic processes.

Weaknesses:

The study lacks deeper connections with previous research, particularly:<br /> • The established role of TAB-TAK1 in AMPK activation during lysosomal damage<br /> • The potential significance of TBK1 in NF-κB signaling pathways

Reviewer #2 (Public review):

Summary:

Govorunova et al present three new anion opsins that have potential applications in silencing neurons. They identify new opsins by scanning numerous databases for sequence homology to known opsins, focusing on anion opsins. The three opsins identified are uncommonly fast, potent, and are able to silence neuronal activity. The authors characterize numerous parameters of the opsins.

Strengths:

This paper follows the tradition of the Spudich lab, presenting and rigorously characterizing potentially valuable opsins. Furthermore, they explore several mutations of the identified opsin that may make these opsins even more useful for the broader community. The opsins AnsACR and FtACR are particularly notable, having extraordinarily fast onset kinetics that could have utility in many domains. Furthermore, the authors show that AnsACR is usable in multiphoton experiments having a peak photocurrent in a commonly used wavelength. Overall, the author's detailed measurements and characterization make for an important resource, both presenting new opsins that may be important for future experiments, and providing characterizations to expand our understanding of opsin biophysics in general.

Weaknesses:

First, while the authors frequently reference GtACR1, a well-used anion opsin, there is no side-by-side data comparing these new opsins to the existing state-of-the-art. Such comparisons are very useful to adopt new opsins.

Next, multiphoton optogenetics is a promising emerging field in neuroscience, and I appreciate that the authors began to evaluate this approach with these opsins. However, a few additional comparisons are needed to establish the user viability of this approach, principally the photocurrent evoked using the 2p process, for given power densities. Comparison across the presented opsins and GtACR1 would allow readers to asses if these opsins are meaningfully activated by 2P.

Reviewer #2 (Public review):

Summary:<br /> The flatworm planarian Schmidtea mediterranea is an excellent model for understanding cell fate specification during tissue regeneration and adult tissue maintenance. Planarian stem cells, known as neoblasts, are continuously deployed to support cellular turnover and repair tissues damaged or lost due to injury. This reparative process requires great precision to recognize the location, timing, and cellular fate of a defined number of neoblast progeny. Understanding the molecular mechanisms driving this process could have important implications for regenerative medicine and enhance our understanding of how form and function are maintained in long-lived organisms such as humans. Unfortunately, the molecular basis guiding cell fate and differentiation remains poorly understood.

In this manuscript, Canales et al. identified the role of the map3k1 gene in mediating the differentiation of progenitor cells at the proper target tissue. The map3k1 function in planarians appears evolutionarily conserved as it has been implicated in regulating cell proliferation, differentiation, and cell death in mammals. The results show that the downregulation of map3k1 with RNAi leads to spatial patterning defects in different tissue types, including the eye, pharynx, and the nervous system. Intriguingly, long-term map3k1-RNAi resulted in ectopic outgrowths consistent with teratomas in planarians. The findings suggest that map3k1 mediates signaling, regulating the timing and location of cellular progenitors to maintain correct patterning during adult tissue maintenance.

Strengths:

The authors provide an entry point to understanding molecular mechanisms regulating progenitor cell differentiation and patterning during adult tissue maintenance.

The diverse set of approaches and methods applied to characterize map3k1 function strengthens the case for conserved evolutionary mechanisms in a selected number of tissue types. The creativity using transplantation experiments is commendable, and the findings with the teratoma phenotype are intriguing and worth characterizing.

Weaknesses:

The article presents a provocative idea related to the importance of positional control for organs and cells, which is at least in part regulated by map3k1. Nonetheless, the role of map3k1 or its potential interaction with regulators of the anterior-posterior, mediolateral axes, and PCGs is somewhat superficial. The authors could elaborate or even speculate more in the discussion section and the different scenarios incorporating these axial modulators into the map3k1 model presented in Figure 8.

The article can be improved by addressing inconsistencies and adding details to the results, including the main figures and supplements. This represents one of the most significant weaknesses of this otherwise intriguing manuscript. Below are some examples of a few figures, but the authors are expected to pay close attention to the remaining figures in the paper.

Details associated with the number of animals per experiment, statistical methods used, and detailed descriptions of figures appear inconsistent or lacking in almost all figures. In some instances, the percentage of animals affected by the phenotype is shown without detailing the number of animals in the experiment or the number of repeats. Figures and their legends throughout the paper lack details on what is represented and sometimes are mislabeled or unrelated. Specifically, the arrows in Figure 1A are different colors. Still, no reasoning is given for this, and in the exact figure, the top side (1A) shows the percentages and the number of animals below. Conversely, in Figures 1B, C, and D, no details on the number of animals or percentages are shown, nor an explanation of why opsin was used in Figure 1A but not 1B. Is Figure 1B missing an image for the respective control? Figure 1C needs details regarding what the two smaller boxes underneath are. Figure 1C could use an AP labeling map in 10 days (e.g., AP6 has one optic cup present). Figure 1C and F counts do not match. In Figure 1C, we do not know the number of animals tested, controls used, the scale bar sizes in the first two images, nor the degree of magnification used despite the pharynx region appearing magnified in the second image. Figure 1C is also shown out of chronological order; 36 days post RNAi is shown before 10 days post RNAi. Moreover, the legends for Figures 1C and 1D are swapped.

Additionally, Figure 1F and many other figures throughout the paper lack overall statistical considerations. Furthermore, Figure 1F has three components, but only one is labeled. Labeling each of them individually and describing them in the corresponding figure legend may be more appropriate.

Figure 2C shows images of gene expression for two genes, but the counts are shown for only one in Figure 2D. It is challenging to follow the author's conclusions without apparent reasoning and by only displaying quantitative considerations for one case but not the other. These inconsistencies are also observed in different figures. In Figure 2D, 24/24 animals were reported to show the phenotype, but only eight were counted (is there a reason for this?). In Figure 2E, the expression for three genes is shown, with some displaying anterior and posterior regions while others only show the anterior picture. Is there a particular reason for this? Also, in Figure 2F, the counts are shown for only the posterior region of two genes out of the three displayed in Figure 2E. It is unclear why the authors do not show counts for the anterior areas considered in Figure 2E. Furthermore, the legend for Figure 2D is missing, and the legend for 2F is mislabeled as a description for Figure 2D.

Supplement Figure 1 B reports data up to 6 weeks, but no text in the manuscript or supplement mentions any experiment going up to 6 weeks. There are no statistics for data in Supplement Figure 1E. Any significance between groups is unclear.

Reviewer #2 (Public review):

Summary:

Chen et al. describe the mechanisms that separate the common pan-sensory progenitor region into individual sensory patches, which presage the formation of the sensory epithelium in each of the inner ear organs. By focusing on the separation of the anterior and then lateral cristae, they find that long supra-cellular cables form at the interface of the pan-sensory domain and the forming cristae. They find that at these interfaces, the cells have a larger apical surface area, due to basal constriction, and Sox2 is down-regulated. Through analysis of Lmx1 mutants, the authors suggest that while Lmx1 is necessary for the complete segregation of the sensory organs, it is likely not necessary for the initial boundary formation, and the down-regulation of Sox2.

Strengths:

The manuscript adds to our knowledge and provides valuable mechanistic insight into sensory organ segregation. Of particular interest are the cell biological mechanisms: The authors show that contractility directed by ROCK is important for the maintenance of the boundary and segregation of sensory organs.

Weaknesses:

The manuscript would benefit from a more in-depth look at contractility - the current images of PMLC are not too convincing. Can the authors look at p or ppMLC expression in an apical view? Are they expressed in the boundary along the actin cables? Does Y-27362 inhibit this expression?

The authors suggest that one role for ROCK is the basal constriction. I was a little confused about basal constriction. Are these the initial steps in the thinning of the intervening non-sensory regions between the sensory organs? What happens to the basally constricted cells as this process continues?

The steps the authors explore happen after boundaries are established. This correlates with a down-regulation of Sox2, and the formation of a boundary. What is known about the expression of molecules that may underlie the apparent interfacial tension at the boundaries? Is there any evidence for differential adhesion or for Eph-Ephrin signalling? Is there a role for Notch signalling or a role for Jag1 as detailed in the group's 2017 paper?

A comment on whether cellular intercalation/rearrangements may underlie some of the observed tissue changes.

The change in the long axis appears to correlate with the expression of Lmx1a (Fig 5d). The authors could discuss this more. Are these changes associated with altered PCP/Vangl2 expression?

Reviewer #2 (Public review):

Summary:

In this paper, the authors defined the "channelome," consisting of 419 predicted human ion channels as well as 48,000 ion channel orthologs from other organisms. Using this information, the ion channels were clustered into groups, which can potentially be used to make predictions about understudied ion channels in the groups. The authors then focused on the CALHM ion channel family, mutating conserved residues and assessing channel function.

Strengths:

The curation of the channelome provides an excellent resource for researchers studying ion channels. Supplemental Table 1 is well organized with an abundance of useful information.

Weaknesses:

There are substantial concerns regarding the analysis of the CALHM channels as detailed below.

(1) There are significant problems with the methodology used for the electrophysiology studies. Pulse protocol is used to assess the current voltage relationship (-100 to +140 mV), which extends far beyond the physiological range; currents for the mutant channels were only assessed at +120 mV. It is also unclear why a holding potential of 0 mV was used for CALHM6 recordings; the channel is already open at this voltage (and in Figure 4, only n = 3 for CALHM6). Further, proper controls were not performed. Inhibitors such as Gd3+ can be used to ensure that only CALHM currents are being measured.

(2) In line 334, the authors state that "expression levels of wild-type proteins and mutants are comparable." However, Western blots showing CALHM protein abundance (Supplementary Figure 3) are not of acceptable quality - in the top blot, WT CALHM1 can't even be seen. Representative blots were not shown for all mutants, and there was no effort to determine if levels were statistically significant compared to the wild-type control. Even if there is more or less protein, what does this mean? The protein could be in an intracellular compartment and not at the plasma membrane. In mammalian cells, CALHM6 is localized to intracellular compartments and only translocates to the plasma membrane upon activating stimulus (Danielli et al, EMBO J, 2023). Thus, if CALHM6 is only intracellular, the protein amount would not change, but the measured current would. Abundant intracellular CALHM1 has also been observed in mammalian cells transfected with this protein (Dreses-Werringloer et al., Cell, 2008). The best way to determine if mutations impact CALHM channel localization is to express GFP-tagged constructs in Xenopus oocytes and look for surface expression.

(3) Since the authors have not definitively shown that there are no defects in localization, they cannot make the claim in lines 346-356 that the mutations "either abolished or markedly reduced channel activity." Further, from their data, there is speculation regarding how these residues impact conformational changes during channel opening and closing. Line 404 - again, there is no concrete evidence that any of these residues play a role in gating function. Lines 406-433 - this entire paragraph is speculation without data to back it up. There is also a lack of specificity with statements such as "all mutants showed either reduced or completely abolished activity." What is meant by activity? Do the authors mean conductance?

(4) Line 303 - 13 aligned amino acids were conserved across all CALHM homologs - are these also aligned in related connexin and pannexin families? It is likely that cysteines and proline in TM2 are since CALHM channels overall share a lot of similarities with connexins and pannexins (Siebert et al, JBC, 2013). As in line 207, it would be expected that pannexins, connexins, and CALHM channel families would group together. Related to this, see Line 406 - in connexins, there is also a proline kink in TM2 that may play a role in mediating conformational changes between channel states (Ri et al, Biophysical Journal, 1999).

Reviewer #2 (Public review):

The manuscript "Domain Coupling in Allosteric Regulation of SthK Measured Using Time-Resolved Transition Metal Ion FRET" by Eggan et al. investigates the energetics of conformational transitions in the cyclic nucleotide-gated (CNG) channel SthK. This lab pioneered transition metal FRET (tmFRET), which has previously provided detailed insights into ion channel conformational changes. Here, the authors analyze tmFRET fluorescence lifetime measurements in the time domain, yielding detailed insights into conformational transitions within the cyclic nucleotide binding domains (CNBDs) of the channel. The integration of tmFRET with time-correlated single-photon counting (TCSPC) represents an advancement of this technique.

The results summarize known conformational transitions of the C-helix and provide distance distributions that agree with predicted values based on available structures. The authors first validated their TCSPC approach using the isolated CNBD construct previously employed for similar experiments. They then study the more complex full-length SthK channel protein. The findings agree with earlier results from this group, demonstrating that the C-helix is more mobile in the closed state than static structures reflect. Upon adding the activating ligand cAMP, the C-helix moves closer to the bound ligand, as indicated by a reduced fluorescence lifetime, suggesting a shorter distance between the donor and acceptor. The observed effects depend on the cAMP concentration, with affinities comparable to functional measurements. Interestingly, a substantial amount of CNBDs appear to be in the activated state even in the absence of cAMP (Figure 6E and F, fA2 ~ 0.4).

This may be attributed to cooperativity among the CNBDs, which the authors could elaborate on further. In this context, the major limitation of this study is that distance distributions are observed only in one domain. While inter-subunit FRET is detected and accounted for, the results focus exclusively on movements within one domain. Thus, the resulting energetic considerations must be assessed with caution. In the absence of the activator, the closed state is favored, while the presence of cAMP favors the open state. This quantifies the standard assumption; otherwise, an activator would not effectively activate the channel. However, the numerical values of approximately 3 kcal/mol are limited by the fact that only one domain is observed in the experiment, and only one distance (C- helix relative to the CNBD) is probed. Additional conformational changes leading to pore opening (including rotation and upward movement of the CNBD, and radial dilation of the tetrameric assembly) are not captured by the current experiments. These limitations should be taken into account when interpreting the results.

Reviewer #2 (Public review):

Summary:

The authors sought to identify the impact skin viscoelasticity has on neural signalling of contact forces that are representative of those experienced during normal tactile behaviour. The evidence presented in the analyses indicate there is a clear effect of viscoelasticity on the imposed skin movements from a force-controlled stimulus. Both skin mechanics and evoked afferent firing were affected based on prior stimulation, which has not previously been thoroughly explored. This study outlines that viscoelastic effects have an important impact on encoding in the tactile system, which should be considered in the design and interpretation of future studies. Viscoelasticity was shown to affect the mechanical skin deflections and stresses/strains imposed by previous and current interaction force, and also the resultant neuronal signalling. The result of this was an impaired coding of contact forces based upon previous stimulation. The authors may be able to strengthen their findings, by using the existing data to further explore the link between skin mechanics and neural signalling, giving a clearer picture than demonstrating shared variability. This is not a critical addition, but I believe would strengthen the work and make it more generally applicable.

Strengths:

-Elegant design of the study. Direct measurements have been made from the tactile sensory neurons to give detailed information on touch encoding. Experiments have been well designed and the forces/displacements have been thoroughly controlled and measured to give accurate measurements of global skin mechanics during a set of controlled mechanical stimuli.<br /> -Analytical techniques used. Analysis of fundamental information coding and information representation in the sensory afferents reveals dynamic coding properties to develop putative models of the neural representation of force. This advanced analysis method has been applied to a large dataset to study neural encoding of force, the temporal dynamics of this, and the variability in this.

Weaknesses:<br /> -Lack of exploration of the variation in neural responses. Although there is a viscoelastic effect which produces variability in the stimulus effects based on prior stimulation, it is a shame that the variability in neural firing and force induced skin displacements have been presented, and are similarly variable, but there has been no investigation of a link between the two. I believe with these data the authors can go beyond demonstrating shared variability. The force per se is clearly not faithfully represented in the neural signal, being masked by stimulation history, and it is of interest if the underlying resultant contact mechanics are.

Validity of conclusions:

The authors have succeeded in demonstrating skin viscoelasticity has an impact on skin contact mechanics with a given force and that this impacts on the resultant neural coding of force. Their study has been well designed and the results support their conclusions. The importance and scope of the work is adequately outlined for readers to interpret the results and significance.

Impact:

This study will have important implications for future studies performing tactile stimulation and evaluating tactile feedback during motor control tasks. In detailed studies of tactile function, it illustrates the necessity to measure skin contact dynamics to properly understand the effects of a force stimulus on the skin and mechanoreceptors.

Reviewer #2 (Public review):

Summary:

The revised paper by Kim et al. reports two disease mutations in proBMP4, S91C and E93G, disrupt the FAM20C phosphorylation site at Ser91, blocking the activation of proBMP4 homodimers, while still allowing BMP4/7 heterodimers to function. Analysis of DMZ explants from Xenopus embryos expressing the proBMP4 S91C or E93G mutants showed reduced expression of pSmad1 and tbxt1. The expert amphibian tissue transplant studies were expanded to in vivo studies in Bmp4S91C/+ and Bmp4E93G/+ mice, highlighting the impact of these mutations on embryonic development, particularly in female mice, consistent with patient studies. Additionally, studies in mouse embryonic fibroblasts (MEFs) demonstrated that the mutations did not affect proBMP4 glycosylation or ER-to-Golgi transport but appeared to inhibit the furin-dependent cleavage of proBMP4 to BMP4. Based on these findings and AI modeling using AlphaFold of proBMP4, the authors speculate that pSer91 influences access of furin to its cleavage site at Arg289AlaLysArg292 in a new "Ideas and Speculation" section. Overall, the authors addressed the reviewers' comments, improving the presentation.

Strengths:

The strengths of this work continue to lie in the elegant Xenopus and mouse studies that elucidate the impact of the S91C and E93G disease mutations on BMP signaling and embryonic development. Including an "Ideas and Speculation" subsection for mechanistic ideas reduces some shortcomings regarding the analysis of the underlying mechanisms.

Reviewer #2 (Public review):

This elegant study provides significant and impactful insights into the factors contributing to the distinct arrangement of sub-membrane microtubules within mouse β-cells of the pancreas. The authors propose that in these cells, the motor protein KIF5B plays a crucial role in sliding existing microtubules toward the cell periphery and aligning them with one another along the plasma membrane. Furthermore, similar to other physiological features of β-cells, high glucose levels enhance this microtubule sliding process. A precise arrangement of microtubules beneath the cell membrane in β-cells is vital for the regulated secretion of pancreatic enzymes and hormones; thus, KIF5B has a significant role in pancreatic activity in both healthy conditions and diseases. The authors support their model by demonstrating that the levels of KIF5B mRNA in MIN6 cells are higher than those of other known kinesins. They show that microtubule sliding becomes less efficient when KIF5B is genetically silenced using two different short hairpin RNAs (shRNAs). Additionally, silencing of KIF5A in the same cells results in a general reorganization of microtubules throughout the cell. Specifically, while control cells exhibit a convoluted and non-radial arrangement of microtubules near the cell membrane, KIF5B-depleted cells display a sparse and less dense sub-membrane array of microtubules. Based on these findings, the authors conclude that the loss of KIF5B strongly affects the localization of microtubules to the cell periphery. Using a dominant-negative approach, the authors also demonstrate that KIF5B facilitates the sliding of microtubules by binding to cargo microtubules through the kinesin-1 tail binding domain. They present evidence suggesting that KIF5B-mediated microtubule sliding is glucose-dependent, similar to the activity levels of kinesin-1, which increase in the presence of glucose. Lastly, they show that this is glucose-dependent.

Strengths:

This study unveils a previously unexplained mechanism that regulates the specific rearrangement of microtubules beneath the cell membrane in pancreatic β-cells. The findings have significant implications because the precise regulation of the microtubule array at the secretion zone plays a critical role in controlling pancreatic function in both healthy and diseased states. The provided data supports the authors' conclusions well, and the study demonstrates the use of state-of-the-art methodologies, including quantification techniques and elegant dominant-negative experiments.

Weaknesses: None

Reviewer #2 (Public review):

This is a short and unpretentious paper. It is an interesting area and therefore, although much of this area of research was pioneered in flies, extending basic findings to butterflies would be worthwhile. Indeed, there is an intriguing observation but it is technically flawed and these flaws are far too serious to allow us to recommend publication

The authors show that mirror is expressed at the back of the wing in butterflies (as in flies). They present some evidence that is required for the proper development of the back of the wing in butterflies (a region dubbed the vannus by the ancient guru Snodgrass). But there are problems with that evidence. First, concerning the method, using CRISP they treat embryos and the expectation is that the mirror gene will be damaged in groups of cell lineages, giving a mosaic animal in which some lines of cells are normal for mirror and others not. We do not know where the clones or patches of cells that are defective for mirror are because they are not marked. Also, we do not know what part of the wing is wildtype and what part is mutant for mirror. When the mirror mutant cells colonise the back of the wing and that butterfly survives (many butterflies fail to develop), the back of the wing is altered in some selected butterflies. This raises a second problem: we do not know whether the rear of the wing is missing or transformed. From the images the appearance of the back of the wing is clearly different from wild type, but is that due to transformation or not? And then I believe we need to know specifically what us difference between the rear of the wing and the main part. What we see is a silvery look at the back that is not present in the main part, is it the structure of the scales? We are not told. There are other problems. Mirror is only part of a group of genes in flies and in flies both iroquois and mirror are needed to make the back of the wing, the alula (Kehl et al). What is known about iro expression in butterflies?

In flies, mirror regulates a late and local expression of dpp that seems to be responsible of making the alula. What happens in butterflies? Would a study of expression of Dpp in wildtype and mirror compromised wings be useful?

Thus, I find the paper to be disappointing for a general journal as it does little more than claim what was discovered in Drosophila is at least partly true in butterflies. Also it fails to explain what the authors mean by "wing domains" and "domain specification". They are not alone, butterfly workers in general appear vague about these concepts, their vagueness allowing too much loose thinking. Since these matters are at the heart of the purpose and meaning of the work reported here, we readers need a paper containing more critical thought and information. I would like to have a better and more logical introduction and discussion.

They do define what they mean by the vannus of the wing. In flies the definition of compartments is clear and abundantly demonstrated, with gene expression and requirement being limited precisely to sets of cells that display lineage boundaries. It is true that domains of gene expression in flies, for example, of the iroquois complex, which includes mirror, can only be related to pattern with difficulty. Some recap of what is known plus the opinion of the authors on how they interpret papers on possible lineage domains in butterflies might also be useful as the reader, is no wiser about what the authors might mean at the end of it!

The references are sometimes inappropriate. The discovery of the AP compartments should not be referred to Guillen et al 1995, but to Morata and Lawrence 1975.

Comments on revisions:

Nearly all the previous criticisms remain valid and are not discussed or overcome in the revision. The authors wish to draw their conclusions and we think they can do that, but they should make clear that key evidence is lacking. Thus their conclusions are speculative. But they present them more or less as facts. This is not justified. Let us suppose that clones lacking mirror do not survive or do not develop properly in the rear part of the wing and what they are seeing is occasional damage due to incomplete regeneration or to regenerative duplication?

Many clones in flies only include parts of one surface of the wing, could this happen here and how would it affect interpretations?

The null phenotype in the wing is not known but deduced from abnormal wings which "even in mKO..... appeared to have a mutant phenotype across the entire posterior region", a nice example of circular logic.

We believe the authors should be more objective and explain that their interpretations are not solid and that they should ideally be tested by finding ways of independently marking the clones. Other clonal mosaic experiments in butterflies have been done (eg https://journals.biologists.com/dev/article/150/18/dev201868/329659/Frizzled2-receives-WntA-signaling-during-butterfly) without cell autonomous independent markers, but they are more solid as transformed spots are made visible cell by cell by scale colour changes etc.

Their deduction that "mirror acts as a selector gene necessary to define the far posterior wing domain" is a speculative hypothesis, not a deduction and the readers should be so informed.

Reviewer #2 (Public review):

Summary:

This paper investigates the neuronal encoding of the relationship between head and body orientations in the brain. Specifically, the authors focus on the angular relationship between the head and body by employing virtual avatars. Neuronal responses were recorded electrophysiologically from two fMRI-defined areas in the superior temporal sulcus and analyzed using decoding methods. They found that: (1) anterior STS neurons encode head-body angle configurations; (2) these neurons distinguish aligned and opposite head-body configurations effectively, whereas mirror-symmetric configurations are more difficult to differentiate; and (3) an upside-down inversion diminishes the encoding of head-body angles. These findings advance our understanding of how visual perception of individuals is mediated, providing a fundamental clue as to how the primate brain processes the relationship between head and body-a process that is crucial for social communication.

Strengths:

The paper is clearly written, and the experimental design is thoughtfully constructed and detailed. The use of electrophysiological recordings from fMRI-defined areas elucidated the mechanism of head-body angle encoding at the level of local neuronal populations. Multiple experiments, control conditions, and detailed analyses thoroughly examined various factors that could affect the decoding results. The decoding methods effectively and consistently revealed the encoding of head-body angles in the anterior STS neurons. Consequently, this study offers valuable insights into the neuronal mechanisms underlying our capacity to integrate head and body cues for social cognition-a topic that is likely to captivate readers in this field.

Weaknesses:

I did not identify any major weaknesses in this paper.

Reviewer #2 (Public review):

The authors used combined blockers/modulators to dissect the potassium currents mediated by inter-subunit heteromeric Kv channels. The method is robust given that the researchers know their limitations. Nevertheless, the authors elegantly tested their hypotheses, making this manuscript friendly to read despite the depth of all aspects they dealt with.

The quality of the data presented will positively impact the science involved in the study heteromeric channels, with clear developments in the field. Finally, the approach presented may unlock new studies related to these channels.

Comments on revisions:

The authors clarified all my points and beyond, specifically by adding some computational work that will also contribute to the subfield of heteromeric Kv channels.

Reviewer #2 (Public review):

Summary:

The authors have made microfluidic arrays of pores and obstacles with a complex shape and studied the swimming of multicellular magnetotactic bacteria through this system. They provide a comprehensive discussion of the relevant parameters of this system and identify one dimensionless parameter, which they call the scattering number and which depends on the swimming speed and magnetic moment of the bacteria as well as the magnetic field and the size of the pores, as the most relevant. They measure the effective speed through the array of pores and obstacles as a function of that parameter, both in their microfluidic experiments and in simulations, with good agreement between the two. They find an optimal scattering number, which they estimate to reflect the parameters of the studied multicellular bacteria in their natural environment. They finally use this knowledge to compare different species. Despite the variability of bacteria parameters, they estimate the scattering number to be rather narrowly distributed, suggesting that their results apply to a broad range of species.

Strengths:

This is a beautiful experimental approach and the observation of an optimal scattering number (likely reflecting an optimal magnetic moment) is very convincing. The results here improve on similar previous work in two respects: On the one hand, the tracking of bacteria does not have the limitations of previous work, and on the other hand, the effective motility is quantified. Both features are enabled by choices of the experimental system: the use the multicellular bacteria which are larger than the usual single-celled magnetotactic bacteria and the design of the obstacle array which allows the quantification of transition rates due to the regular organization as well as the controlled release of bacteria into this array through a clever mechanism.

Weaknesses:

Some of the key experimental choices on which the strength of the approach is based also come at a price and impose some limitations, namely the use of a non-culturable organism and the regular, somewhat unrealistic artificial obstacle array, but the advantages of these choices outweigh the drawbacks.

Comments on revisions:

The paper has been improved with respect to presentation and content. In particular, I appreciate the new plots comparing the simulation and experiments directly and the estimate of the scattering number for different species. In my opinion, all issues raised by the reviewers have been addressed in a productive way.

Reviewer #2 (Public review):

This is a great paper. Yoshida et al. convincingly show that DnaA does not exclusively do loading of the replicative helicase at the E. coli oriC, but that PriC can also perform this function. Importantly, PriC seems to contribute to helicase loading even in wt cells albeit to a much lesser degree than DnaA. On the other hand, PriC takes a larger role in helicase loading during aberrant initiation, i.e. when the origin sequence is truncated or when the properties of initiation proteins is suboptimal. Here highlighted by mutations in dnaA or dnaC.

This a major finding because it clearly demonstrates that the two roles of DnaA in the initiation process can be separated into initially forming an open complex at the DUE region by binding/nucleation onto DnaA-boxes and second in loading of the helicase. Whereas these two functions are normally assumed to be coupled, the present data clearly show that they can be separated and that PriC can perform at least part of the helicase loading provided that an area of duplex opening is formed by DnaA.<br /> This puts into questions the interpretation of a large body of previous work on mutagenesis of oriC and dnaA to find a minimal oriC/DnaA complex in many bacteria. In other words, mutants in which oriC is truncated/mutated may support initiation of replication and cell viability only in the presence of PriC. Such mutants are capable to generate single strand opening but may fail to load the helicase in absence of PriC. Similarly, dnaA mutants may generate aberrant complex on oriC that trigger strand opening but are incapable of loading DnaB unless PriC is present.

In the present work, the sequence of experiments presented is logical and the manuscript is clearly written and easy to follow. The very last part regarding PriC in cSDR replication does not add much to the story and may be omitted.

I have a few specific questions/comments

The partial complementation of the dnaC2 strain by PriC seems quite straightforward since this particular mutation leads to initiation arrest at the open complex stage and this sets the stage for PriC to load the helicase. The situation is somewhat different for dnaA46. Why is this mutation partly complemented by PriC at 37C? DnaA46 binds neither ATP nor ADP, yet it functions in initiation at permissive temperature. At nonpermssive temperature, it binds oriC as well but does not lead to initiation. Does the present data imply that the true initiation defect of DnaA46 lies in helicase loading? The authors need to comment on this in the text.

Relating to the above. In Fig. 3 it is shown that the pFH plasmid partly complement dnaA46 in a PriC dependent manner. Again, it would be nice to know the nature of the DnaA46 protein defect. It would be interesting to see how a pING1-dnaA46 plasmid performs in the experiment presented in Fig. 3.

Reviewer #2 (Public review):

Summary:

The authors use ligands (inverse agonists, partial agonists) for PPAR, and coactivators and corepressors, to investigate how ligands and cofactors interact in a complex manner to achieve functional outcomes (repressive vs. activating).

Strengths:<br /> The data (mostly biophysical data) are compelling from well-designed experiments. Figures are clearly illustrated. The conclusions are supported by these compelling data. These results contribute to our fundamental understanding of the complex ligand-cofactor-receptor interactions.

Weaknesses:

This is not the weakness of this particular paper, but the general limitation in using simplified models to study a complex system.

Reviewer #2 (Public review):

A long-standing debate in the field of Pavlovian learning relates to the phenomenon of timescale invariance in learning i.e. that the rate at which an animal learns about a Pavlovian CS is driven by the relative rate of reinforcement of the cue (CS) to the background rate of reinforcement. In practice, if a CS is reinforced on every trial, then the rate of acquisition is determined by the relative duration of the CS (T) and the ITI (C = inter-US-interval = duration of CS + ITI), specifically the ratio of C/T. Therefore, the point of acquisition should be the same with a 10s CS and a 90s ITI (T = 10; C = 90 + 10 = 100, C/T = 100/10 = 10) and with a 100s CS and a 900s ITI (T = 100; C = 900 + 100 = 1000, C/T = 1000/100 = 10). That is to say, the rate of acquisition is invariant to the absolute timescale as long as this ratio is the same. This idea has many other consequences, but is also notably different from more popular prediction-error based associative learning models such as the Rescrola-Wagner model. The initial demonstrations that the ratio C/T predicts the point of acquisition across a wide range of parameters (both within and across multiple studies) was conducted in Pigeons using a Pavlovian autoshaping procedure. What has remained under contention is whether or not this relationship holds across species, particularly in the standard appetitive Pavlovian conditioning paradigms used in rodents. The results from rodent studies aimed at testing this have been mixed, and often the debate around the source of these inconsistent results focuses on the different statistical methods used to identify the point of acquisition for the highly variable trial-by-trial responses at the level of individual animals.<br /> The authors successfully replicate same effect found in pigeon autoshaping paradigms decades ago (with almost identical model parameters) in a standard Pavlovian appetitive paradigm in rats. They achieve this through a clever change the experimental design, using a convincingly wide range of parameters across 14 groups of rats, and by a thorough and meticulous analysis of these data. It is also interesting to note that the two author's have published on opposing sides of this debate for many years, and as a result have developed and refined many of the ideas in this manuscript through this process.

Main findings

(1) The present findings demonstrate that the point of initial acquisition of responding is predicted by the C/T ratio.

(2) The terminal rates of responding to the CS appears to be related to the reinforcement rate of the CS (T; specifically, 1/T) but not its relation to the reinforcement rate of the context (i.e. C or C/T). In the present experiment, all CS trials were reinforced so it is also the case that the terminal rate of responding was related to the duration of the CS.

(3) An unexpected finding was that responding during the ITI was similarly related to the rate of contextual reinforcement (1/C). This novel finding suggests that the terminal rate of responding during the ITI and the CS are related to their corresponding rates of reinforcement. This finding is surprising as it suggests that responding during the ITI is not being driven by the probability of reinforcement during the ITI.

(4) Finally, the authors characterised the nature of increased responding from the point of initial acquisition until responding peaks at a maximum. Their analyses suggest that nature of this increase was best described as linear in the majority of rats, as opposed to the non-linear increase that might be predicted by prediction error learning models (e.g. Rescorla-Wagner). However, more detailed analyses revealed that these changes can be quite variable across rats, and more variable when the CS had lower informativeness (defined as C/T).

Strengths and Weaknesses:

There is an inherent paradox regarding the consistency of the acquisition data from Gibbon & Balsam's (1981) meta-analysis of autoshaping in pigeons, and the present results in magazine response frequency in rats. This consistency is remarkable and impressive, and is suggestive of a relatively conserved or similar underlying learning principle. However, the consistency is also surprising given some significant differences in how these experiments were run. Some of these differences might reasonably be expected to lead to differences in how these different species respond. For example:

- The autoshaping procedure commonly used in the pigeons from these data were pretrained to retrieve rewards from a grain hopper with an instrumental contingency between head entry into the hopper and grain availability. During Pavlovian training, pecking the key light also elicited an auditory click feedback stimulus, and when the grain hopper was made available the hopper was also illuminated.

- In the present experimental procedure, the rats were not given contextual exposure to the pellet reinforcers in the magazine (e.g. a magazine training session is typically found in similar rodent procedures). The Pavlovian CS was a cue light within the magazine itself.

These design features in the present rodent experiment are clearly intentional. Pretraining with the reinforcer in the testing chambers would reasonably alter the background rate of reinforcement (parameter), so it make sense not to include this but differs from the paradigm used in pigeons. Having the CS inside the magazine where pellets are delivered provides an effective way to reduce any potential response competition between CS and US directed responding and combines these all into the same physical response. This makes the magazine approach response more like the pecking of the light stimulus in the pigeon autoshaping paradigm. However, the location of the CS and US is separated in pigeon autoshaping, raising questions about why the findings across species are consistent despite these differences.

Intriguingly, when the insertion of a lever is used as a Pavlovian cue in rodent studies, CS directed responding (sign-tracking) often develops over training such that eventually all animals bias their responding towards the lever than towards the US (goal-tracking at the magazine). However, the nature of this shift highlights the important point that these CS and US directed responses can be quite distinct physically as well as psychologically. Therefore, by conflating the development of these different forms of responding, it is not clear whether the relationship between C/T and the acquisition of responding describes the sum of all Pavlovian responding or predominantly CS or US directed responding.

Another interesting aspect of these findings is that there is a large amount of variability that scales inversely with C/T. A potential account of the source of this variability is related to the absence of preexposure to the reward pellets. This is normally done within the animals' homecage as a form of preexposure to reduce neophobia. If some rats take longer to notice and then approach and finally consume the reward pellets in the magazine, the impact of this would systematically differ depending on the length of the ITI. For animals presented with relatively short CSs and ITIs, they may essentially miss the first couple of trials and/or attribute uneaten pellets accumulating in the magazine to the background/contextual rate of reinforcement. What is not currently clear is whether this was accounted for in some way by confirming when the rats first started retrieving and consuming the rewards from the magazine.

While the generality of these findings across species is impressive, the very specific set of parameters employed to generate these data raise questions about the generality of these findings across other standard Pavlovian conditioning parameters. While this is obviously beyond the scope of the present experiment, it is important to consider that the present study explored a situation with 100% reinforcement on every trial, with a variable duration CS (drawn form a uniform distribution), with a single relatively brief CS (maximum of 122s) CS and a single US. Again, the choice of these parameters in the present experiment is appropriate and very deliberately based on refinements from many previous studies from the authors. This includes a number of criteria used to define magazine response frequency that includes discarding specific responses (discussed and reasonably justified clearly in the methods section). Similarly, the finding that terminal rates of responding are reliably related to 1/T is surprising, and it is not clear whether this might be a property specific to this form of variable duration CS, the use of a uniform sampling distribution, or the use of only a single CS. However, it is important to keeps these limitations in mind when considering some of the claims made in the discussion section of this manuscript that go beyond what these data can support.

The main finding demonstrating the consistent findings across species is presented in Figure 3. In the analysis of these data, it is not clear why the correlations between C, T, and C/T and the measure of acquisition in Figure 3A were presented as r values, whereas the r2 values were presented in the discussion of Figure 3B, and no values were provided in discussing Figure 3C. The measure of acquisition in Figure 3A is based on a previously established metric, whereas the measure in Figure 3B employs the relatively novel nDKL measure that is argued to be a better and theoretically based metric. Surprisingly, when r and r2 values are converted to the same metric across analyses, it appears that this new metric (Figure 3B) does well but not as well as the approach in Figure 3A. This raises questions about why a theoretically derived measure might not be performing as well on this analysis, and whether the more effective measure is either more reliable or tapping into some aspect of the processes that underlie acquisition that is not accounted for by the nDKL metric. Unfortunately, the new metric is discussed and defined at great length but its utility is not considered.<br /> An important analysis issue that is unclear in the present manuscript is exactly how the statistics were run (how the model was defined, were individual subjects or group medians used, what software was used etc...). For example, it is not clear whether the analyses conducted in relation to Figure 3 used the data from individual rats or the group medians. Similarly, it appears that each rat contributes four separate data points, and a single regression line was fit to all these data despite the highly likely violation of the assumption independent observations (or more precisely, the assumption of uncorrelated errors) in this analysis. Furthermore, it is claimed that the same regression line fit the IT and CS period data in this figure, however this<br /> If the data in figure 3 were analyzed with log(ITI) or log(C/ITI) i.e. log(C/(T-C)), would this be a better fit for these data? Is it the case that the ratio of C/T the best predictor of the trial/point of acquisition, or is it the case that another metric related to reinforcement rates provides a better fit?

Based on the variables provided in Supplementary file 3, containing the acquisition data, I was unable to reproduce the values reported in the analysis of Figure 3.<br /> In relation to Figure 3: I am curious about whether the authors would be able to comment on whether the individual variability in trials to acquisition would be expected to scale differently based on C/T, or C, or (if a less restricted range was used) T?<br /> It is not clear why Figure 3C is presented but not analyzed, and why the data presented in Figure 4 to clarify the spread of the distribution of the data observed across the plots in Figure 3 uses the data from Figure 3C. This would seem like the least representative data to illustrate the point of Figure 4. It also appears to my eye that the data actually plotted in Figure 4 correspond to Figure 3A and 3B rather than the odds 10:1 data indicated in text.

What was the decision criteria used to decide on averaging the final 5 conditioning sessions as terminal responding for the analyses in Figure 5? This is an oddly specific number. Was this based on consistency with previous work, or based on the greatest number of sessions where stable data for all animals could be extracted?<br /> In the analysis corresponding to Figures 7-8: If I understand the description of this analysis correctly, for each rat the data are the cumulative response data during the CS, starting from the trial on which responding to the CS > ITI (t = 1), and ending at the trial on which CS responding peaked (maximum over 3 session moving average window; t = end). This analysis does not seem to account for changes (decline) in the ITI response rates over this period of acquisition, and it is likely that responding during the ITI is still declining after t=1. Are the 4 functions that were fit to these data to discriminate between different underlying generative processes still appropriate on total CS responding instead of conditional CS responding after accounting for changes in baseline response rates during ITI?

Page 27, Procedure, final sentence: The magazine responding during the ITI is defined as the 20s period immediately before CS onset. The range of ITI values (Table 1) always starts as low as 15s in all 14 groups. Even in the case of an ITI on a trial that was exactly 20s, this would also mean that the start of this period overlaps with the termination of the CS from the previous trial and delivery (and presumably consumption) of a pellet. Please indicate if the definition of the ITI period was modified on trials where the preceding ITI was <20s, and if any other criteria were used to define the ITI.

Were the rats exposed to the reinforcers/pellets in their home cage prior to acquisition? Please indicate whether rats where pre-exposed to the reward pellets in their home cages e.g. as is often done to reduce neophobia. Given the deliberate absence of a magazine-training phase, this information is important when assessing the experienced contingency between the CS and the US.

For all the analyses, please provide the exact models that were fit and the software used. For example, it is not necessarily clear to the reader (particularly in the absence of degrees of freedom) that the model fits discussed in Figure 3 are fit on the individual subject data points or the group medians. Similarly, in Figure 6 there is no indication of whether a single regression model was fit to all the plotted data or whether tests of different slopes for each of the conditions were compared. With regards to the statistics in Figure 6, depending on how this was run, it is also a potential problem that the analyses does not correct for the potentially highly correlated multiple measurements from the same subjects i.e. each rat provides 4 data points which are very likely not to be independent observations.

A number of sections of the discussion are speculative or not directly supported by the present experimental data (but may well be supported by previous findings that are not the direct focus of the present experiment). For example, Page 19, Paragraph 2: this entire paragraph is not really clearly explained and is presenting an opinion rather than a strong conclusion that follows directly from the present findings. Evidence for an aspect of RET in the present paper (i.e. the prediction of time scale invariance on the initial point of acquisition, but not necessarily the findings regarding the rate of terminal acquisition) - while supportive - does not necessarily provide unconditional evidence for this theory over all the alternatives.

Similarly, the Conclusion section (Page 23) makes the claim that "the equations have at most one free parameter", which may be an oversimplification that is conditionally true in the narrow context of the present experiment where many things were kept constant between groups and run in a particular way to ensure this is the case. While the equations do well in this narrow case, it is unlikely that additional parameters would not need to be added to account for more general learning situations. To clarify, I am not contending that this kind of statement is necessarily untrue, merely that it is being presented in a narrow context and may require a deeper discussion of much more of the literature to qualify/support properly - and the discussion section of the present experiment/manuscript may not be the appropriate place for this.

- Consider taking advantage of an "Ideas and Speculation" subsection within the Discussion that is supported by eLife [ https://elifesciences.org/inside-elife/e3e52a93/elife-latest-including-ideas-and-speculation-in-elife-papers ]. This might be more appropriate to qualify the tone of much of the discussion from page 19 onwards.

It seems like there are entire analyses and new figures being presented in the discussion e.g. Page 20: Information-Theoretic Contingency. These sections might be better placed in the methods section or a supplementary section/discussion.

Reviewer #2 (Public review):

Summary:

This study by Leong and colleagues examines the role of the TCF7L2 transcription factor in the Wnt signaling pathway as a regulator of colon/small intestinal cancers and cachexia. Investigators utilize a Tet off repressor genetic system in mice under Dox regulation to silence TCF7L2. Results show DSS-treated APCMin/+ mice lose body weight that can be rapidly rescued by Dox treatment and suppression of TCF7L2 expression. Reduction of TCF7L2 rescues features of cachexia, including body weight, gastrocnemius muscle and adipose mass, as well as molecular markers of cachexia such as the E3 Ub ligases, MuRF1, and Atrogin-1. The most significant finding in the study is that loss of TCF7L2 reduces but does not eliminate tumor progression, as tumors go from adenomas to adenocarcinomas over time while mice are treated with Dox, yet cachexia persists. This implies that TCF7L2 has a direct effect on cachexia. Overall, the study provides insight into the role of TCFL2 in the development of intestinal cancers and muscle atrophy in cachexia.

Strengths:

The study uses an elegant genetic mouse model to provide significant new insight into the role of TCFL2 in colon and small intestinal cancers. In addition, the authors describe the role of TCF7L2 as a regulator of muscle wasting in cachexia. This, too, can be viewed as a new finding for the cachexia field.

Weaknesses:

However, in its current form, the study lacks sufficient data to support the authors' claim regarding the relevance of TCF7L2 as a regulator of cachexia.

Reviewer #2 (Public review):

Summary:

The authors wanted to investigate how cells defend against intracellular pathogens, such as Shigella and Burkholderia species, that co-opt the host actin machinery to spread from cell-to-cell. Previous work has identified IFNg-inducible GTPase of the Guanylate Binding Protein (GBP) family in cytosolic defence against Gram-negative bacteria. By forming a coat around Shigella, human GBP1 suppresses actin-based motility by displacing IcsA, which is the actin-polymerising virulence factor present at bacterial poles. In addition, GBP1 recruits the cytosolic LPS-sensor, caspase-4, to the bacterial surface, which results in the removal of bacterial replicative niches via pyroptotic cell death. Here, they followed up their finding that GBP1 can reduce actin-based motility of Shigella in HeLa cells and, surprisingly, fails to do so during Burkholderia infection. In contrast, in T24 bladder epithelial cells, GBP1 is competent in blocking Burkholderia actin-tails. They therefore wanted to identify the GBP1-independent factor that blocks actin-based motility in IFNg-treated cells that is absent in HeLa cells.

Major strengths and weaknesses of the methods and results:

The authors report a second IFNg-dependent pathway involving the protein product of the gene GVIN1, which was previously thought to be a pseudogene. GVIN1 (GTPase, very large interferon inducible 1) is thus the first human member of this family of ~250 kDa putative GTPases to be demonstrated to be functional and have potential antimicrobial roles. The discovery that GVIN1 is indeed functional, forms coats on Burkholderia in an LPS O-antigen-dependent manner, and limits actin-dependent motility are the main strengths of this paper. The authors use CRISPR/Cas9-based knockouts in HeLa and T24 cells, and complement them to demonstrate that GBP1 and GVIN1 are both required to inhibit actin-based motility.

An appraisal of whether the authors achieved their aims and whether the results support their conclusions:

The authors achieved their main goals through well-planned experiments and unbiased screens. They succeeded in finding the factor that blocks actin-based motility independently of GBP1. This is driven by GVIN1, which coats bacteria and limits actin-tail formation by reducing the expression of BimA through currently unknown mechanisms. Further, they found that an O-antigen mutant can escape coating by GVIN1, indicating the requirement for these polysaccharides in GVIN1-dependent bacterial sensing. However, the authors have not investigated whether GVIN1, which has two GTPase-domains, does indeed have GTPase activity and whether GVIN1 and GBP1 together completely block cell-to-cell spread by Burkholderia and thereby restrict bacterial numbers over the infection time course. They also do not show whether GBP1 and GVIN1 target the same bacterial cell or different populations of bacteria.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

This work uncovers the antimicrobial actions of a member of yet another family of IFNg-induced GTPases, which potentially acts against other intracellular pathogens. GVIN1 appears to operate independently and in parallel to GBP1, pointing to the breadth and complexity of the IFNg-inducible GTPase families.

Reviewer #2 (Public review):

This observational study investigates the efficacy of intracameral injected human stems cells as a means to re-functionalize the trabecular meshwork for the restoration of intraocular pressure homeostasis. Using a murine model of glaucoma, human adipose-derived mesenchymal stem cells are shown to be biologically safer and functionally superior at eliciting a sustained reduction in intraocular pressure (IOP). The authors conclude that the use of magnetically-steered human adipose-derived mesenchymal stem cells has potential for long-term treatment of ocular hypertension in glaucoma.

Comments on revisions: Previously noted concerns have been thoughtfully and sincerely considered by the authors and are now clearly addressed in the revised manuscript. No further concerns/comments.

Reviewer #2 (Public review):

Summary:

The present study sets out to examine the impact of counterconditioning (CC) and extinction on conditioned threat responses in humans, particularly looking at neural mechanisms involved in threat memory suppression. By combining behavioral, physiological, and neuroimaging (fMRI) data, the authors aim to provide a clear picture of how CC might engage unique neural circuits and coding dynamics, potentially offering a more robust reduction in threat responses compared to traditional extinction.

Strengths:

One major strength of this work lies in its thoughtful and unique design - integrating subjective, physiological, and neuroimaging measures to capture the variouse aspects of counterconditiong (CC) in humans. Additionally, the study is centered on a well-motivated hypothesis and the findings have potentials for improving the current understanding of pathways associated with emotional and cognitive control.

The data presentation is systematic, and the results on behavioral and physiological measures fit well with the hypothesized outcomes. The neuroimaging results also provide strong support for distinct neural mechanisms underlying CC versus extinction.

Weaknesses:

Overall, this study is a well-conducted and thought-provoking investigation into counterconditioning, with strong potential to advance our understanding of threat modulation mechanisms. Two minor weaknesses concern the scope and decisions regarding analysis choices. First, while the findings are solid, the topic of counterconditioning is relatively niche and may have limited appeal to a broader audience. Expanding the discussion to connect counterconditioning more explicitly to widely studied frameworks in emotional regulation or cognitive control would enhance the paper's accessibility and relevance to a wider range of readers. This broader framing could also underscore the generalizability and broader significance of the results. In addition, detailed steps in the statistical procedures and analysis parameters seem to be missing. This makes it challenging for readers to interpret the results in light of potential limitations given the data modality and/or analysis choices.

Comments on revisions: My previous concerns and questions have been sufficiently addressed.

Reviewer #2 (Public review):

Summary:

This manuscript presents a new approach for non-invasive, MRI-based, measurements of cerebral blood volume (CBV). Here, the authors use ferumoxytol, a high-contrast agent and apply specific sequences to infer CBV. The authors then move to statistically compare measured regional CBV with known distribution of different types of neurons, markers of metabolic load and others. While the presented methodology captures and estimated 30% of the vasculature, the authors corroborated previous findings regarding lack of vascular compartmentalization around functional neuronal units in the primary visual cortex.

Strengths:

Non-invasive methodology geared to map vascular properties in vivo.

Implementation of a highly sensitive approach for measuring blood volume.

Ability to map vascular structural and functional vascular metrics to other types of published data.

Weaknesses:

The key issue here is the underlying assumption about the appropriate spatial sampling frequency needed to captures the architecture of the brain vasculature. Namely, ~7 penetrating vessels / mm2 as derived from Weber et al 2008 (Cer Cor). The cited work, begins by characterizing the spacing of penetrating arteries and ascending veins using vascular cast of 7 monkeys (Macaca mulatta, same as in the current paper). The ~7 penetrating vessels / mm2 is computed by dividing the total number of identified vessels by the area imaged. The problem here is that all measurements were made in a "non-volumetric" manner and only in V1. Extrapolating from here to other brain areas is therefore not possible without further exploration with independent methodologies.

Please note that these are comments on the revised version.

Reviewer #2 (Public review):

Summary:

The authors generated three mouse lines harboring ASD, Schizophrenia, and Bi-polar-associated variants in the TRIO gene. Anatomical, behavioral, physiological, and biochemical assays were deployed to compare and contrast the impact of these mutations in these animals. In this undertaking the authors sought to identify and characterize the cellular and molecular mechanisms responsible for ASD, Schizophrenia, and Bi-polar disorder development.

Strengths:

The establishment of TRIO dysfunction in the development of ASD, Schizophrenia, and Bi-polar disorder is very recent and of great interest. Disorder-specific variants have been identified in the TRIO gene, and this study is the first to compare and contrast the impact of these variants in vivo in preclinical models. The impact of these mutations was carefully examined using an impressive host of methods. The authors achieved their goal of identifying behavioral, physiological, and molecular alterations that are disorder/variant specific. The impact of this work is extremely high given the growing appreciation of TRIO dysfunction in a large number of brain-related disorders. This work is very interesting in that it begins to identify the unique and subtle ways brain function is altered in ASD, Schizophrenia, and Bi-polar disorder.

Weaknesses:

(1) Most assays were performed in older animals and perhaps only capture alterations that result from homeostatic changes resulting from prodromal pathology that may look very different.

(2) Identification of upregulated (potentially compensating) genes in response to these disorder specific Trio variants is extremely interesting. However, a functional demonstration of compensation is not provided.

(3) There are instances where data is not shown in the manuscript. See "data not shown". All data collected should be provided even if significant differences are not observed.

I consider weaknesses 1 and 2 minor. While they would very interesting to explore, these experiments might be more appropriate for a follow up study. The missing data in 3 should be provided in the supplemental material.

Revised Manuscript:

All of my above concerns were well addressed by the authors in the revised submission.

Reviewer #2 (Public review):

Summary:

This study examines the role of TRPV1 signaling in the recruitment of monocyte-derived macrophages and the promotion of angiogenesis during tympanic membrane (TM) wound healing. The authors use a combination of genetic mouse models, macrophage depletion, and transcriptomic approaches to suggest that neuronal TRPV1 activity contributes to macrophage-driven vascular responses necessary for tissue repair.

Strengths:

(1) The topic of neuroimmune interactions in tissue regeneration is of interest and underexplored in the context of the TM, which presents a unique model due to its anatomical features.

(2) The use of reporter mice and bone marrow chimeras allows for some dissection of immune cell origin.

(3) The authors incorporate transcriptomic data to contextualize inflammatory and angiogenic processes during wound healing.

Weaknesses:

(1) The primary claims of the manuscript are not convincingly supported by the evidence presented. Most of the data are correlative in nature, and no direct mechanistic experiments are included to establish causality between TRPV1 signaling and macrophage recruitment or function.

(2) Functional validation of key molecular players (such as Tac1 or Spp1) is lacking, and their roles are inferred primarily from gene expression data rather than experimentally tested.

(3) The reuse of publicly available scRNA-seq data is not sufficiently integrated or extended to yield new biological insights, and it remains largely descriptive.

(4) The macrophage depletion model (CX3CR1CreER; iDTR) lacks specificity, and possible off-target or systemic effects are not addressed.

(5) Several interpretations of the data appear overstated, particularly regarding the necessity of TRPV1 for monocyte recruitment and wound healing.

(6) Overall, the study appears to apply known concepts - namely, TRPV1-mediated neurogenic inflammation and macrophage-driven angiogenesis - to a new anatomical site without providing new mechanistic insight or advancing the field substantially.

Overall:

While the study addresses an interesting topic, the current version does not provide sufficiently strong or novel evidence to support its major conclusions. Additional mechanistic experiments and more rigorous validation would be necessary to substantiate the proposed model and clarify the relevance of the findings beyond this specific tissue context.

Reviewer #2 (Public review):

Summary:

The current study aims to shed light on why previous work on perceptual rhythmicity has led to inconsistent results. They propose that the differences may stem from conceptual and methodological issues. In a series of experiments, the current study reports perceptual rhythmicity in different frequency bands that differ between different ear stimulations and behavioral measures. The study suggests challenges regarding the idea of universal perceptual rhythmicity in hearing.

Strengths:

The study aims to address differences observed in previous studies about perceptual rhythmicity. This is important and timely because the existing literature provides quite inconsistent findings. Several experiments were conducted to assess perceptual rhythmicity in hearing from different angles. The authors use sophisticated approaches to address the research questions.

Weaknesses:

(1) Conceptional concerns:

The authors place their research in the context of a rhythmic mode of perception. They also discuss continuous vs rhythmic mode processing. Their study further follows a design that seems to be based on paradigms that assume a recent phase in neural oscillations that subsequently influence perception (e.g., Fiebelkorn et al.; Landau & Fries). In my view, these are different facets in the neural oscillation research space that require a bit more nuanced separation. Continuous mode processing is associated with vigilance tasks (work by Schroeder and Lakatos; reduction of low frequency oscillations and sustained gamma activity), whereas the authors of this study seem to link it to hearing tasks specifically (e.g., line 694). Rhythmic mode processing is associated with rhythmic stimulation by which neural oscillations entrain and influence perception (also, Schroeder and Lakatos; greater low-frequency fluctuations and more rhythmic gamma activity). The current study mirrors the continuous rather than the rhythmic mode (i.e., there was no rhythmic stimulation), but even the former seems not fully fitting, because trials are 1.8 s short and do not really reflect a vigilance task. Finally, previous paradigms on phase-resetting reflect more closely the design of the current study (i.e., different times of a target stimulus relative to the reset of an oscillation). This is the work by Fiebelkorn et al., Landau & Fries, and others, which do not seem to be cited here, which I find surprising. Moreover, the authors would want to discuss the role of the background noise in resetting the phase of an oscillation, and the role of the fixation cross also possibly resetting the phase of an oscillation. Regardless, the conceptional mixture of all these facets makes interpretations really challenging. The phase-reset nature of the paradigm is not (or not well) explained, and the discussion mixes the different concepts and approaches. I recommend that the authors frame their work more clearly in the context of these different concepts (affecting large portions of the manuscript).

(2) Methodological concerns:

The authors use a relatively unorthodox approach to statistical testing. I understand that they try to capture and characterize the sensitivity of the different analysis approaches to rhythmic behavioral effects. However, it is a bit unclear what meaningful effects are in the study. For example, the bootstrapping approach that identifies the percentage of significant variations of sample selections is rather descriptive (Figures 5-7). The authors seem to suggest that 50% of the samples are meaningful (given the dashed line in the figure), even though this is rarely reached in any of the analyses. Perhaps >80% of samples should show a significant effect to be meaningful (at least to my subjective mind). To me, the low percentage rather suggests that there is not too much meaningful rhythmicity present. I suggest that the authors also present more traditional, perhaps multi-level, analyses: Calculation of spectra, binning, or single-trial analysis for each participant and condition, and the respective calculation of the surrogate data analysis, and then comparison of the surrogate data to the original data on the second (participant) level using t-tests. I also thought the statistical approach undertaken here could have been a bit more clearly/didactically described as well.

The authors used an adaptive procedure during the experimental blocks such that the stimulus intensity was adjusted throughout. In practice, this can be a disadvantage relative to keeping the intensity constant throughout, because, on average, correct trials will be associated with a higher intensity than incorrect trials, potentially making observations of perceptual rhythmicity more challenging. The authors would want to discuss this potential issue. Intensity adjustments could perhaps contribute to the observed rhythmicity effects. Perhaps the rhythmicity of the stimulus intensity could be analyzed as well. In any case, the adaptive procedure may add variance to the data.

Additional methodological concerns relate to Figure 8. Figures 8A and C seem to indicate that a baseline correction for a very short time window was calculated (I could not find anything about this in the methods section). The data seem very variable and artificially constrained in the baseline time window. It was unclear what the reader might take from Figure 8.

Motivation and discussion of eye-movement/pupillometry and motor activity: The dual task paradigm of Experiment 4 and the reasons for assessing eye metrics in the current study could have been better motivated. The experiment somehow does not fit in very well. There is recent evidence that eye movements decrease during effortful tasks (e.g., Contadini-Wright et al. 2023 J Neurosci; Herrmann & Ryan 2024 J Cog Neurosci), which appears to contradict the results presented in the current study. Moreover, by appealing to active sensing frameworks, the authors suggest that active movements can facilitate listening outcomes (line 677; they should provide a reference for this claim), but it is unclear how this would relate to eye movements. Certainly, a person may move their head closer to a sound source in the presence of competing sound to increase the signal-to-noise ratio, but this is not really the active movements that are measured here. A more detailed discussion may be important. The authors further frame the difference between Experiments 1 and 2 as being related to participants' motor activity. However, there are other factors that could explain differences between experiments. Self-paced trials give participants the opportunity to rest more (inter-trial durations were likely longer in Experiment 2), perhaps affecting attentional engagement. I think a more nuanced discussion may be warranted.

Discussion:

The main data in Figure 3 showed little rhythmicity. The authors seem to glance over this fact by simply stating that the same phase is not necessary for their statistical analysis. Previous work, however, showed rhythmicity in the across-participant average (e.g., Fiebelkorn's and similar work). Moreover, one would expect that some of the effects in the low-frequency band (e.g., 2-4 Hz) are somewhat similar across participants. Conduction delays in the auditory system are much smaller than the 0.25-0.5 s associated with 2-4 Hz. The authors would want to discuss why different participants would express so vastly different phases that the across-participant average does not show any rhythmicity, and what this would mean neurophysiologically.

An additional point that may require more nuanced discussion is related to the rhythmicity of response bias versus sensitivity. The authors could discuss what the rhythmicity of these different measures in different frequency bands means, with respect to underlying neural oscillations.

Figures:

Much of the text in the figures seems really small. Perhaps the authors would want to ensure it is readable even for those with low vision abilities. Moreover, Figure 1A is not as intuitive as it could be and may perhaps be made clearer. I also suggest the authors discuss a bit more the potential monoaural vs binaural issues, because the perceptual rhythmicity is much slower than any conduction delays in the auditory system that could lead to interference.

Reviewer #2 (Public review):

Summary:

This paper investigates the evolution of pesticide resistance in the two-spotted spider mite following the introduction of an SDHI acaricide, cyatpyrafen, in China. The authors make use of cyatpyrafen-naive populations collected before that pesticide was first used, as well as more recent populations (both sensitive and resistant) to conduct comparative population genomics. They report 15 different mutations in the insecticide target site from resistant populations, many reported here for the first time, and look at the mutation and selection processes underlying the evolution of resistance, through GWAS, haplotype mapping, and testing for loss of diversity indicating selective sweeps. None of the target site mutations found in resistant populations was found in pre-exposure populations, suggesting that the mutations may have arisen de novo rather than being present as standing variation, unless initially present at very low frequencies; a de novo origin is also supported by evidence of selective sweeps in some resistant populations. Furthermore, there is no significant evidence of migration of resistant genotypes between the sampled field populations, indicating multiple origins of common mutations. Overall, this indicates a very high mutation rate and a wide range of mutational pathways to resistance for this target site in this pest species. The series of population genomic analyses carried out here, in addition to the evolutionary processes that appear to underlie resistance development in this case, could have implications for the study of resistance evolution more widely.

Strengths:

This paper combines phenotypic characterisation with extensive comparative population genomics, made possible by the availability of multiple population samples (each with hundreds of individuals) collected before as well as after the introduction of the pesticide cyatpyrafen, as well as lab-evolved lines. This results in findings of mutation and selection processes that can be related back to the pesticide resistance trait of concern. Large numbers of mites were tested phenotypically to show the levels of resistance present, and the authors also made near-isogenic lines to confirm the phenotypic effects of key mutations. The population genomic analyses consider a range of alternative hypotheses, including mutations arising by de novo mutation or selection from standing genetic variation, and mutations in different populations arising independently or arriving by migration. The claim that mutations most likley arose by multiple repeated de novo mutations is therefore supported by multiple lines of evidence: the direct evidence of none of the mutations being found in over 2000 individuals from naive populations, and the indirect evidence from population genomics showing evidence of selective sweeps but not of significant migration between the sampled populations.

Weaknesses:

As acknowledged within the discussion, whilst evidence supports a de novo origin of the resistance-associated mutations, this cannot be proven definitively as mutations may have been present at a very low frequency and therefore not found within the tested pesticide-naive population samples.

Near-isofemale lines were made to confirm the resistance levels associated with five of the 15 mutations, but otherwise, the genotype-phenotype associations are correlative, as confirmation by functional genetics was beyond the scope of this study.

Reviewer #2 (Public review):

Summary:

This study focuses on Orf9b, a SARS-COV1/2 protein that regulates innate signaling through interaction with Tom70. San Felipe et al use a combination of biophysical methods to characterize the coupling between lipid-binding, dimerization, conformational change, and protein-protein-interaction equilibria for the Orf9b-Tom70 system. Their analysis provides a detailed explanation for previous observations of Orf9b function. In a cellular context, they find other factors may also be important for the biological functioning of Orf9b.

Strengths:

San Felipe et al elegantly combine structural biology, biophysics, kinetic modelling, and cellular assays, allowing detailed analysis of the Orf9b-Tom70 system. Such complex systems involving coupled equilibria are prevalent in various aspects of biology, and a quantitative description of them, while challenging, provides a detailed understanding and prediction of biological outcomes. Using SPR to guide initial estimates of the rate constants for solution measurements is an interesting approach.

Weaknesses:

This study would benefit from a more quantitative description of uncertainties in the numerous rate constants of the models, either through a detailed presentation of the sensitivity analysis or another approach such as MCMC. Quantitative uncertainty analysis, such as MCMC is not trivial for ODEs, particularly when they involve many parameters and are to be fitted to numerous data points, as is the case for this study. The authors use sensitivity analysis as an alternative, however, the results of the sensitivity analysis are not presented in detail, and I believe the authors should consider whether there is a way to present this analysis more quantitatively. For example, could the residuals for each +/-10% parameter change for the peptide model be presented as a supplementary figure, and similarly for the more complex models? Further details of the range of rate constants tested would be useful, particularly for the ka and kB parameters.

The authors build a model that incorporates an α-helix-β-sheet conformational change, but the rate constant for the conversion to the α-helix conformation is required to be second order. Although the authors provide some rationale, I do not find this satisfactorily convincing given the large number of adjustable parameters in the model and the use of manual model fitting. The authors should discuss whether there is any precedence for second-order rate constants for conformational changes in the literature. On page 14, the authors state this rate constant "had to be non-linear in the monomer β-sheet concentration" - how many other models did the authors explore? For example, would αT↔α↔αα↔ββ (i.e., conformational change before dimer dissociation) or α↔βαT↔ββ (i.e., Tom70 binding driving dimer dissociation) be other plausible models for the conformational change that do not require assumptions of second-order rate constants for the conformational change?

Overall, this study progresses the analysis of coupled equilibria and provides insights into Orf9b function.

Reviewer #2 (Public review):

Summary:

The authors were to investigate the functional role of IL10 on mucosal immunity in chickens. CRISPR technology was employed to generate IL10 knock-out chickens in both exon and putative enhancer regions. IL10 expressions were either abolished (knockout in exon) or reduced (enhancer knock-out). IL-10 plays an important role in the composition of the caecal microbiome. Through various enteric pathogen challenges, deficient IL10 expression was associated with enhanced pathogen clearance, but with more severe lesion scores and body weight loss.

Strengths:

Both in vitro and in vivo knock-out abolished and reduced IL10 expression, and broad enteric pathogens were challenged in vivo, and various parameters were examined to evaluate the functional role of IL10 on mucosal immunity.

Weaknesses:

Overexpression of IL-10 either in vitro or in vivo may further support the findings from this study.

Reviewer #2 (Public review):

Summary:

How corticostriatal synaptic connectivity gives rise to SPN encoding of sensory information is an important and currently unanswered question. The authors utilize a clever slice preparation in combination with electrophysiology and glutamate uncaging to dissect the synaptic connectivity between barrel cortex and individual striatal SPNs. In addition to mapping connectivity across major anatomical axes and cortical layers, the authors provide data showing that SPNs uniquely integrate sparse input from variable stretches across barrel cortex.

Strengths:

The methodology shows impressive rigor, and the data robustly support the authors' conclusions. Overall, the manuscript addresses its core question, provides valuable insights into corticostriatal architecture, and is a welcome addition to the field.

Weaknesses:

A few minor changes to the figures and text could be made to improve clarity.

Reviewer #2 (Public review):

Summary:

The authors attempt to estimate the heritability of brain activity evoked from a naturalistic fMRI paradigm. No new data were collected; the authors analyzed the publicly available and well-known data from the Human Connectome Project. The paper has 3 main pieces, as described in the Abstract:

(1) Heritability of movie-evoked brain activity and connectivity patterns across the cortex.

(2) Decomposition of this heritability into genetic similarity in "where" vs. "how" sensory information is processed.

(3) Heritability of brain activity patterns, as partially explained by the heritability of neural timescales.

Strengths:

The authors investigate a very relevant topic that concerns how heritable patterns of brain activity among individuals subjected to the same kind of naturalistic stimulation are. Notably, the authors complement their analysis of movie-watching data with resting-state data.

Weaknesses:

The paper has numerous problems, most of which stem from the statistical analyses. I also note the lack of mapping between the subsections within the Methods section and the subsections within the Results section. We can only assess results after understanding and confirming the methods are valid; here, however, Methods and Results, as written, are not aligned, so we can't always be sure which results are coming from which analysis.

(A) Intersubject correlation (ISC) (section that starts from line 143): "We used non-parametric permutation testing to quantify average differences in ISC for each parcel in the Schaefer 400 atlas for each day of data collection across three groups: MZ dyads, DZ dyads, and unrelated (UR) dyads, where all UR dyads were matched for gender and age in years." ... "some participants contributed to ISC values for multiple dyads (thus violating independence assumptions)"

This is an indirect attempt to demonstrate heritability. And it's also incorrect since, as the authors themselves point out, some subjects contribute to more than one dyad.

Permutation tests don't quantify "average differences", they provide a measure of evidence about whether differences observed are sufficient to reject a hypothesis of no difference.

Matching subjects is also incorrect as it artificially alters the sample; covarying for age and sex, as done in standard analyses of heritability, would have been appropriate.

It isn't clear why the authors went through the trouble of implementing their own non-parametric test if HCP recommends using PALM, which already contains the validated and documented methods for permutation tests developed precisely for HCP data.

The results from this analysis, in their current form, are likely incorrect.

(B) Functional connectivity (FC) (section that starts from line 159): Here the authors compute two 400x400 FC matrix for each subject, one for rest, one for movie-watching, then correlate the correlations within each dyad, then compared the average correlation of correlations for MZ, DZ, and UR. In addition to the same problems as the previous analysis, here it is not clear what is meant by "averaging correlations [...] within a network combination". What is a "network combination"? Further, to average correlations, they need to be r-to-z transformed first. As with the above, the results from this analysis in its current form are likely incorrect.

(C) ISC and FC profile heritability analyses (section that starts from line 175): Here, the authors use first a valid method remarkably similar to the old Haseman-Elston approach to compute heritability, complemented by a permutation test. That is fine. But then they proceed with two novel, ill-described, and likely invalid methods to (1) "compare the heritability of movie and rest FC profiles" and (2) to "determine the sample size necessary for stable multidimensional heritability results". For (1), they permute, seemingly under the alternative, rest and movie-watching timeseries, and (2), by dropping subjects and estimating changes in the distribution.

The (1) might be correct, but there are items that are not clearly described, so the reader cannot be sure of what was done. What are the "153 unique network combinations"? Why do the authors separate by day here, whereas the previous analyses concatenated both days? Were the correlations r-to-z transformed before averaging?

The (2) is also not well described, and in any case, power can be computed analytically; it isn't clear why the authors needed to resort to this ad hoc approach, the validity of which is unknown. If the issue is the possibility that the multidimensional phenotypic correlation matrix is rank-deficient, it suffices that there are more independent measurements per subject than the number of subjects.

(D) Frequency-dependent ISC heritability analysis (from line 216): Here, the authors decompose the timeseries into frequency bands, then repeat earlier analyses, thus bringing here the same earlier problems and questions of non-exchangability in the permutations given the dyads pattern, r-z transforms, and sex/age covariates.

(E) FC strength heritability analysis (from line 236): Here, the authors use the univariate FC to compute heritability using valid and well-established methods as implemented in SOLAR. There is no "linkage" being done here (thus, the statement in line 238 is incorrect in this application. SOLAR already produces SEs, so it's unclear why the authors went out of their way to obtain jackknife estimates. If the issue is non-normality, I note that the assumption of normality is present already at the stage in which parameters themselves are estimated, not just the standard errors; for non-normal data, a rank-based inverse-normal transformation could have been used. Moreover, typically, r-to-z transformed values tend to be fairly normally distributed. So, while the heritabilities might be correct, the standard errors may not be (the authors don't demonstrate that their jackknife SE estimator is valid). The comparison of h2 between dyads raises the same questions about permutations, age/sex covariates, and r-z transforms as above.

(F) Hyperalignment (from line 245): It isn't clear at this point in the manuscript in what way hyperalignment would help to decompose heritability in "where vs. how" (from the Abstract). That information and references are only described much later, from around line 459. The description itself provides no references, and one cannot even try to reproduce what is described here in the Methods section. Regardless, it isn't entirely clear why this analysis was done: by matching functional areas, all heritabilities are going to be reduced because there will be less variance between subjects. Perhaps studying the parameters that drive the alignment (akin to what is done in tensor-based and deformation-based morphometry) could have been more informative. Plus, the alignment process itself may introduce errors, which could also reduce heritability. This could be an alternative explanation for the reduced heritability after hyperalignment and should be discussed. An investigation of hyperaligment parameters, their heritability, and their co-heritability with the BOLD-phenotypes can inform on this.

(G) Relationships between parcel area and heritability (from line 270): As under F), how much the results are distorted likely depends on the accuracy of the alignment, and the error variance (vs heritable variance) introduced by this.

(H) Neural timescale analyses (from line 280): Here, a valid phenotype (NT) is assessed with statistical methods with the same limitations as those previously (exchangability of dyads, age/sex covariates, and r-z transforms). NT values are combined across space and used as covariates in "some multivariate analyses". As a reader, I really wanted to see the results related to NT, something as simple as its heritability, but these aren't clearly shown, only differences between types of dyads.

(I) Significance testing for autocorrelated brain maps and FC matrices (from line 310): Here, the authors suddenly bring up something entirely different: reliability of heritability maps, and then never return to the topic of reliability again. As a reader, I find this confusing. In any case, analyses with BrainSMASH with well-behaved, normally distributed data are ok. Whether their data is well behaved or whether they ensured that the data would be well behaved so that BrainSMASH is valid is not described. As to why Spearman correlations are needed here, Mantel tests, or whether the 1000 "surrogate" maps are valid realizations of the data under the null, remains undemonstrated.

(J) Global signal was removed, and the authors do not acknowledge that this could be a limitation in their analyses, nor offer a side analysis in which the global signal is preserved.

(K) FDR is used to control the error rate, but in many cases, as it's applied to multiple sets of p-values, the amount of false discoveries is only controlled across all tests, but not within each set. The number of errors within any set remains unknown.

(L) Generally, when studying the heritability of a trait, the trait must be defined first. Here, multiple traits are investigated, but are never rigorously defined. Worse, the trait being analyzed changes at every turn.

Reviewer #2 (Public review):

Summary:

This study shows that when excitatory DREADD receptors are expressed in the ventral area of the cervical spinal cord containing phrenic motoneurons, systemic administration of the DREADD ligand J60 increases diaphragm EMG activity without altering respiratory rate. The authors took a non-selective expression approach in wild-type mice, as well as a more selective Cre-dependent approach in Chat-Cre mice and Chat-Cre rats to stimulate cervical motoneurons in the spinal cord. This is a proof of principle study that supports the use of DREADD technology to stimulate the motor output to the diaphragm.

Strengths:

The strengths of the study lie in the use of both mice and rats to test whether the chomogenetic activation of phrenic motoneurons with multiple experimental approaches increases diaphragm EMG activity (both tonic and phasic) and tidal volume.

Comments on revisions:

Thanks for addressing my comments. One last comment that could be discussed or addressed is :

Line 295- was the time post-infection, which varies considerably between groups and across samples, taken into consideration when comparison of response was made between ChatCre mice (4-9 weeks post-infection) and WT mice (four to five weeks post-infection)?

Reviewer #1 (Public review):

Summary:

This study provides an in-depth analysis of syncytiotrophoblast (STB) gene expression at the single-nucleus (SN) and single-cell (SC) levels, using both primary human placental tissues and two trophoblast organoid (TO) models. The authors compare the older TO model, where STB forms internally (STBin), with a newer model where STB forms externally (STBout). Through a series of comparative analyses, the study highlights the necessity of using both SN and SC techniques to fully understand placental biology. The findings demonstrate that the STBout model shows more differentiated STBs with higher expression of canonical markers and hormones compared to STBin. Additionally, the study identifies both conserved and distinct gene expression profiles between the TO models and human placenta, offering valuable insights for researchers using TOs to study STB and CTB differentiation.

Strengths:

The study offers a comprehensive SC- and SN-based characterization of trophoblast organoid models, providing a thorough validation of these models against human placental tissues. By comparing the older STBin and newer STBout models, the authors effectively demonstrate the improvements in the latter, particularly in the differentiation and gene expression profiles of STBs. This work serves as a critical resource for researchers, offering a clear delineation of the similarities and differences between TO-derived and primary STBs. The use of multiple advanced techniques, such as high-resolution sequencing and trajectory analysis, further enhances the study's contribution to the field.

Weaknesses were addressed during the revision.

The authors effectively addressed my critiques in the rebuttal letter and made corresponding changes in the manuscript. Specifically, they: 1) emphasized the importance of TO orientation in influencing STB nuclear subtype differentiation by adding text to the introduction; 2) clarified the differences in cluster numbers and names between primary tissue and TO data, explaining that each dataset was analyzed independently with separate clustering algorithms and adding clarifying text to the results section; 3) included additional rationale for using SN over SC sequencing, particularly for studying the multinucleated STB; 4) acknowledged that their original evidence was insufficient to definitively determine STBout nuclei differentiation status and removed language suggesting STB-3 as a terminally differentiated subtype, presenting alternative hypotheses in the discussion; and 5) incorporated new figures and clarifications, including RNA-FISH experiments, to validate subtype-specific marker gene expression. Overall, the authors' revisions strengthened the manuscript and aligned well with my critiques.

Reviewer #2 (Public review):

Summary:

This article presents a study on a mutant form of RNA polymerase I (RNAPI) in yeast, referred to as SuperPol, which demonstrates increased rRNA production compared to the wild-type enzyme. While rRNA production levels are elevated in the mutant, RNAPI occupancy as detected by CRAC is reduced at the 5' end of rDNA transcription units. The authors interpret these findings by proposing that the wild-type RNAPI pauses in the external transcribed spacer (ETS), leading to premature transcription termination (PTT) and degradation of truncated rRNAs by the RNA exosome (Rrp6). They further show that SuperPol's enhanced activity is linked to a lower frequency of PTT events, likely due to altered elongation dynamics and reduced RNA cleavage activity, as supported by both in vivo and in vitro data.

The study also examines the impact of BMH-21, a drug known to inhibit Pol I elongation, and shows that SuperPol is less sensitive to this drug, as demonstrated through genetic, biochemical, and in vivo approaches. The authors show that BMH-21 treatment induces premature termination in wild-type Pol I, but only to a lesser extent in SuperPol. They suggest that BMH-21 promotes termination by targeting paused Pol I complexes and propose that PTT is an important regulatory mechanism for rRNA production in yeast.

The data presented are of high quality and support the notion that 1) premature transcription termination occurs at the 5' end of rDNA transcription units; 2) SuperPol has an increased elongation rate with reduced premature termination; and 3) BMH-21 promotes both pausing and termination. The authors employ several complementary methods, including in vitro transcription assays. These results are significant and of interest for a broad audience.

Beyond the minor points listed below, my main criticism concerns the interpretation of data in relation to termination. While it is possible that the SuperPol mutation affects the wild-type Pol I's natural propensity for termination, it is also possible that premature termination is simply a consequence of natural or BMH-21-induced Pol I pausing. SuperPol may elongate more efficiently than the wild-type enzyme, pause less frequently, and thus terminate less often. In this light, the notion that termination "regulates" rRNA production might be an overstatement, with pausing as the primary event. Claiming a direct effect on termination by both the mutation and BMH-21 would require showing that with equivalent levels of pausing, termination occurs more or less efficiently, which would be challenging and should not be expected in this study. The authors address this point in the last two paragraphs of the discussion. My suggestion is to temper the claims regarding termination as a regulatory mechanism.

Significance:

These results are significant and of interest for a basic research audience.

Reviewer #3 (Public review):

In the present study, the author revealed that cardiomyocyte-specific deletion of mouse AARS2 exhibited evident cardiomyopathy with impaired cardiac function, notable cardiac fibrosis, and cardiomyocyte apoptosis. Cardiomyocyte-specific AARS2 overexpression in mice improved cardiac function and reduced cardiac fibrosis after myocardial infarction (MI), without affecting cardiomyocyte proliferation and coronary angiogenesis. Mechanistically, AARS2 overexpression suppressed cardiomyocyte apoptosis and mitochondrial reactive oxide species production, and changed cellular metabolism from oxidative phosphorylation toward glycolysis in cardiomyocytes, thus leading to cardiomyocyte survival from ischemia and hypoxia stress. Ribo-Seq revealed that AARS2 overexpression increased pyruvate kinase M2 (PKM2) protein translation and the ratio of PKM2 dimers to tetramers that promote glycolysis. Additionally, PKM2 activator TEPP-46 reversed cardiomyocyte apoptosis and cardiac fibrosis caused by AARS2 deficiency. Thus, this study demonstrates that AARS2 plays an essential role in protecting cardiomyocytes from ischemic pressure via fine-tuning PKM2-mediated energy metabolism, and presents a novel cardiac protective AARS2-PKM2 signaling during the pathogenesis of MI.

Comments on revised version:

The authors addressed all the issues, no more comments.

Reviewer #2 (Public review):

This work by Manley and Vaziri identify brain networks that are associated with trial-to-trial variability during prey-capture and predator avoidance behaviors. However, mixing of signals across space and time make it difficult to interpret the data generated and relate the data to findings from prior work.

Comments on revisions:'

In their response to prior reviewer comments, Manley and Vaziri have now provided helpful methodological clarity and additional analyses. The additional work makes clear that the claims of variability and mixing of sensory, motor, and internal variables at the single-cell level are not well supported.

RESOLUTION<br /> - The new information provided regarding resolution may not be very relevant as this was from an experiment in air. It would be much more informative to show how PSF degrades in the brain with depth.

DEPTH<br /> - It is helpful to see the registered light-field and confocal images. Both appear to provide poor or little information in regions >200 below the surface (like the hypothalamus), making the claim that whole brain data is being collected at cellular resolution difficult to justify.

MERGING<br /> - The typical soma at these ages has a radius of 2.5 microns, which corresponds to a volume of 65 microns^3. Given the close packing of most cells, this means that a typical ROI of 750 microns^3 contains more than 10 neurons. Therefore, the authors should not claim they are reporting activity at cellular resolution.<br /> - Furthermore, the fact that these ROIs contains tens of cells brings into question the degree of variability at the single-cell level. For example, if every cluster of 10 cells has one variable cell, then all clusters might be labeled as exhibiting variability even though only 10% of the cells show variability.

SLOW CALCIUM DYNAMICS<br /> - Convolution/Deconvolution with the inappropriate kernel both have problems, some of which the authors have noted. However, by not deconvolving, the authors are significantly obscuring the interpretation of their data by mixing together signals across time.<br /> - Also, the claim that "neurons highly tuned to a particular stimulus exhibited variability in their responses across multiple presentations of the same stimuli" should be clarified or qualified. It is not clear from what has been shown if the responses are indeed variable, or rather if there is additional activity (or apparent activity) occasionally present that shifts the pre-stimulus baseline around (for example, 3J suggests that in many cases the visual signal from the prior trial is still present when a new trial begins).<br /> - Figure 3A should show when the stimulus occurs, should show some of the prestimulus period, and ideally be off-set corrected so all traces in a given panel start at the same y-value at the beginning of the stimulus period.

ORTHOGONALITY<br /> - It is now clearer that the visual signal and noise vectors were determined for the entire time series with all trials. Therefore, the concern that sources of activation in advance of a given trial were being ignored is alleviated. The concern remains, however, that these sources are being properly accounted for given potential kernel variations and nonlinearity. Nonetheless, it is recognized that the GCaMP filtering most likely would lead to a decrease in the disparity between two populations.<br /> - The authors' clarification that the analyzed ROIs consist of cell clusters raises the trivial possibility that the observed orthogonality between the visual signal and leading noise vectors is explained by noise simply reflecting the activation of different motor or motor-planning related neurons in an ROI, neurons that are separate from visually-encoding neurons in the same cluster.

SOURCES of VARIABILITY<br /> - The data presented in Supplemental Figure 3Ei actually is suggestive that eye movements are a significant contributor to the reported variability. Notice how in (1 4) vs (1 5) and (4 7) vs (4 8) there is a notable difference in the distribution of responses. Adding eye kinematic variables to the analysis of Figure S4 could be clarifying.-

Reviewer #2 (Public review):

Summary:

This manuscript describes a workflow and software package, SMARTR, for mapping and analyzing neuronal ensembles tagged using activity-dependent methods. They showcase this pipeline by analyzing ensembles tagged during the learned helplessness paradigm. This is an impressive effort, and I commend the authors for developing open-source software to make whole-brain analyses more feasible for the community. After peer-review, the authors addressed reviewer suggestions and concerns regarding the usability and maintainability of the SMARTTR package, ensuring that the package will be published on CRAN, improving documentation, and including unit tests to ensure code stability. Overall, this software package will prove to have a broad impact on the field.

Reviewer #2 (Public review):

Summary:

This paper investigates the general concept that avian and mammalian pallium specifications share similar mechanisms. To explore that idea, the authors focus their attention on the role of miR-19b as a key controlling factor in the neuronal proliferation/differentiation balance. To do so, the authors checked the expression and protein level of several genes involved in neuronal differentiation, such as NeuroD1 or E2f8, genes also expressed in mammals after conducting their functional gene manipulation experiments. The work also shows a dysregulation in the number of neurons from lower and upper layers when miR-19b expression is altered.

To test it, the authors conducted a series of functional experiments of gain and loss of function (G&LoF) and enhancer-reporter assays. The enhancer-reporter assays demonstrate a direct relationship between miR-19b and NeuroD1 and E2f8 which is also validated by the G&LoF experiments. It´s also noteworthy to mention that the way miR-19b acts is maintaining the progenitor cells from the ventricular zone in an undifferentiated stage, thus promoting them into a stage of cellular division.

Overall, the paper argues that the expression of miR-19b in the ventricular zone promotes the cells in a proliferative phase and inhibits the expression of differentiation genes such as E2f8 and NeurD1. The authors claim that a decrease in the progenitor cell pool leads to an increase and decrease in neurons in the lower and upper layers, respectively.

Strengths:

(1) Novelty Contribution<br /> The paper offers strong arguments to prove that the neurodevelopmental basis between mammals and birds is quite the same. Moreover, this work contributes to a better understanding of brain evolution along the animal evolutionary tree and will give us a clearer idea about the roots of how our brain has been developed. This stands in contrast to the conventional framing of mammal brain development as an independent subject unlinked to the "less evolved species". The authors also nicely show a concept that was previously restricted to mammals - the role of microRNAs in development.

(2) Right experimental approach<br /> The authors perform a set of functional experiments correctly adjusted to answer the role of miR-19b in the control of neuronal stem cell proliferation and differentiation. Their histological, functional, and genetic approach gives us a clear idea about the relations between several genes involved in the differentiation of the neurons in the avian pallium. In this idea, they maintain the role of miR-19b as a hub controller, keeping the ventricular zone cells in an undifferentiated stage to perpetuate the cellular pool.

(3) Future directions<br /> The findings open a door to future experiments, particularly to a better comprehension of the role of microRNAs and pallidal genetic connections. Furthermore, this work also proves the use of avians as a model to study cortical development due to the similarities with mammals.

Weaknesses:

While there are questions answered, there are still several that remain unsolved. The experiments analyzed here lead us to speculate that the early differentiation of the progenitor cells from the ventricular zone entails a reduction in the cellular pool, affecting thereafter the number of latter-born neurons (upper layers). The authors should explore that option by testing progenitor cell markers in the ventricular zone, such as Pax6. Even so, it remains possible that miR-19b is also changing the expression pattern of neurons that are going to populate the different layers, instead of their numbers, so the authors cannot rule that out or verify it. Since the paper focuses on the role of miR-19b in patterning, I think the authors should check the relationship and expression between progenitors (Pax6) and intermediate (Tbr2) cells when miR-19b is affected. Since neuronal expression markers change so fast within a few days (HH24-HH35), I don't understand why the authors stop the functional experiments at different time points.

Reviewer #2 (Public review):

Summary:

In this study, the authors test whether intuitive biological causal knowledge is embedded in domain-specific semantic networks, primarily focusing on the precuneus as part of the animacy semantic network. They do so tanks to an fMRI task, by comparing brain activity elicited by participants' exposure to written situations suggesting a plausible cause of illness with brain activity in linguistically equivalent situations suggesting a plausible cause of mechanical failure or damage and non-causal situations. These contrasts confirm the PC as the main "culprit" in whole-brain and fROIs univariate analyses. In turn, inferring causes of mechanical failure engages mostly the PPA. The authors further test whether the content-specificity has to do with inferences about animates in general, or if there are some distinctions between reasoning about people's bodies versus mental states. To answer this question, the authors localize the mentalizing network and study the relation between brain activity elicited by Illness-Causal > Mech-Causal and Mentalizing > Physical stories. They conclude that inferring about the causes of illness partially differentiates from reasoning about people's states of mind. The authors finally test the alternative yet non-mutually exclusive hypothesis that both types of implicit causal inferences (illness and mechanical) depend on shared neural machinery. Good candidates are language and logic, which justifies the use of a language/logic localizer. No evidence of commonalities across causal inferences versus non-causal situations are found.

Strengths:

(1) This study introduces a useful paradigm and well-designed set of stimuli to test for implicit causal inferences.<br /> (2) Another important methodological advance is the addition of physical stories to the original mentalizing protocol.<br /> These tools pave the way for further investigation of domain-specific causal inference.<br /> (3) The authors have significantly improved the manuscript, addressing previous concerns and incorporating additional analyses that strengthen their conclusions.

Key improvements:<br /> (1) The revised introduction makes the study's contribution more explicit and resolves initial ambiguities regarding its scope.<br /> (2) The rationale for focusing primarily on the precuneus is now clearer and the additional analysis in the fusiform face area provides a valuable comparison.<br /> (3) The revised manuscript now includes a more detailed examination of the searchlight MVPA results, showing that illness and mechanical inferences elicit spatially distinct neural patterns in key regions, including the left PC, anterior PPA, and lateral occipitotemporal cortex.<br /> (4) The authors' justification for using an implicit inference task, arguing that explicit tasks introduce executive function confounds, is convincing.<br /> (5) The authors now acknowledge that while their results support a content-specific neural basis for implicit causal inference, domain-general mechanisms may still play a role in other contexts.

I have no major remaining concerns.

Reviewer #2 (Public review):

The data is largely electrophysiological recordings coupled with behavioral measurements (technically impressive) and some gain-of-function experiments in freely walking flies. Loss-of-function was tested but has minimal effect, which is not surprising in a system with partially redundant control mechanisms. The data is also consistent with/complementary to subsequent manuscripts (Yang 2023, Feng 2024, and Ros 2024) showing additional descending neurons with contributions to steering in walking and flying.

The experiments are well executed, the results interesting, and the description clear. Some hypotheses based on connectome anatomy are tested: the insights on the pre-synaptic side - how sensory and central complex heading circuits converge onto these DNs is stronger than the suggestions about biomechanical mechanisms for how turning happens on the motor side.

Of particular interest is the idea that different sensory cues can converge on a common motor program. The turn-toward or turn-away mechanism is initiated by valence rather than whether the stimulus was odor or temperature or memory of heading. The idea that animals chose a direction based on external sensory information and then maintain that direction as a heading through a more internal, goal-based memory mechanism, is interesting but it is hard to separate conclusively.

The "see-saw", where left-right symmetry is broken to allow a turn, presumably by excitation on one side and inhibition of the other leg motor modules, is interesting but not well explained here. How hyperpolarization affects motor outputs is not clear.

The statement near Figure 5B that "DNa02 activity was higher on the side ipsilateral to the attractive stimulus, but contralateral to the aversive stimulus" is really important - and only possible to see because of the dual recordings.

Comments on revisions:

I am happy that the revised manuscript addresses all reviewers' concerns.

Reviewer #2 (Public review):

Strengths:

The article addresses a topic of significant importance, focusing on early life growth faltering in low-income countries-a key marker of undernutrition-and its impact on brain functional connectivity (FC) and cognitive development. The study's strengths include the laborious data collection process, as well as the rigorous data preprocessing methods employed to ensure high data quality. The use of cutting-edge preprocessing techniques further enhances the reliability and validity of the findings, making this a valuable contribution to the field of developmental neuroscience and global health.

Weaknesses:

The study fails to fully leverage its longitudinal design to explore neurodevelopmental changes or trajectories, as highlighted by all three reviewers. The revised manuscript still primarily focuses on FC values at a single age stage (i.e., 24 months) rather than utilizing the longitudinal data to investigate how FC evolves over time or predicts cognitive development. Although the authors acknowledge that analyzing changes in FC (ΔFC) would reduce degrees of freedom (to ~30) and risk interpretability, they do not report or discuss these results, even as exploratory findings.

Furthermore, the study lacks specificity in identifying which specific brain networks are affected by growth faltering, as the current exploratory analyses mainly provide an overall conclusion that infant brain network development is impacted without pinpointing the precise neural mechanisms or networks involved.

Reviewer #2 (Public review):

Summary:

Communication between sensory and motor corticies is likely to be important for many aspects of behavior, and in this study the authors carefully analyse neuronal spiking activity in S1 and M1 evoked by peripheral paw stimulation finding clear evidence for sensory responses in both cortical regions

Strengths:

The experiments and data analyses appear to have been carefully carried out and clearly represented.

Weaknesses:

The revised manuscript addressed the minor weaknesses I noted relating to the first submission.

Reviewer #2 (Public review):

Summary:

The manuscript offers an important contribution to the field of virology, especially concerning NNV entry mechanisms. The major strength of the study lies in the identification of MmMYL3 as a functional receptor for RGNNV and its role in macropinocytosis, mediated by the IGF1R-Rac1/Cdc42 signaling axis. This represents a significant advance in understanding NNV entry mechanisms beyond previously known receptors such as HSP90ab1 and HSC70. The data, supported by comprehensive in vitro and in vivo experiments, strongly justify the authors' claims about MYL3's role in NNV infection in marine medaka.

Strengths:

(1) The identification of MmMYL3 as a functional receptor for RGNNV is a significant contribution to the field. The study fills a crucial gap in understanding the molecular mechanisms governing NNV entry into host cells.

(2) The work highlights the involvement of IGF1R in macropinocytosis-mediated NNV entry and downstream Rac1/Cdc42 activation, thus providing a thorough mechanistic understanding of NNV internalization process. This could pave the way for further exploration of antiviral targets.

Comments on revisions:

The authors have addressed the concerns from reviewers. This manuscript can be published in the current form.

Reviewer #2 (Public review):

Summary:

In this work, the investigators isolated one Lacticaseibacillus rhamnosus strain (P118), and determined this strain worked well against Salmonella Typhimurium infection. Then, further studies were performed to identify the mechanism of bacterial resistance, and a list of confirmatory assays were carried out to test the hypothesis.

Strengths:

The authors provided details regarding all assays performed in this work, and this reviewer trusted that the conclusion in this manuscript is solid. I appreciate the efforts of the authors to perform different types of in vivo and in vitro studies to confirm the hypothesis.

Weaknesses:

I have mainly two questions for this work.

Main point-1:<br /> The authors provided the below information about the sources from which Lacticaseibacillus rhamnosus was isolated. More details are needed. What are the criteria to choose these samples? Where were these samples originate from? How many strains of bacteria were obtained from which types of samples?

Lines 486-488: Lactic acid bacteria (LAB) and Enterococcus strains were isolated from the fermented yoghurts collected from families in multiple cities of China and the intestinal contents from healthy piglets without pathogen infection and diarrhoea by our lab.

Lines 129-133: A total of 290 bacterial strains were isolated and identified from 32 samples of the fermented yoghurt and piglet rectal contents collected across diverse regions within China using MRS and BHI medium , which consist s of 63 Streptococcus strains, 158 Lactobacillus/ Lacticaseibacillus Limosilactobacillus strains and 69 Enterococcus strains.

Main-point-2:<br /> As probiotics, Lacticaseibacillus rhamnosus has been widely studied. In fact, there are many commercially available products, and Lacticaseibacillus rhamnosus is the main bacteria in these products. There are also ATCC type strain such as 53103.

I am sure the authors are also interested to know if P118 is better as a probiotics candidate than other commercially available strains. Also, would the mechanism described for P118 apply to other Lacticaseibacillus rhamnosus strains?

It would be ideal if the authors could include one or two Lacticaseibacillus rhamnosus which are currently commercially used, or from the ATCC. Then, the authors can compare the efficacy and antibacterial mechanisms of their P118 with other strains. This would open the windows for future work.

Reviewer #2 (Public review):

Summary:

Shin et al aim to identify in a very extensive piece of work a mechanism that contributes to dynamic regulation of synaptic output in the rat cortex at the second time scale. This mechanism is related to a new powerful model and is well versed to test if the pool of SV ready for fusion is dynamically scaled to adjust supply demand aspects. The methods applied are state-of-the-art and both address quantitative aspects with high signal to noise. In addition, the authors examine both excitatory output onto glutamatergic and GABAergic neurons, which provides important information on how general the observed signals are in neural networks. The results are compellingly clear and show that pool regulation may be predominantly responsible. Their results suggests that a regulation of release probability, the alternative contender for regulation, is unlikely to be involved in the observed short term plasticity behavior (but see below). Besides providing a clear analysis pof the underlying physiology, they test two molecular contenders for the observed mechanism by showing that loss of Synaptotagmin7 function and the role of the Ca dependent phospholipase activity seems critical for the short term plasticity behavior. The authors go on to test the in vivo role of the mechanism by modulating Syt7 function and examining working memory tasks as well as overall changes in network activity using immediate early gene activity. Finally, they model their data, providing strong support for their interpretation of TS pool occupancy regulation.

Strengths: