Reviewer #1 (Public Review):

I'll begin by summarizing what I understand from the results presented, and where relevant how my understanding seems to differ from the authors' claims. I'll then make specific comments with respect to points raised in my previous review (below), using the same numbering. Because this is a revision I'll try to restrict comments here to the changes made, which provide some clarification, but leave many issues incompletely addressed.

As I understand it the main new result here is that certain recurrent network architectures promote emergence of coordinated grid firing patterns in a model previously introduced by Kropff and Treves (Hippocampus, 2008). The previous work very nicely showed that single neurons that receive stable spatial input could 'learn' to generate grid representations by combining a plasticity rule with firing rate adaptation. The previous study also showed that when multiple neurons were synaptically connected their grid representations could develop a shared orientation, although with the recurrent connectivity previously used this substantially reduced the grid scores of many of the neurons. The advance here is to show that if the initial recurrent connectivity is consistent with that of a line attractor then the network does a much better job of establishing grid firing patterns with shared orientation.

Beyond this point, things become potentially confusing. As I understand it now, the important influence of the recurrent dynamics is in establishing the shared orientation and not in its online generation. This is clear from Figure S3, but not from an initial read of the abstract or main text. This result is consistent with Kropff and Treves' initial suggestion that 'a strong collateral connection... from neuron A to neuron B... favors the two neurons to have close-by fields... Summing all possible contributions would result in a field for neuron B that is a ring around the field of neuron A.' This should be the case for the recurrent connections now considered, but the evidence provided doesn't convincingly show that attractor dynamics of the circuit are a necessary condition for this to arise. My general suggestion for the authors is to remove these kind of claims and to keep their interpretations more closely aligned with what the results show.

Major (numbered according to previous review)

(1) Does the network maintain attractor dynamics after training? Results now show that 'in a trained network without feedforward Hebbian learning the removal of recurrent collaterals results in a slight increase in gridness and spacing'. This clearly implies that the recurrent collaterals are not required for online generation of the grid patterns. This point needs to be abundantly clear in the abstract and main text so the reader can appreciate that the recurrent dynamics are important specifically during learning.<br /> (2) Additional controls for Figure 2 to test that it is connectivity rather than attractor dynamics (e.g. drawing weights from Gaussian or exponential distributions). The authors provide one additional control based on shuffling weights. However, this is far from exhaustive and it seems difficult on this basis to conclude that it is specifically the attractor dynamics that drive the emergence of coordinated grid firing.<br /> (3) What happens if recurrent connections are turned off? The new data clearly show that the recurrent connections are not required for online grid firing, but this is not clear from the abstract and is hard to appreciate from the main text.<br /> (4) This is addressed, although the legend to Fig. S2D could provide an explanation / definition for the y-axis values.<br /> (5) Given the 2D structure of the network input it perhaps isn't surprising that the network generates 2D representations and this may have little to do with its 1D connectivity. The finding that the networks maintain coordinated grids when recurrent connections are switched off supports my initial concern and the authors explanation, to me at least, remain confusing. I think it would be helpful to consider that the connectivity is specifically important for establishing the coordinated grid firing, but that the online network does not require attractor dynamics to generate coordinated grid firing.<br /> (6) Clarity of the introduction. This is somewhat clearer, but I wonder if it would be hard for someone not familiar with the literature to accurately appreciate the key points.<br /> (7) Remapping. I'm not sure why this is ill posed. It seems the proposed model can not account for remapping results (e.g. Fyhn et al. 2007). Perhaps the authors could just clearly state this as a limitation of the model (or show that it can do this).

Previous review:

This study investigates the impact of recurrent connections on grid fields generated in networks trained by adjusting the strength of feedforward spatial inputs. The main result is that if the recurrent connections in the network are given a 1D continuous attractor architecture, then aligned grid firing patterns emerge in the network following training. Detailed analyses of the low dimensional dynamics of the resulting networks are then presented. The simulations and analyses appear carefully carried out.

The feedforward model investigated by the authors (previously introduced by Kropff & Treves, 2008) is an interesting and important alternative to models that generate grid firing patterns through 2-dimensional continuous attractor network (CAN) dynamics. However, while both classes of model generate grid fields, in making comparisons the manuscript is insufficiently clear about their differences. In particular, in the CAN models grid firing is a direct result of their 2-D architecture, either a torus structure with a single activity bump (e.g. Guanella et al. 2007, Pastoll et al. 2013), or sheet with multiple local activity bumps (Fuhs & Touretzky, Burak & Fiete, 2009). In these models, spatial input can anchor the grid representations but is not necessary for grid firing. By contrast, in the feedforward models neurons transform existing spatial inputs into a grid representation. Thus, the two classes of model implement different computations; CANs path integrate, while the feedforward models transform spatial representations. A demonstration that a 1D CAN generates coordinated 2D grid fields would be surprising and important, but its less clear why coordination between grids generated by the feedforward mechanism would be surprising. As written, it's unclear which of these claims the study is trying to make. If the former, then the conclusion doesn't appear well supported by the data as presented, if the latter then the results are perhaps not so unexpected, and the imposed attractor dynamics may still not be relevant.

Whichever claim is being made, it could be helpful to more carefully evaluate the model dynamics given predictions expected for the different classes of model. Key questions that are not answered by the manuscript include:

- At what point is the 1D attractor architecture playing a role in the models presented here? Is it important specifically for training or is it also contributing to computation in the fully trained network?

- Is an attractor architecture required at all for emergence of population alignment and gridness? Key controls missing from Figure 2 include training on networks with other architectures. For example, one might consider various architectures with randomly structured connectivity (e.g. drawing weights from exponential or Gaussian distributions).

- In the trained models do the recurrent connections substantially influence activity in the test conditions? Or after training are the 1D dynamics drowned out by feedforward inputs?

- What is the low dimensional structure of the input to the network? Can the apparent discrepancy between dimensionality of architecture and representation be resolved by considering structure of the inputs, e.g. if the input is a 2 dimensional representation of location then is it surprising that the output is too?

- What happens to representations in the trained networks presented when place cells remap? Is the 1D manifold maintained as expected for CAN models, or does it reorganise?

Reviewer #1 (Public Review):

Summary:

This study examined the role of statistical learning in pain perception, suggesting that individuals' expectations about a sequence of events influence their perception of pain intensity. They incorporated the components of volatility and stochasticity into their experimental design and asked participants (n = 27) to rate the pain intensity, their prediction, and their confidence level. They compared two different inference strategies: Bayesian inference vs. heuristic-employing Kalman filters and model-free reinforcement learning. They showed that the expectation-weighted Kalman filter best explained the temporal pattern of participants' ratings. These results provide evidence for a Bayesian inference perspective on pain, supported by a computational model that elucidates the underlying process.

Strengths:

- Their experimental design included a wide range of input intensities and the levels of volatility and stochasticity. With elaborated computational models, they provide solid evidence that statistical learning shapes pain.

Weaknesses:

- Relevance to clinical pain: While the authors underscore the relevance of their findings to chronic pain, they did not include data pertaining to clinical pain.

Reviewer #1 (Public Review):

Summary:

This paper investigates the neural population activity patterns of the medial frontal cortex in rats performing a nose poking timing task using in vivo calcium imaging. The results showed neurons that were active at the beginning and end of the nose poking and neurons that formed sequential patterns of activation that covaried with the timed interval during nose poking on a trial-by-trial basis. The former were not stable across sessions, while the latter tended to remain stable over weeks. The analysis on incorrect trials suggests the shorter non-rewarded intervals were due to errors in the scaling of the sequential pattern of activity.

Strengths:

This study measured stable signals using in vivo calcium imaging during experimental sessions that were separated by many days in animals performing a nose poking timing task. The correlation analysis on the activation profile to separate the cells in the three groups was effective and the functional dissociation between beginning and end, and duration cells was revealing. The analysis on the stability of decoding of both the nose poking state and poking time was very informative. Hence, this study dissected a neural population that formed sequential patterns of activation that encoded timed intervals.

Weaknesses:

It is not clear whether animals had enough simultaneously recorded cells to perform the analyzes of Figures 2-4. In fact, rat 3 had 18 responsive neurons which probably is not enough to get robust neural sequences for the trial-by-trial analysis and the correct and incorrect trial analysis. In addition, the analysis of behavioral errors could be improved. The analysis in Figure 4A could be replaced by a detailed analysis on the speed, and the geometry of neural population trajectories for correct and incorrect trials. In the case of Figure 4G is not clear why the density of errors formed two clusters instead of having a linear relation with the produce duration. I would be recommendable to compute the scaling factor on neuronal population trajectories and single cell activity or the computation of the center of mass to test the type III errors.

Due to the slow time resolution of calcium imaging, it is difficult to perform robust analysis on ramping activity. Therefore, I recommend downplaying the conclusion that: "Together, our data suggest that sequential activity might be a more relevant coding regime than the ramping activity in representing time under physiological conditions."

Reviewer #1 (Public Review):

Summary:

This work studies spatio-temporal patterns of structure-function coupling in developing brains, using a large set of imaging data acquired from children aged 5-22. Magnetic resonance imaging data of brain structure and function were obtained from a publicly available database, from which structural and functional features and measures were derived. The authors examined the spatial patterns of structure-function coupling and how they evolve with brain development. This work further sought correlations of brain structure-function coupling with behavior and explored evolutionary, microarchitectural and genetic bases that could potentially account for the observed patterns.

Strength:

The strength of this work is the use of currently available state-of-the-art analysis methods, along with a large set of high-quality imaging data, and comprehensive examinations of structure-function coupling in developing brains. The results are comprehensive and illuminating.

Weakness:

As with most other studies, transcriptomic and cellular architectures of structure-function coupling were characterized only on the basis of a common atlas in this work.

The authors have achieved their aims in this study, and the findings provide mechanistic insights into brain development, which will inspire further basic and clinical studies along this line.

Reviewer #1 (Public Review):

Summary:

In this study, the authors provide a new computational platform called Vermouth to automate topology generation, a crucial step that any biomolecular simulation starts with. Given a wide arrange of chemical structures that need to be simulated, varying qualities of structural models as inputs obtained from various sources, and diverse force fields and molecular dynamics engines employed for simulations, automation of this fundamental step is challenging, especially for complex systems and in case that there is a need to conduct high-throughput simulations in the application of computer-aided drug design (CADD). To overcome this challenge, the authors develop a programing library composed of components that carry out various types of fundamental functionalities that are commonly encountered in topological generation. These components are intended to be general for any type of molecules and not to depend on any specific force field and MD engines. To demonstrate the applicability of this library, the authors employ those components to re-assemble a pipeline called Martinize2 used in topology generation for simulations with a widely used coarse-grained model (CG) MARTINI. This pipeline can fully recapitulate the functionality of its original version Martinize but exhibit greatly enhanced generality, as confirmed by the ability of the pipeline to faithfully generate topologies for two high-complexity benchmarking sets of proteins.

Strengths:

The main strength of this work is the use of concepts and algorithms associated with induced subgraph in graph theory to automate several key but non-trivial steps of topology generation such as the identification of monomer residue units (MRU), the repair of input structures with missing atoms, the mapping of topologies between different resolutions, and the generation of parameters needed for describing interactions between MRUs. In addition, the documentation website provided by the authors is very informative, allowing users to get quickly started with Vermouth.

Weaknesses:

Although the Vermouth library is designed as a general tool for topology generation for molecular simulations, only its applications with MARTINI have been demonstrated in the current study. Thus, the claimed generality of Vermouth remains to be exmained. The authors may consider to point out this in their manuscript.

Reviewer #1 (Public Review):

Summary:

The authors demonstrate with a simple stochastic model that the initial composition of the community is important in achieving a target frequency during the artificial selection of a community.

Strengths:

To my knowledge, the intra-collective selection during artificial selection has not been seriously theoretically considered. However, in many cases, the species dynamics during the incubation of each selection cycle are important and relevant to the outcome of the artificial selection experiment. Stochasticity from birth and death (demographic stochasticity) plays a big role in these species' abundance dynamics. This work uses a simple framework to tackle this idea meticulously.

This work may or may not be related to hysteresis (path dependency). If this is true, maybe it would be nice to have a discussion paragraph talking about how this may be the case. Then, this work would even attract the interest of people studying dynamic systems.

Weaknesses:

(1) Connecting structure and function

In typical artificial selection literature, most of them select the community based on collective function. Here in this paper, the authors are selecting a target composition. Although there is a schematic cartoon illustrating the relationship between collective function (y-axis) and the community composition in the main Figure 1, there is no explicit explanation or justification of what may be the origin of this relationship. I think giving the readers a naïve idea about how this structure-function relationship arises in the introduction section would help. This is because the conclusion of this paper is that the intra-collective selection makes it hard to artificially select a community that has an intermediate frequency of f (or s). If there is really evidence or theoretical derivation from this framework that indeed the highest function comes from the intermediate frequency of f, then the impact of this paper would increase because the conclusions of this stochastic model could allude to the reasons for the prevalent failures of artificial selection in literature.

(2) Explain intra-collective and inter-collective selection better for readers.

The abstract, the introduction, and the result section use these terms or intra-collective and inter-collective selection without much explanation. A clear definition in the beginning would help the audience grasp the importance of this paper, because these concepts are at the core of this work.

(3) Achievable target frequency strongly depending on the degree of demographic stochasticity.

I would expect that the experimentalists would find these results interesting and would want to consider these results during their artificial selection experiments. The main Figure 4 indicates that the Newborn size N0 is a very important factor to consider during the artificial selection experiment. This would be equivalent to how much bottleneck is imposed on the artificial selection process in every iteration step (i.e., the ratio of serial dilution experiment). However, with a low population size, all target frequencies can be achieved, and therefore in these regimes, the initial frequency now does not matter much. It would be great for the authors to provide what the N0 parameter actually means during the artificial selection experiments. Maybe relative to some other parameter in the model. I know this could be very hard. But without this, the main result of this paper (initial frequency matters) cannot be taken advantage of by the experimentalists.

(4) Consideration of environmental stochasticity.

The success (gold area of Figure 2d) in this framework mainly depends on the size of the demographic stochasticity (birth-only model) during the intra-collective selection. However, during experiments, a lot of environmental stochasticity appears to be occurring during artificial selection. This may be out of the scope of this study. But it would definitely be exciting to see how much environmental stochasticity relative to the demographic stochasticity (variation in the Gaussian distribution of F and S) matters in succeeding in achieving the target composition from artificial selection.

(5) Assumption about mutation rates

If setting the mutation rates to zero does not change the result of the simulations and the conclusion, what is the purpose of having the mutation rates \mu? Also, is the unidirectional (S -> F -> FF) mutation realistic? I didn't quite understand how the mutations could fit into the story of this paper.

(6) Minor points

In Figure 3b, it is not clear to me how the frequency difference for the Intra-collective and the Inter-collective selection is computed.

In Figure 5b, the gold region (success) near the FF is not visible. Maybe increase the size of the figure or have an inset for zoom-in. Why is the region not as big as the bottom gold region?

Reviewer #1 (Public Review):

Summary:

In this study, Kume et al examined the role of the protein Semaphorin 4a in steady-state skin homeostasis and how this relates to skin changes seen in human psoriasis and imiquimod-induced psoriasis-like disease in mice. The authors found that human psoriatic skin has reduced expression of Sema4a in the epidermis. While Sema4a has been shown to drive inflammatory activation in different immune populations, this finding suggested Sema4a might be important for negatively regulating Th17 inflammation in the skin. The authors go on to show that Sema4a knockout mice have skin changes in key keratinocyte genes, increased gdT cells, and increased IL-17 similar to differences seen in non-lesional psoriatic skin, and that bone marrow chimera mice with WT immune cells and Sema4a KO stromal cells develop worse IMQ-induced psoriasis-like disease, further linking expression of Sema4a in the skin to maintaining skin homeostasis. The authors next studied downstream pathways that might mediate the homeostatic effects of Sema4a, focusing on mTOR given its known role in keratinocyte function. As with the immune phenotypes, Sema4a KO mice had increased mTOR activation in the epidermis in a similar pattern to mTOR activation noted in non-lesional psoriatic skin. The authors next targeted the mTOR pathway and showed rapamycin could reverse some of the psoriasis-like skin changes in Sema4a KO mice, confirming the role of increased mTOR in contributing to the observed skin phenotype.

Strengths:

The most interesting finding is the tissue-specific role for Sema4a, where it has previously been considered to play a mostly pro-inflammatory role in immune cells, this study shows that when expressed by keratinocytes, Sema4a plays a homeostatic role that when missing leads to the development of psoriasis-like skin changes. This has important implications in terms of targeting Sema4a pharmacologically. It also may yield a novel mouse model to study mechanisms of psoriasis development in mice separate from the commonly used IMQ model. The included experiments are well-controlled and executed rigorously.

Weaknesses:

A weakness of the study is the lack of tissue-specific Sema4a knockout mice (e.g. in keratinocytes only). The authors did use bone marrow chimeras, but only in one experiment. This work implies that psoriasis may represent a Sema4a-deficient state in the epidermal cells, while the same might not be true for immune cells. Indeed, in their analysis of non-lesional psoriasis skin, Sema4a was not significantly decreased compared to control skin, possibly due to compensatory increased Sema4a from other cell types. Unbiased RNA-seq of Sema4a KO mouse skin for comparison to non-lesional skin might identify other similarities besides mTOR signaling. Indeed, targeting mTOR with rapamycin reveres some of the skin changes in Sema4a KO mice, but not skin thickness, so other pathways impacted by Sema4a may be better targets if they could be identified. Utilizing WTKO chimeras in addition to global KO mice in the experiments in Figures 6-8 would more strongly implicate the separate role of Sema4a in skin vs immune cell populations and might more closely mimic non-lesional psoriasis skin.

Reviewer #1 (Public Review):

Summary:

This study investigated the role of CD47 and TSP1 in extramedullary erythropoiesis by utilization of both global CD47-/- mice and TSP1-/- mice.

Strengths:

Flow cytometry combined with spleen bulk and single cell transcriptomics were employed. The authors found that stress-induced erythropoiesis markers were increased in CD47-/- spleen cells, particularly genes that are required for terminal erythroid differentiation. Moreover, CD47 dependent erythroid precursors population was identified by spleen scRNA sequencing. In contrast, the same cells were not detected in TSP1-/- spleen. These findings provide strong evidence to support the conclusion that differential role of CD47 and TSP1 in extramedullary erythropoiesis in mouse spleen. Furthermore, the relevance of the current finding to the prevalent side effect (anemia) of anti-CD47 mediated cancer therapy has been discussed in the Discussion section.

Reviewer #1 (Public Review):

Summary:

This study by Wang et al. identifies a new type of deacetylase, CobQ, in Aeromonas hydrophila. Notably, the identification of this deacetylase reveals a lack of homology with eukaryotic counterparts, thus underscoring its unique evolutionary trajectory within the bacterial domain.

Strengths:

The manuscript convincingly illustrates CobQ's deacetylase activity through robust in vitro experiments, establishing its distinctiveness from known prokaryotic deacetylases. Additionally, the authors elucidate CobQ's potential cooperation with other deacetylases in vivo to regulate bacterial cellular processes. Furthermore, the study highlights CobQ's significance in the regulation of acetylation within prokaryotic cells.

Weaknesses:

While the manuscript is generally well-structured, some clarification and some minor corrections are needed.

Reviewer #1 (Public Review):

Summary:

Plasmodium vivax can persist in the liver of infected individuals in the form of dormant hypnozoites, which cause malaria relapses and are resistant to most current antimalarial drugs. This highlights the need to develop new drugs active against hypnozoites that could be used for radical cure. Here, the authors capitalize on an in vitro culture system based on primary human hepatocytes infected with P. vivax sporozoites to screen libraries of repurposed molecules and compounds acting on epigenetic pathways. They identified a number of hits, including hydrazinophthalazine analogs. They propose that some of these compounds may act on epigenetic pathways potentially involved in parasite quiescence. To provide some support to this hypothesis, they document DNA methylation of parasite DNA based on 5-methylcytosine immunostaining, mass spectrometry, and bisulfite sequencing.

Strengths:<br /> -The drug screen itself represents a huge amount of work and, given the complexity of the experimental model, is a tour de force.<br /> -The screening was performed in two different laboratories, with a third laboratory being involved in the confirmation of some of the hits, providing strong support that the results were reproducible.<br /> -The screening of repurposing libraries is highly relevant to accelerate the development of new radical cure strategies.

Weaknesses:

-The manuscript is composed of two main parts, the drug screening itself and the description of DNA methylation in Plasmodium pre-erythrocytic stages. Unfortunately, these two parts are loosely connected. First, there is no evidence that the identified hits kill hypnozoites via epigenetic mechanisms. The hit compounds almost all act on schizonts in addition to hypnozoites, therefore it is unlikely that they target quiescence-specific pathways. At least one compound, colforsin, seems to selectively act on hypnozoites, but this observation still requires confirmation. Second, while the description of DNA methylation is per se interesting, its role in quiescence is not directly addressed here. Again, this is clearly not a specific feature of hypnozoites as it is also observed in P. vivax and P. cynomolgi hepatic schizonts and in P. falciparum blood stages. Therefore, the link between DNA methylation and hypnozoite formation is unclear. In addition, DNA methylation in sporozoites may not reflect epigenetic regulation occurring in the subsequent liver stages.

-The mode of action of the hit compounds remains unknown. In particular, it is not clear whether the drugs act on the parasite or on the host cell. Merely counting host cell nuclei to evaluate the toxicity of the compounds is probably acceptable for the screen but may not be sufficient to rule out an effect on the host cell. A more thorough characterization of the toxicity of the selected hit compounds is required.

-There is no convincing explanation for the differences observed between P. vivax and P. cynomolgi. The authors question the relevance of the simian model but the discrepancy could also be due to the P. vivax in vitro platform they used.

-Many experiments were performed only once, not only during the screen (where most compounds were apparently tested in a single well) but also in other experiments. The quality of the data would be increased with more replication.

-While the extended assay (12 days versus 8 days) represents an improvement of the screen, the relevance of adding inhibitors of core cytochrome activity is less clear, as under these conditions the culture system deviates from physiological conditions.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors established an in vitro triple co-culture BBB model and demonstrated its advantages compared with the mono or double co-culture BBB model. Further, the authors used their established in vitro BBB model and combined it with other methodologies to investigate the specific mechanism that co-culture with astrocytes but also neurons enhanced the integrity of endothelial cells.

Strengths:

The results persuasively showed the established triple co-culture BBB model well mimicked several important characteristics of BBB compared with the mono-culture BBB model, including better barrier function and in vivo/in vitro correlation. The human-derived immortalized cells used made the model construction process faster and more efficient, and have a better in vivo correlation without species differences. This model is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.

Based on the previous experimental results, detailed studies investigated how co-culture with neurons and astrocytes promoted claudin-5 and VE-cadherin in endothelial cells, and the specific signaling mechanisms were also studied. Interestingly, the authors found that neurons also released GDNF to promote barrier properties of brain endothelial cells, as most current research has focused on the promoting effect of astrocytes-derived GDNF on BBB. Meanwhile, the author also validated the functions of GDNF for BBB integrity in vivo by silencing GDNF in mouse brains. Overall, the experiments and data presented support their claim that, in addition to astrocytes, neurons also have a promoting effect on the barrier function of endothelial cells through GDNF secretion.

Weaknesses:

Although the authors demonstrated a highly usable for predicting the BBB permeability, recorded TEER measurements are still far from the human BBB in vivo reported measurements of TEER, and expression of transporters was not promoted by co-culture, which may lead to the model being unsuitable for studying drug transport mediated by transporters on BBB.

Reviewer #1 (Public Review):

The paper has shown the expression of RGS10 is related to the molecular subtype, distant metastasis, and survival status of breast cancer. The study utilizes bioinformatic analyses, human tissue samples, and in vitro and in vivo experiments which strengthen the data. RGS10 was validated to inhibit EMT through a novel mechanism dependent on LCN2 and miR-539-5p, thereby reducing cancer cell proliferation, colony formation, invasion, and migration. The study elaborated on the function of RGS10 in influencing the prognosis and biological behavior which could be considered as a potential drug target in breast cancer.

Reviewer #1 (Public Review):

Summary:

This paper is focused on the role of Cadherin Flamingo (Fmi) - also called Starry night (stan) - in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that express activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which makes continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind.

The authors show that clonal loss of Fmi by an allele or by RNAi in the RasV12, scrib-i tumors suppresses their growth in both the eye disc (continuous clones) and wing disc (discrete clones). The authors attributed this result to less killing of WT neighbors when Myc over-expressing clones lacking Fmi, but another interpretation (that Fmi regulates clonal growth) is equally as plausible with the current results. Next, the authors show that scrib-RNAi clones that are normally out-competed by WT cells prior to adult stages are present in higher numbers when WT cells are depleted for Fmi. They then examine death in RasV12, scrib-i ey-FLP clones, or in discrete hs-FLP UAS-Myc clones. They state that they see death in WT cells neighboring RasV12, scrib-i clones in the eye disc (Figures 4A-C). Next, they write that RasV12, scrib-I cells become losers (i.e., have apoptosis markers) when Fmi is removed. Neither of these results are quantified and thus are not compelling. They state that a similar result is observed for Myc over-expression clones that lack Fmi, but the image was not compelling, the results are not quantified and the controls are missing (Myc over-expressing clones alone and Fmi clones alone). They then want to test whether Myc over-expressing clones have more proliferation. They show an image of a wing disc that has many small Myc overexpressing clones with and without Fmi. The pHH3 results support their conclusion that Myc overexpressing clones have more pHH3, but I have reservations about the many clones in these panels (Figures 5L-N). They show that the cell competition roles of Fmi are not shared by another PCP component and are not due to the Cadherin domain of Fmi. The authors appear to interpret their results as Fmi is required for winner status. Overall, some of these results are potentially interesting and at least partially supported by the data, but others are not supported by the data.

Strengths:

Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

Weaknesses:

(1) In the Myc over-expression experiments, the increased size of the Myc clones could be because they divide faster (but don't outcompete WT neighbors). If the authors want to conclude that the bigger size of the Myc clones is due to out-competition of WT neighbors, they should measure cell death across many discs of with these clones. They should also assess if reducing apoptosis (like using one copy of the H99 deficiency that removes hid, rpr, and grim) suppresses winner clone size. If cell death is not addressed experimentally and quantified rigorously, then their results could be explained by faster division of Myc over-expressing clones (and not death of neighbors). This could also apply to the RasV12, scrib-i results.

(2) This same comment about Fmi affecting clone growth should be considered in the scrib RNAi clones in Figure 3.

(3) I don't understand why the quantifications of clone areas in Figures 2D, 2H, 6D are log values. The simple ratio of GFP/RFP should be shown. Additionally, in some of the samples (e.g., fmiE59 >> Myc, only 5 discs and fmiE59 vs >Myc only 4 discs are quantified but other samples have more than 10 discs). I suggest that the authors increase the number of discs that they count in each genotype to at least 20 and then standardize this number.

(4) There is a typo when referring to Figures 3C-D. It should be Figure 2C-D.

(5) Figure 4 - shows examples of cell death. Cas3 is written on the figure but Dcp-1 is written in the results. Which antibody was used? The authors need to quantify these results. They also need to show that the death of cells is part of the phenotype, like an H99 deficiency, etc (see above).

(6) It is well established that clones overexpressing Myc have increased cell death. The authors should consider this when interpreting their results.

(7) A better characterization of discrete Fmi clones would also be helpful. I suggest inducing hs-flp clones in the eye or wing disc and then determining clone size vs twin spot size and also examining cell death etc. If such experiments have already been done and published, the authors should include a description of such work in the preprint.

(8) We need more information about the expression pattern of Fmi. Is it expressed in all cells in imaginal discs? Are there any patterns of expression during larval and pupal development?

(9) Overall, the paper is written for specialists who work in cell competition and is fairly difficult to follow, and I suggest re-writing the results to make it accessible to a broader audience.

Reviewer #1 (Public Review):

Summary:

This study shows a new mechanism of GS regulation in the archaean Methanosarcina maze and clarifies the direct activation of GS activity by 2-oxoglutarate, thus featuring another way in which 2-oxoglutarate acts as a central status reporter of C/N sensing.

Mass photometry and single particle cryoEM structure analysis convincingly show the direct regulation of GS activity by 2-OG promoted formation of the dodecameric structure of GS. The previously recognized small proteins GlnK1 and Sp26 seem to play a subordinate role in GS regulation, which is in good agreement with previous data. Although these data are quite clear now, there remains one major open question: how does 2-OG further increase GS activity once the full dodecameric state is achieved (at 5 mM)? This point needs to be reconsidered.

Strengths:

Mass photometry reveals a dynamic mode of the effect of 2-OG on the oligomerization state of GS. Single particle Cryo-EM reveals the mechanism of 2-OG mediated dodecamer formation.

Weaknesses:

It is not entirely clear, how very high 2-OG concentrations activate GS beyond dodecamer formation.

The data presented in this work are in stark contrast to the previously reported structure of M. mazei GS by the Schumacher lab. This is very confusing for the scientific community and requires clarification. The discussion should consider possible reasons for the contradictory results.

Importantly, it is puzzling how Schumacher could achieve an apo-structire of dodecameeric GS? If 2-OG is necessary for dodecameric formation, this should be discussed. If GlnK1 doesn't form a complex with the dodecameric GS, how could such a complex be resolved there?

In addition, the text is in principle clear but could be improved by professional editing. Most obviously there is insufficient comma placement.

Reviewer #1 (Public Review):

The paper meticulously explores various conformations and states of the ribosome-translocon complex. Employing advanced techniques such as cryoEM structural determination and AlphaFold modeling, the study delves into the dynamic nature of the ribosome-translocon complex. The findings from these analyses unveil crucial insights, significantly advancing our understanding of the co-translational translocation process in cellular mechanisms.

To begin with, the authors employed a construct comprising the first two transmembrane domains of rhodopsin as a model for studying protein translocation. They conducted in vitro translation, followed by the purification of the ribosome-translocon complex, and determined its cryoEM structures. An in-depth analysis of their ribosome-translocon complex structure revealed that the nascent chain can pass through the lateral gate of translocon Sec61, akin to the behavior of a Signaling Peptide. Additionally, Sec61 was found to interact with 28S rRNA helix 24 and the ribosomal protein uL24. In summary, their structural model aligns with the through-pore model of insertion, contradicting the sliding model.

Secondly, the authors successfully identified RAMP4 in their ribosome-translocon complex structure. Notably, the transmembrane domain of RAMP4 mimics the binding of a Signaling Peptide at the lateral gate of Sec61, albeit without unplugging. Intriguingly, RAMP4 is exclusively present in the non-multipass translocon ribosome-translocon complex, not in those containing multipass translocon. This observation suggests that co-translational translocation specifically occurs in the Sec61 channel that includes bound RAMP4. Additionally, the authors discovered an interaction between the C-tail of ribosomal proteins uL22 and the translocon Sec61, providing valuable insights into the nascent chain's behavior.

Moving on to the third point, the focused classification unveiled TRAP complex interactions with various components. The authors propose that the extra density observed in their novel ribosome-translocon complex can be attributed to calnexin, a major binder of TRAP according to previous studies. Furthermore, the new structure reveals a TRAP-OSTA interaction. This newly identified TRAP-OSTA interaction offers a potential explanation for why patients with TRAP delta defects exhibit congenital disorders of glycosylation.

In conclusion, this paper presents a robust contribution to the field with its thorough structural and modeling analyses. The significance of the findings is evident, providing valuable insights into the intricate mechanisms of protein co-translational translocation. The well-crafted writing, meticulous analyses, and clear figures collectively contribute to the overall strength of the paper.

Reviewer #1 (Public Review):

Summary:

The authors have previously described a way to boost WNT/CTNNB1 signaling in a tissue-specific manner, by directing an RSPO2 mutant protein (RSPO2RA) to a liver-specific receptor (ASGR1/2). This is done by fusing the RSPO2RA to an antibody that binds ASGR1/2.

Here the authors describe two new antibodies, 8M24 and 8G8, with similar effects. 8M24 shows specificity for ASGR1, while 8G8 has broader affinity for mouse/human ASGR1/2.<br /> The authors resolve and describe the crystal structure of the hASGR1CRD:8M24 complex and the hASGR2CRD:8G8 complex in great detail, which help explain the specificities of the 8M24 and 8G8 antibodies. Their epitopes are non-overlapping.<br /> Upon fusion of the antibodies to an RSPO2RA (an RSPO mutant), these antibodies are able to enhance WNT signaling by promoting the ASGR1-mediated clearance of ZNRF3/RNF43, thereby increasing cell surface expression of FZD. This has previously also been shown to be the case for RSPO2RA fused to an anti-ASGR1 antibody 4F3 - and the paper also tests how the antibodies compare to the 4F3 fusion.

Strengths:

(1) One challenge in treating diseases, is the fact that one would like therapeutics to be highly specific - not just in terms of their target (e.g. aimed at a specific protein of interest) but also in terms of tissue specificity (i.e. affecting only tissue X but leaving all others unaffected). This study broadens the collection of antibodies that can be used for this purpose and thus expands a potential future clinical toolbox.

(2) The authors have addressed questions raised after a first round of review, e.g. by showing that ASGR1 is itself indeed ubiquitinated.

Weaknesses:

(1) Some questions remain as to how 8M24 and 8G8 compare to 4F3.

(2) Some questions remain as to the specificity of the approach: the initial goal was not to also downregulate ASGR1 per se, so this targeting to a specific receptor/membrane protein is not trivial and/or neutral.

Reviewer #1 (Public Review):

The manuscript presents novel results on the regulation of Drosophila wing growth by the protocadherins Ds and Fat. The manuscript performs a more careful analysis of disc volume, larval size, and the relationship between the two, in normal and mutant larvae, and after localized knockdown or overexpression of Fat and Ds. Not all of the results are equally surprising given the previous work on Fat, Ds, and their regulation of disc growth, pupariation, and the Hippo pathway, but the presentation and detail of the presented data is new. The most novel results concern the scaling of gradients of Fat and Ds protein during development, a largely unstudied gradient of Fat protein, and using overexpression of Ds to argue that changes in the Ds gradient do not underlie the slowing and halting of cell divisions during development.

Reviewer #1 (Public Review):

Muscle models are important tools in the fields of biomechanics and physiology. Muscle models serve a wide variety of functions, including validating existing theories, testing new hypotheses, and predicting forces produced by humans and animals in health and disease. This paper attempts to provide an alternative to Hill-type muscle models that includes contributions of titin to force enhancement over multiple time scales. Due to the significant limitations of Hill-type models, alternative models are needed and therefore the work is important and timely.

The effort to include a role for titin in muscle models is a major strength of the methods and results. The results clearly demonstrate the weaknesses of Hill models and the advantages of incorporating titin into theoretical treatments of muscle mechanics. Another strength is to address muscle mechanics over a large range of time scales.

The authors succeed in demonstrating the need to incorporate titin in muscle models, and further show that the model accurately predicts in situ force of cat soleus (Kirsch et al. 1994; Herzog & Leonard, 2002) and rabbit posts myofibrils (Leonard et al. 2010). However, it remains unclear whether the model will be practical for use with data from different muscles or preparations. Several ad hoc modifications were described in the paper, and the degree to which the model requires parameter optimization for different muscles, preparations and experiment types remains unclear.

Reviewer #1 (Public Review):

Summary:<br /> In the paper by Choi et al., the authors aimed to develop base editing strategies to convert CAG repeats to CAA repeats in the huntingtin gene (HTT), which causes Huntington's disease (HD). They hypothesized that this conversion would delay disease onset by shortening the uninterrupted CAG repeat. Using HEK-293T cells as a model, the researchers employed cytosine base editors and guide RNAs (gRNAs) to efficiently convert CAG to CAA at various sites within the CAG repeat. No significant indels, off-target edits, transcriptome alterations, or changes in HTT protein levels were detected. Interestingly, somatic CAG repeat expansion was completely abolished in HD knock-in mice carrying CAA-interrupted repeats.

Strengths:<br /> This study represents the first proof-of-concept exploration of the cytosine base editing technique as a potential treatment for HD and other repeat expansion disorders with similar mechanisms.

Weaknesses:<br /> Given that HD is a neurodegenerative disorder, it is crucial to determine the efficiency of the base editing strategies tested in this manuscript and their feasibility in relevant cells affected by HD and the brain, which needed to be improved in this manuscript.

Reviewer #1 (Public Review):

Summary:

In this study, the authors used a multi-alternative decision task and a multidimensional signal-detection model to gain further insight into the cause of perceptual impairments during the attentional blink. The model-based analyses of behavioural and EEG data show that such perceptual failures can be unpacked into distinct deficits in visual detection and discrimination, with visual detection being linked to the amplitude of late ERP components (N2P and P3) and discrimination being linked to the coherence of fronto-parietal brain activity.

Strengths:

The main strength of this paper lies in the fact that it presents a novel perspective on the cause of perceptual failures during the attentional blink. The multidimensional signal-detection modelling approach is explained clearly, and the results of the study show that this approach offers a powerful method to unpack behavioural and EEG data into distinct processes of detection and discrimination.

Weaknesses:

While the model-based analyses are compelling, the paper also features some analyses that seem misguided, or, at least, insufficiently motivated and explained. Specifically, in the introduction, the authors raise the suggestion that the attentional blink could be due to a reduction in sensitivity or a response bias. The suggestion that a response bias could play a role seems misguided, as any response bias would be expected to be constant across lags, while the attentional blink effect is only observed at short lags. Thus, it is difficult to understand why the authors would think that a response bias could explain the attentional blink.

A second point of concern regards the way in which the measures for detection and discrimination accuracy were computed. If I understand the paper correctly, a correct detection was defined as either correctly identifying T2 (i.e., reporting CW or CCW if T2 was CW or CCW, respectively, see Figure 2B), or correctly reporting T2's absence (a correct rejection). Here, it seems that one should also count a misidentification (i.e., incorrect choice of CW or CCW when T2 was present) as a correct detection, because participants apparently did detect T2, but failed to judge/remember its orientation properly in case of a misidentification. Conversely, the manner in which discrimination performance is computed also raises questions. Here, the authors appear to compute accuracy as the average proportion of T2-present trials on which participants selected the correct response option for T2, thus including trials in which participants missed T2 entirely. Thus, a failure to detect T2 is now counted as a failure to discriminate T2. Wouldn't a more proper measure of discrimination accuracy be to compute the proportion of correct discriminations for trials in which participants detected T2?

My last point of critique is that the paper offers little if any guidance on how the inferred distinction between detection and discrimination can be linked to existing theories of the attentional blink. The discussion mostly focuses on comparisons to previous EEG studies, but it would be interesting to know how the authors connect their findings to extant, mechanistic accounts of the attentional blink. A key question here is whether the finding of dissociable processes of detection and discrimination would also hold with more meaningful stimuli in an identification task (e.g., the canonical AB task of identifying two letters shown amongst digits). There is evidence to suggest that meaningful stimuli are categorized just as quickly as they are detected (Grill-Spector & Kanwisher, 2005; Grill-Spector K, Kanwisher N. Visual recognition: as soon as you know it is there, you know what it is. Psychol Sci. 2005 Feb;16(2):152-60. doi: 10.1111/j.0956-7976.2005.00796.x. PMID: 15686582.). Does that mean that the observed distinction between detection and discrimination would only apply to tasks in which the targets consist of otherwise meaningless visual elements, such as lines of different orientations?

Reviewer #1 (Public Review):

Summary:

This manuscript from So et al. describes what is suggested to be an improved protocol for single-nuclei RNA sequencing (snRNA-seq) of adipose tissue. The authors provide evidence that modifications to the existing protocols result in better RNA quality and nuclei integrity than previously observed, with ultimately greater coverage of the transcriptome upon sequencing. Using the modified protocol, the authors compare the cellular landscape of murine inguinal and perigonadal white adipose tissue (WAT) depots harvested from animals fed a standard chow diet (lean mice) or those fed a high-fat diet (mice with obesity).

Strengths:

Overall, the manuscript is well-written, and the data are clearly presented. The strengths of the manuscript rest in the description of an improved protocol for snRNA-seq analysis. This should be valuable for the growing number of investigators in the field of adipose tissue biology that are utilizing snRNA-seq technology, as well as those other fields attempting similar experiments with tissues possessing high levels of RNAse activity.

Moreover, the study makes some notable observations that provide the foundation for future investigation. One observation is the correlation between nuclei size and cell size, allowing for the transcriptomes of relatively hypertrophic adipocytes in perigonadal WAT to be examined. Another notable observation is the identification of an adipocyte subcluster (Ad6) that appears "stressed" or dysfunctional and likely localizes to crown-like inflammatory structures where pro-inflammatory immune cells reside.

Weaknesses:

Analogous studies have been reported in the literature, including a notable study from Savari et al. (Cell Metabolism). This somewhat diminishes the novelty of some of the biological findings presented here. Moreover, a direct comparison of the transcriptomic data derived from the new vs. existing protocols (i.e. fully executed side by side) was not presented. As such, the true benefit of the protocol modifications cannot be fully understood.

Reviewer #1 (Public Review):

Summary:

This paper by Beath et. al. identifies a potential regulatory role for proteins involved in cytoplasmic streaming and maintaining the grouping of paternal organelles: holding sperm contents in the fertilized embryos away from the oocyte meiotic spindle so that they don't get ejected into the polar body during meiotic chromosome segregation. The authors show that by time-lapse video, paternal mitochondria (used as a readout for sperm and its genome) is excluded from yolk granules and maternal mitochondria, even when moving long distances by cytoplasmic streaming. To understand how this exclusion is accomplished, they first show that it is independent of both internal packing and the engulfment of the paternal chromosomes by maternal endoplasmic reticulum creating an impermeable barrier. They then test whether the control of cytoplasmic steaming affects this exclusion by knocking down two microtubule motors, Katanin and kinesis I. They find that the ER ring, which is used as a proxy for paternal chromosomes, undergoes extensive displacement with these treatments during anaphase I and interacts with the meiotic spindle, supporting their hypothesis that the exclusion of paternal chromosomes is regulated by cytoplasmic streaming. Next, they test whether a regulator of maternal ER organization, ATX-2, disrupts sperm organization so that they can combine the double depletion of ATX-2 and KLP-7, presumably because klp-7 RNAi (unlike mei-1 RNAi) does not affect polar body extrusion and they can report on what happens to paternal chromosomes. They find that the knockdown of both ATX-2 and KLP-7 produces a higher incidence of what appears to be the capture of paternal chromosomes by the meiotic spindle (5/24 vs 1/25). However, this capture event appears to halt the cell cycle, preventing the authors from directly observing whether this would result in the paternal chromosomes being ejected into the polar body.

Strengths:

This is a useful, descriptive paper that highlights a potential challenge for embryos during fertilization: when fertilization results in the resumption of meiotic divisions, how are the paternal and maternal genomes kept apart so that the maternal genome can undergo chromosome segregation and polar body extrusion without endangering the paternal genome? In general, the experiments are well-executed and analyzed. In particular, the authors' use of multiple ways to knock down ATX-2 shows rigor.

Weaknesses:

The paper makes a case that this regulation may be important but the authors should do some additional work to make this case more convincing and accessible for those outside the field. In particular, some of the figures could include greater detail to support their conclusions, they could explain the rationale for some experiments better and they could perform some additional control experiments with their double depletion experiments to better support their interpretations. Also, the authors' inability to assess the functional biological consequences of the capture of the sperm genome by the oocyte spindle should be discussed, particularly in light of the cell cycle arrest that they observe.

Joint Public Review:

Summary:

The study identified biallelic variants of DNAH3 in four unrelated Han Chinese infertile men through whole-exome sequencing, which contributes to abnormal sperm flagellar morphology and ultrastructure. To investigate the importance of DNAH3 in male infertility, the authors generated crispant DNAH3 knockout (KO) male mice. They observed that KO mice are also infertile, showing a severe reduction in sperm movement with abnormal IDA (inner dynein arms) and mitochondrion structure. Moreover, nonfunctional DNAH3 expression decreased the expression of IDA-associated proteins in the spermatozoa of patients and KO mice, which are involved in the disruption of sperm motility. Interestingly, the infertility of patients and KO mice was rescued by intracytoplasmic sperm injection (ICSI). Taken together, the authors propose that DNAH3 is a novel pathogenic gene for asthenoterozoospermia and male infertility.

Strengths:

This work investigates the role of DNAH3 in sperm mobility and male infertility and utilised gold-standard molecular biology techniques, showing strong evidence of its role in male infertility. All aspects of the study design and methods are well described and appropriate to address the main question of the manuscript. The conclusions drawn are consistent with the analyses conducted and supported by the data.

Weaknesses:

(1) The manuscript lacks a comparison with previous studies on DNAH3 in the Discussion section.

(2) The variants of DNAH3 in four infertile men were identified through whole-exome sequencing. Providing an overview of the WES data would be beneficial to offer additional insights into whether other variants may contribute the infertility. This could also help explain why ICSI only works for two out of four patients with DNAH3 variants.

(3) Quantification of images would help substantiate the conclusions, particularly in Figures 2, 3, 4, and 6. Improved images in Figures 3A, 4B, and 4C, would help increase confidence in the claims made.

Reviewer #1 (Public Review):

The goal of Knudsen-Palmer et al. was to define a biological set of rules that dictate the differential RNAi-mediated silencing of distinct target genes, motivated by facilitating the long-term development of effective RNAi-based drugs/therapeutics. To achieve this, the authors use a combination of computational modeling and RNAi function assays to reveal several criteria for effective RNAi-mediated silencing. This work provides insights into how (1) cis-regulatory elements influence the RNAi-mediated regulation of genes; (2) it is determined that genes can "recover" from RNAi-silencing signals in an animal; and 3) pUGylation occurs exclusively downstream of the dsRNA trigger sequence, suggesting 3º siRNAs are not produced. In addition, the authors show that the speed at which RNAi-silencing is triggered does not correlate with the longevity of the silencing. These insights are significant because they suggest that if we understand the rules by which RNAi pathways effectively silence genes with different transcription/processing levels then we can design more effective synthetic RNAi-based therapeutics targeting endogenous genes. The conclusions of this study are mostly supported by the data, but there are some aspects that need to be clarified.

(1) The methods do not describe the "aged RNAi plates feeding assay" in Figure 2E. The figure legend states that "aged RNAi plates" were used to trigger weaker RNAi, but the detail explaining the experiment is insufficient. How aged is aged? If the goal was to effectively reduce the dsRNA load available to the animals, why not quantitatively titrate the dsRNA provided? Were worms previously fed on the plates, or was simply a lawn of bacteria grown until presumably the IPTG on the plate was exhausted?

(2) Is the data presented in Figure 2F completed using the "aged RNAi plates" to achieve the partial silencing of dpy-7 observed? Clarification of this point would be helpful.

(3) Throughout the manuscript the authors refer to "non-dividing cells" when discussing animals' ability to recover from RNA silencing. It is not clear what the authors specifically mean with the phrase "non-dividing cells", but as this is referred to in one of their major findings, it should be clarified. Do they mean the cells are somatic cells in aged animals, thus if they are "non-dividing" the siRNA pools within the cells cannot be diluted by cell division? Based on the methods, the animals of RNAi assays were L4/Young adults that were scored over 8 days after the initial pulse of dsRNA feeding. If this is the case, wouldn't these animals be growing into gravid adults after the feeding, and thus have dividing cells as they grew?

(4) What are the typical expression levels/turnover of unc-22 and bli-1? Based on the results from the altered cis-regulatory regions of bli-1 and unc-22 in Figure 5, it seems like the transcription/turnover rates of each of these genes could also be used as a proof of principle for testing the model proposed in Figure 4. The strength of the model would be further increased if the RNAi sensitivity of unc-22 reflects differences in its transcription/turnover rates compared to bli-1.

Reviewer #1 (Public Review):

Summary:

Clostridium thermocellum serves as a model for consolidated bioprocess (CBP) in lignocellulosic ethanol production, but yet faces limitations in solid contents and ethanol titers achieved by engineered strains thus far. The primary ethanol production pathway involves the enzyme aldehyde-alcohol dehydrogenase (AdhE), which forms long oligomeric structures known as spirosomes, previously characterized via the 3.5 Å resolution E. coli AdhE structure using single-particle cryo-EM. The present study describes the cryo-EM structure of the C. thermocellum ortholog, sharing 62% sequence identity with E. coli AdhE, resolved at 3.28 Å resolution. Detailed comparative structural analysis, including the Vibrio cholerae AdhE structure, was conducted. Integrating cryo-EM data with molecular dynamics simulations indicated that the aldehyde intermediate resides longer in the channel of the extended form, supporting the hypothesis that the extended spirosome represents the active form of AdhE.

Strengths:

The study conducts a comprehensive structural comparative analysis of oligomerization interfaces and the acetaldehyde channel across compact and extended conformations. Structural and computational results suggest the extended spirosome as the most likely active state of AdhE.

Weaknesses:

The overall resolution of the C. thermocellum structure is similar to the E. coli ortholog, which shares 62% sequence identity, and the oligomerization interfaces and the acetaldehyde channel were previously described.

Reviewer #1 (Public Review):

In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient.

The authors observed that NMIIA is required for durotaxis and, building on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis.

The experiments support the main message of the paper regarding durotaxis by amoeboid cells. In my opinion, a few clarifications on the mechanism proposed to explain this phenomenon could strengthen this research:

(1) According to your model, the rear end of the cell, which is in contact with softer substrates, will have slower diffusion rates of MNIIA. Does this mean that bigger cells will durotax better than smaller cells because the stiffness difference between front and rear is higher? Is it conceivable to attenuate the slope of the durotactic gradient to a degree where smaller cells lose their ability to durotact, while longer cells retain their capacity for directional movement?

(2) Where did you place the threshold for soft, middle, and stiff regions (Figure 6)? Is it possible that you only have a linear rigidity gradient in the center of your gel and the more you approach the borders, the flatter the gradient gets? In this case, cells would migrate randomly on uniform substrates. Did you perform AFM over the whole length of the gel or just in the central part?

(3) In which region (soft, middle, stiff) did you perform all the cell tracking of the previous figures?

(4) What is the level of confinement experienced by the cells? Is it possible that cells on the soft side of the gels experience less confinement due to a "spring effect" whereby the coverslips descending onto the cells might exert diminished pressure because the soft hydrogels act as buffers, akin to springs? If this were the case, cells could migrate following a confinement gradient.

Reviewer #1 (Public Review):

Summary:

The authors address cellular mechanisms underlying the early stages of Sjogren's syndrome, using a mouse model in which 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA) is applied to stimulate the interferon gene (STING) pathway. They show that, in this model, salivary secretion in response to neural stimulation is greatly reduced, even though individual secretory cell calcium responses were enhanced. They attribute the secretion defect to reduced activation of Ca2+ -activated Cl- channels (TMEM16a), due to an increased distance between Ca2+ release channels (IP3 receptors) and TMEM16a which is expected to reduce the [Ca2+] sensed by TMEM16a. A variety of disruptions in mitochondria were also observed after DMXAA treatment, including reduced abundance, altered morphology, depolarization, and reduced oxygen consumption rate. The results of this study shed new light on some of the early events leading to the loss of secretory function in Sjogren's syndrome, at a time before inflammatory responses cause the death of secretory cells.

Strengths:

Two-photon microscopy enabled Ca2+ measurements in the salivary glands of intact animals in response to physiological stimuli (nerve stimulation). This approach has been shown previously by the authors as necessary to preserve the normal spatiotemporal organization of calcium signals that lead to secretion under physiological conditions.

Superresolution (STED) microscopy allowed precise measurements of the spacing of IP3R and TMEM16a and the cell membranes that would otherwise be prevented by the diffraction limit. The measured increase of distance (from 84 to 155 nm) would be expected to reduce [Ca2+] at the TMEM16a channel.

The authors effectively ruled out a variety of alternative explanations for reduced secretion, including changes in AQP5 expression, TMEM16a expression, localization, and Ca2+ sensitivity as indicated by Cl- current in response to defined levels of Ca2+.

Weaknesses:

While the Ca2+ distribution in the cells was less restricted to the apical region in DMXAA-treated cells, it is not clear that this is relevant to the reduced activation of TMEM16a. The way in which the change in Ca2+ distribution is quantified (apical/basal ratio) is not informative, as this is not what activates TMEM16a, but rather the local [Ca2+] at the channel.

Despite the decreased level of secretion, Ca2+ signal amplitudes were higher in the treated cells, raising the question of how much this might compensate for the increased distance between IP3R and TMEM16a. The authors assume that the increased separation of IP3R and TMEM16a (and the resulting decrease in local [Ca2+]) outweighed the effect of higher global [Ca2+], but this important point was not addressed.

The description of mitochondrial changes in abundance, morphology, membrane potential, and oxygen consumption rate were not well integrated into the rest of the paper. While they may be a facet of the multiple effects of STING activation and may occur during Sjogren's syndrome, their possible role in reducing secretion was not examined. As it stands, the mitochondrial results are largely descriptive and there is no evidence here that they contribute to the secretory phenotype.

Contact outside of the classroom is difficult for both commuters in on-ground courses and online environments. Also it's becoming an issue for traditional students who are working more and more hours to reduce overall costs of education.

Reviewer #1 (Public Review):

Summary:

The current manuscript provides an extensive in vivo analysis of two guidance pathways identifying multiple mechanisms that shape the bifurcation of DRG axons when forming the dorsal funiculus in the DREZ.

Strengths:

Multiple mouse mutant lines were used, together with complementary techniques; the results are very clear and compelling.<br /> The findings are very significant and clearly move forward our understanding of the regulation of axonal development at the DREZ.

Weaknesses:

No major weaknesses were found. As it is I have no recommendations that would increase the clarity or quality of the manuscript.

Reviewer #2 (Public Review):

The authors sought to establish the role played by N343 glycosylation on the SARS-CoV-2 S receptor binding domain structure and binding affinity to the human host receptor ACE2 across several variants of concern. The work includes both computational analysis in the form of molecular dynamics simulations and experimental binding assays between the RBD and ganglioside receptors.

The work extensively samples the conformational space of the RBD beginning with atomic coordinates representing both the bound and unbound states and computes molecular dynamics trajectories until equilibrium is achieved with and without removing N343 glycosylation. Through comparison of these simulated structures, the authors are able to demonstrate that N343 glycosylation stabilizes the RBD. Prior work had demonstrated that glycosylation at this site plays an important role in shielding the RBD core and in this work the authors demonstrate that removal of this glycan can trigger a conformational change to reduce water access to the core without it. This response is variant dependent and variants containing interface substitutions which increase RBD stability, including Delta substitution L452R, do not experience the same conformational change when the glycan is removed. The authors also explore structures corresponding to Alpha and Beta in which no structure-reinforcing substitutions were identified and two Omicron variants in which other substitutions with an analogous effect to L452R are present.

The authors experimentally assessed these inferred structural changes by measuring the binding affinity of the RBD for the oligosaccharides of the monosialylated gangliosides GM1os and GM2os with and without the glycan at N343. While GM1os and GM2os binding is influenced by additional factors in the Beta and Omicron variants, the comparison between Delta and Wuhan-hu-1 is clear: removal of the glycan abrogated binding for Wuhan-hu-1 and minimally affected Delta as predicted by structural simulations.

In summary, these findings suggest, in the words of the authors, that SARS-CoV-2 has evolved to render the N-glycosylation site at N343 "structurally dispensable". This study emphasizes how glycosylation impacts both viral immune evasion and structural stability which may in turn impact receptor binding affinity and infectivity. Mutations which stabilize the antigen may relax the structural constraints on glycosylation opening up avenues for subsequent mutations which remove glycans and improve immune evasion. This interplay between immune evasion and receptor stability may support complex epistatic interactions which may in turn substantially expand the predicted mutational repertoire of the virus relative to expectations which do not take into account glycosylation.

Reviewer #1 (Public Review):

Summary:

The study by Seo et al highlights knowledge gaps regarding the role of cerebellar complex spike (CS) activity during different phases of learning related to optokinetic reflex (OKR) in mice. The novelty of the approach is twofold: first, specifically perturbing the activity of climbing fibers (CFs) in the flocculus (as opposed to disrupting communication between the inferior olive (IO) and its cerebellar targets globally); and second, examining whether disruption of the CS activity during the putative "consolidation phase" following training affects OKR performance.

The first part of the results provides adequate evidence supporting the notion that optogenetic disruption of normal CF-Purkinje neuron (PN) signaling results in the degradation of OKR performance. As no effects are seen in OKR performance in animals subjected to optogenetic irradiation during the memory consolidation or retrieval phases, the authors conclude that CF function is not essential beyond memory acquisition. However, the manuscript does not provide a sufficiently solid demonstration that their long-term activity manipulation of CF activity is effective, thus undermining the confidence of the conclusions.

Strengths:

The main strength of the work is the aim to examine the specific involvement of the CF activity in the flocculus during distinct phases of learning. This is a challenging goal, due to the technical challenges related to the anatomical location of the flocculus as well as the IO. These obstacles are counterbalanced by the use of a well-established and easy-to-analyse behavioral model (OKR), that can lead to fundamental insights regarding the long-term cerebellar learning process.

Weaknesses:

The impact of the work is diminshed by several methodological shortcomings.

Most importantly, the key finding that prolonged optogenetic inhibition of CFs (for 30 min to 6 hours after the training period) must be complemented by the demonstration that the manipulation maintains its efficacy. In its current form, the authors only show inhibition by short-term optogenetic irradiation in the context of electrical-stimulation-evoked CSs in an ex vivo preparation. As the inhibitory effect of even the eNpHR3.0 is greatly diminished during seconds-long stimulations (especially when using the yellow laser as is done in this work (see Zhang, Chuanqiang, et al. "Optimized photo-stimulation of halorhodopsin for long-term neuronal inhibition." BMC biology 17.1 (2019): 1-17. ), we remain skeptical of the extent of inhibition during the long manipulations. In short, without a demonstration of effective inhibition throughout the putative consolidation phase (for example by showing a significant decrease in CS frequency throughout the irradiation period), the main claim of the manuscript of phase-specific involvement of CF activity in OKR learning can not be considered to be based on evidence.

Second, the choice of viral targeting strategy leaves gaps in the argument for CF-specific mechanisms. CaMKII promoters are not selective for the IO neurons, and even the most precise viral injections always lead to the transfection of neurons in the surrounding brainstem, many of which project to the cerebellar cortex in the form of mossy fibers (MF). Figure 1Bii shows sparsely-labelled CFs in the flocculus, but possibly also MFs. While obtaining homogenous and strong labeling in all floccular CFs might be impossible, at the very least the authors should demonstrate that their optogenetic manipulation does not affect simple spiking in PNs.

Finally, while the paper explicitly focuses on the effects of CF-evoked complex spikes in the PNs and not, for example, on those mediated by molecular layer interneurons or via direct interaction of the CF with vestibular nuclear neurons, it would be best if these other dimensions of CF involvement in cerebellar learning were candidly discussed.

Reviewer #1 (Public Review):

Summary:

In this paper, the authors used target agnostic MBC sorting and activation methods to identify B cells and antibodies against sexual stages of Plasmodium falciparum. While they isolated some Mabs against PFs48/45 and PFs230, two well-known candidates for "transmission blocking" vaccines, these antibodies' efficacies, as measured by TRA, did not perform as well as other known antibodies. They also isolated one cross-reactive mAb to proteins containing glutamic acid-rich repetitive elements, that express at different stages of the parasite life cycle. They then determined the structure of the Fab with the highest protein binder they could determine through protein microarray, RESA, and observed homotypic interactions.

Strengths:

- Target agnostic B cell isolation (although not a novel methodology).<br /> - New cross-reactive antibody and mechanism (homotypic interactions) as demonstrated by structural data and other biophysical data.

Weaknesses:

The paper lacks clarity at times and could benefit from more transparency (showing all the data) and explanations.<br /> In particular:<br /> -define SIFA<br /> -define TRAbs<br /> -it is not possible to read the Supplementary Figure 6B and C panels.

Reviewer #1 (Public Review):

Throughout the paper, the authors do a fantastic job of highlighting caveats in their approach, from image acquisition to analysis. Despite this, some conclusions and viewpoints portrayed in this study do not appear well-supported by the provided data. Furthermore, there are a few technical points regarding the analysis that should be addressed.

(1) Analysis of signaling traces

- Relevance of "modeled signaling level": It is not clear whether this added complexity and potential for error (below) provides benefits over a more simple analysis such as taking the derivative (shown in Figure 3C). Could the authors provide evidence for the benefits? For example, does the "maximal response" given a simpler metric correlate less well with cell fate than that calculated from the fitted response?

- Assumptions for "modeled signaling level": According to equation (1) Kaede levels are monotonically increasing. This is assumed given the stability of the fluorescent protein. However, this only holds for the "totally produced Kaede/fluorescence". Other metrics such as mean fluorescence can very well decrease over time due to growth and division. Does "intensity" mean total fluorescence? Visual inspection of the traces shown in Figure 2 suggests that "fluorescence intensity" can decrease. What does this mean for the inferred traces?

- Estimation of Kaede reporter half-live: It is not clear how the mRNA stability of Kaede is estimated. It sounds like it was just assessed visually, which seems not entirely appropriate given the quantitative aspects of the rest of the study. Also, given that Shh signaling was inhibited on the level of Smoothened, it is not obvious how the dynamics of signaling shutdown affect the estimate. Most results in Figure 7 seem to be quite robust to the estimate of the half-live. That they are, might suggest that the whole analysis is unnecessary in the first place. However, not all are. Thus, it would be important to make this estimate more quantitative.

(2) Assignment of fates and correlations

- Error estimate for cell-type assignment: Trying to correlate signaling traces to cell fate decisions requires accurate cell fate assignment post-tracking. The provided protocol suggests a rather manual, expert-directed process of making those decisions. Can the authors provide any error-bound on those decisions, for example comparing the results obtained by two experts or something comparable? I am particularly concerned about the results regarding the higher degree of variability in the correlation between signaling dynamics and cell fate in the posterior neural tube. Here, the expression of Olig2 does not seem to segregate between different assigned fates, while it does so nicely in the anterior neural tube. This would suggest to me that cells in the posterior neural tube might not yet be fully committed to a fate or that there could be a relatively high error rate in assigning fates. Thus, the results could emerge from technical errors or differences in pure timing. Could the authors please comment on these possibilities?

- Clustering and fates: One approach the authors use to analyze the correlation between signaling and fate is clustering of cell traces and comparison of the fate distributions in those clusters. There is a large number of clusters with only single traces, suggesting that the data (number of traces) might not be sufficient for this analysis. Furthermore, I am skeptical about clustering cells of different anterior-posterior identities together, given potential differences in the timing of signal reception and signaling. I am not convinced that this analysis reveals enough about how signaling maps to fate given the heterogeneity in traces in large clusters and the prevalence of extremely small clusters.

- Signaling vector and hand-picked metrics: As an alternative approach, that might be better suited for their data, the authors then pick three metrics (based on their model-predicted signaling dynamics) and show that the maximal response is a very good predictor of fate for different anterior-posterior identities. Previous information-theoretic analysis of signaling dynamics has found that a whole time-vector of signaling can carry much more information than individual metrics (Selimkhanov et al, 2014, PMID: 25504722). Have the authors tried to use approaches that make use of the whole trace (such as simple classifiers (Granados et al, 2018, PMID: 29784812), or can comment on why this is not feasible for their data? The authors should at least make clear that their results present a lower bound to how accurately cells can make cell-fate decisions based on signaling dynamics.

(3) Consequences of signaling heterogeneity

The authors focus heavily on portraying that signaling dynamics are highly variable, which seems visually true at first glance. However, there is no metric used or a description given of what this actually means. Mainly, the variability seems to relate to the correlation between signaling and fate. However, given the data and analysis, I would argue that the decoding of signaling dynamics into fate is surprisingly accurate. So signaling dynamics that seem quite noisy and variable by visual inspection can actually be very well discriminated by cells, which to me appears very exciting.

Indeed, simple features of signaling traces can predict cell fate as well as position (for anterior progenitors). Given that signaling should be a function of position, it naively seems as if signaling read-out could be almost perfect. It might be interesting to plot dorsal-ventral position vs the signaling metrics, to also investigate how Shh concentration/position maps to signaling dynamics, this would give an even more comprehensive view of signal transmission.

There remains the discrepancy between signaling traces and fate in the posterior neural tube. The authors point towards differences in tissue architecture and difficulties in interpreting a "small" Shh gradient. However, the data seems consistent with differences in timing of cell-fate decisions between anterior and posterior cells. The authors show that fate does initially not correlate well with position in the posterior neural tube. So, signaling dynamics should likely also not, as they should rather be a function of position, given they are downstream of the Shh gradient. As mentioned above, not even Olig2 expression does segregate the assigned fates well. All this points towards a difference in the time of fate assignment between the anterior and posterior. Given likely delays in reporter protein production and maturation, it can thus not be expected that signaling dynamics correlate better with cell fate than the reporter "83%". Can the authors please discuss this possibility in the paper?

Thus, while this paper represents an example of what the community needs to do to gain a better understanding of robust patterning under variability, the provided data is not always sufficient to make clear conclusions regarding the functional consequences of signaling dynamics.

Reviewer #1 (Public Review):

Summary:

There is a long-standing idea that choices influence evaluation: options we choose are re-evaluated to be better than they were before the choice. There has been some debate about this finding, and the authors developed several novel methods for detecting these re-evaluations in task designs where options are repeatedly presented against several alternatives. Using these novel methods the authors clearly demonstrate this re-evaluation phenomenon in several existing datasets.

Strengths:

The paper is well-written and the figures are clear. The authors provided evidence for the behaviour effect using several techniques and generated surrogate data (where the ground truth is known) to demonstrate the robustness of their methods.

Weaknesses:

The description of the results of the fMRI analysis in the text is not complete: weakening the claim that their re-evaluation algorithm better reveals neural valuation processes.

Reviewer #1 (Public Review):

Summary:

Drosophila is one of the most studied model organisms to understand how neural circuits form and function to control intricate animal behaviors. The ventral nerve cord (VNC) part of the fly's CNS serves as a sensory processing and motor output center just like our spinal cord. Over the last decade, the VNC has become a fruitful platform to understand neural circuits responsible for motor behavior such as walking and flying. The missing resource was the complete connectome of the VNC neurons. This study provides this needed resource. The authors documented their approaches on how to generate the data from tissue preparation to computer-assisted reconstruction in a simple manner and left the in-depth analysis of the network features of the connecting neurons to two other well-written companion articles.

Strengths:<br /> Unlike many other previously published EM datasets, the authors presented a ready-to-view connectome dataset of the adult fly VNC. Readers, without needing permission, can access the dataset to find their neurons of interest and determine their synaptic partners with a few clicks. The authors also share their novel approaches in a detailed manner for others to reproduce similar EM volumes for other tissues.

Weaknesses:

The reconstruction completion, around 50%, might be considered a weakness. However, the data appear to have ~ %50 completion across all different neuropils suggesting that sampling is homogenous and does not induce bias. Nevertheless, a higher percentage will give a more complete picture.

DOI: 10.1186/s12861-020-00217-1

Resource: RRID:BDSC_31501

Curator: @abever99

SciCrunch record: RRID:BDSC_31501

What is this?

Reviewer #1 (Public Review):

This is an important and very well conducted study providing novel evidence on the role of zinc homeostasis for the control of infection with the intracellular bacterium S. typhimurium also disentangling the underlying mechanisms and providing clear evidence on the importance of spatio-temporal distribution of (free) zinc within the cell.

Comments:

It would be important to provide more information on the genotype of mice. It is rather unlikely that C57Bl6 mice survive up to two weeks after i.p. injection of 1x10E5 bacteria.

To be sure that macrophages Slc30A1 fl/fl LysMcre mice really have an impaired clearance of bacteria it would be important to rule out an effect of Slc30A1 deletion of bacterial phagocytosis and containment (f.e. evaluation of bacterial numbers after 30 min of infection).

Does the addition of zinc to macrophages negatively affect iNOS transcription as previously observed for the divalent metal iron and is a similar mechanism also employed (CEBPß/NF-IL6 modulation) (Dlaska M et al. J Immunol 1999)?

How does Zinc or TPEN supplementation to bacteria in LB medium affect the log growth of Salmonella?

Reviewer #1 (Public Review):

Sertonin is an important neurotransmitter and it synaptic concentration is controlled by re-uptake by the sodium-coupled serotonin transporter SERT. In this paper, some 6000 mutations of SERT were made and tested for surface expression and uptake of a serotonin analogue APP+. The SERT mutants were analysed and compared to the SERT structure and dynamics based on MD simulations. The authors have concluded that mutations located on surface exposed regions are tolerated whilst those involved in packing and structural integrity are not. Gain-of-function mutations map onto regions that in most cases favour opening of a solvent-exposed intracellular vestibule. Closure of the intracellular gate is thought to be rate-limiting to the transport cycle, and thus the evolutionary-based screen is consistent with the clustering of gain-of-function mutations.

Strengths:<br /> This paper using a large unbiased data-set to probe the evolution of the serotonin transporter SERT for the substrate APP+. They have been able to compare both localisation and transport data, which is an interesting data-set. Using MD simulations they are further able to provide some rationale basis for the gain-of-function mutants.

Weaknesses:<br /> They can only detect surface expression of myc-tagged SERT based on conjugation with a fluorescent anti-myc antibody. As such, they cannot distinguish between SERT mutants that abolish expression vs. those that are no longer trafficking to the plasma membrane. This is a downside, as it would have been interesting to know the fraction of SERT mutations disrupt trafficking. Indeed, the relationship between misfolding and targeting is poorly understood beyond the calnexin- calreticulin cycle. Furthermore, there seems to be a gap between the large-scale mutagenesis data and the MD simulations in which the main mechanistic conclusions seem to be based on (carried out in a separate publication). Thus, overall while the mutation data-set is impressive its not clear how this aids to our mechanistic understanding of SERT.

Reviewer #1 (Public Review):

The authors report a high-quality genome assembly for a member of Xenacoelomorpha, a taxon that is at the center of the last remaining great controversies in animal evolution. The taxon and the species in question have "jumped around" the animal tree of life over the past 25 years, and seemed to have found their place as a sister-group to all remaining bilaterians. This hypothesis posits that the earliest split within Bilateria includes Xenacoelomorpha on the one hand and a clade known as Nephrozoa (Protostomia + Deuterostomia) on the other, and is thus referred to as the Nephrozoa hypothesis. Nephrozoa is supported by phylogenomic evidence, by a number of synapomorphic morphological characters in the Nephrozoa (namely, the presence of nephridia) and lack of some key bilaterian characters in Xenacoelomorpha, and by the presence of unique miRNAs in Nephrozoa.

The Nephrozoa hypothesis has been challenged several times by the authors' groups who alternatively suggest placing Xenacoelomorpha within Deuterostomia as a sister group to a clade known as Ambulacraria. This hypothesis (the Xenambulacraria hypothesis) is supported by alternative phylogenomic datasets and by the shared presence of a number of unique molecular signatures. In this contribution, the authors aim to strengthen their case by providing full genome data for Xenoturbella bocki.<br /> The actual sequencing and analysis are technically and methodologically excellent. Some of the analyses were done several years ago using approaches that may now seem obsolete, but there is no reason not to include them. As a detailed report of a newly sequenced genome, the manuscript meets the highest standards.

The authors emphasize a number of key findings. One is the fact that the genome is not as simple as one might expect from a "basal" taxon, and is on par with other bilaterian genomes and even more complex than the genome of secondarily simplified bilaterians. There is an implicit expectation here that the sister group to all Bilateria would represent the primitive state. This is of course not true, and the authors are aware of this, but it sometimes feels as though they are using this implicit assumption as a straw dog argument to say that since the genome is not as simple as expected, X. bocki must be nested within Bilateria. The authors get around this by acknowledging that their finding is consistent with a "weak version of the Nephrozoa hypothesis", which is essentially the Nephrozoa phylogenetic hypothesis without implicit assumptions of simplicity.

Another finding is a refutation of the miRNA data supporting Nephrozoa. This is an important finding although it is somewhat flogging a dead horse, since there is already a fair amount of skepticism about the validity of the miRNA data (now over 20 years old) for higher-level phylogenetics.

The finding that the authors feel is most important is gene presence-absence data that recovers a topology in which X. bocki is sister to Abulacraria. The problem is that the same tree does not support the monophyly of Xenacoelomorpha. This may be an artifact of fast evolving acoel genomes, as the authors suggest, but it still raises questions about the robustness of the data.

In sum, the authors' results and analyses leave an open window for the Xenambulacraria hypothesis, but do not refute the Nephrozoa hypothesis. The manuscript is a valuable contribution to the debate but does not go a significant way towards its resolution.<br /> The manuscript has gone through several rounds of review and revision on a preprint server and is thus fairly clear of typos, inconsistencies and lack of clarity. The authors are honest and open in their interpretation of the results and their strengths.

Reviewer #1 (Public Review):

Rubin et al. study chondrocyte columns in the prenatal and postnatal growth plate in 3D for the first time, using a novel analysis pipeline in which Confetti clones in the murine growth plate are analysed morphometrically. Prenatal chondrocytes were found not to be organised in columns parallel to the main orientation of the long bone, but rather, prenatal chondrocytes were commonly organised perpendicular to the main direction of growth. In the postnatal (P40) growth plate there was a diverse arrangement of columns, but more of the columns were vertically aligned

I enjoyed reading the work and the analysis is rigorous. However, I think that it is not valid to state that columns do not form in the embryo. The data only supports the finding that strictly vertical columns do not form in the embryo, as the cells are still organised into columns, albeit with a range of orientations. I do not like the term "typically" aligned, as how can we know what is "typical" when orientation has never before been assessed in 3D... And the authors' data demonstrates that it is certainly not "typical" for chondrocyte to organise into vertical columns prenatally.

It would be very interesting to delve deeper into the reason for the change in orientation of columns between pre- and post-natal. For example, does more circumferential growth happen prenatally as compared to postnatally? Is the rate of circumferential vs longitudinal growth different between prenatal and postnatal, and could the change in column orientation be responsible for a (possible) shift in the balance between longitudinal vs circumferential growth before vs after birth? The first sentence of the Discussion refers to the role of chondrocyte columns in driving bone elongation, but aren't they also involved in driving bone morphology?

I feel describing the activity of the cells as "mis-rotations" which implies the orientations are not intentional. It is likely not accidental or mistaken that the chondrocytes align in the ways they do- the diaphysis is largely for longitudinal growth while the epiphyses, and lateral expansion of the joint is also important. I find the data in Figure 4 fascinating, especially the variation in orientations between the regions of the growth plate (from proximal to distal), with the most lateral orientation at the most proximal and distal ends- it would be nice to see more discussion of these variations and what they may be contributing to.

The abstract focuses solely on the analysis of columns prenatally and would benefit from the inclusion of the data from the postnatal growth plate and from the chondrocyte rotations.

Reviewer #1 (Public Review):

This manuscript presents, for the first time, the utilization of PRV viral transneuronal tracing to elucidate the central coding and control mechanisms governing sympathetic nervous system (SNS) efferent signals to bone. This groundbreaking work not only holds promising research prospects but also establishes a robust foundation for understanding the neural regulation of bone metabolism.

Reviewer #1 (Public Review):

Garcia-Saldivar and colleagues present a manuscript investigating connections between diffusion-weighted imaging (DWI) parameters and paced finger tapping measures. A cohort of human participants (n=32) performed a paced finger tapping task with a synchronization-continuation paradigm, in which they were required to listen to a paced metronome, begin tapping in synchrony with it, and then continue tapping at the same rate without it. Both auditory and visual metronomes were used, at a range of intervals. All subjects received structural scans measuring DWI, with an emphasis on superficial and deep white matter structures. This latter analysis was the most innovative, as it allowed the authors to examine microstructural effects in short-range cortical connections.

Behaviorally, the authors replicated some well-known effects in paced finger tapping, with better performance for auditory over visual rhythms, negative lag-1 autocorrelations, and best performance at a range of ~1.5Hz. For the DWI analyses, a large number of correlations were observed across a wide variety of connections with various brain regions. The most salient effects observed were a connection between asynchrony, only for the auditory condition, and connections between the right auditory and motor systems, around the duration of peak performance, as well as a "chronotopic" organization across parts of the corpus callosum, most notably in areas linking motor regions between hemispheres.

Overall, this paper provides a critical missing link between measures of structural connectivity and rhythmic tapping abilities, pointing to some interesting possibilities for how tapping synchronization (at least for auditory intervals) is carried out. Negative aspects of the paper come from the largely exploratory aspects of the analysis, as well as potential biases from the low sample size.

Reviewer #1 (Public Review):

The manuscript demonstrates an analysis of the synaptic organization within the motor thalamus, emphasizing the interplay between the ventrolateral (VL) and ventroanterior (VA) nuclei and their respective inputs. The primary aim is to unravel the complexities of synaptic interactions among the motor cortex's layer 5 (M1L5), the cerebellum (Cb), and the basal ganglia output nuclei (GPi and SNr), which converge upon the VA/VL nuclei of the motor thalamus. This examination is executed using a combination of anatomical tracing, optogenetics, and electrophysiological recordings in mouse brain slices, which together yield novel insights into the motor control circuitry.

The study uncovers that contrary to traditional models that presumed segregation, some motor thalamic neurons simultaneously integrate inputs from the cerebellum and basal ganglia. Furthermore, a subset of these neurons also receive convergent inputs from M1L5 and basal ganglia, underscoring the complexity of these synaptic networks. Notably, the study reveals that both M1L5 and Cb inputs exhibit driver-type synaptic properties, suggesting a significant impact on thalamic relay neurons.

The functional implications of this synaptic convergence suggest a complex gating mechanism by the inhibitory outputs of the basal ganglia, which could modulate information flow within the motor thalamus. This modulation is significant not only for transthalamic information processing but also for the integration of cerebellar inputs to the motor cortex. The study also highlights direct projections from M1L5 to the motor thalamus, indicating a potential direct influence on thalamic activity, in addition to the known indirect influence through the cortico-basal ganglia-thalamo-cortical loop.

The manuscript suggests that the traditional understanding of motor thalamic connectivity requires reconsideration, and it emphasizes the necessity of further investigation to understand fully the functional implications of this synaptic convergence. Future research may focus on more direct demonstrations of triple-input convergence and its behavioral consequences, as well as cross-species comparative studies to enhance the findings' applicability.

While the study provides valuable contributions to our knowledge of the motor thalamus, illuminating the intricate synaptic architecture of the motor thalamus and setting the stage for future explorations that will deepen our comprehension of motor control and thalamic function.

Reviewer #1 (Public Review):

This manuscript presents an exciting new method for separating insulin secretory granules using insulator-based dielectrophoresis (iDEP) of immunolabeled vesicles. The method has the advantage of being able to separate vesicles by subtle biophysical differences that do not need to be known by the experimenter, and hence could in principle be used to separate any type of organelle in an unbiased way. Any individual organelle ("particle") will have a characteristic ratio of electrokinetic to dielectrophoretic mobilities (EKMr) that will determine where it migrates in the presence of an electric field. Particles with different EKMr will migrate differently and thus can be separated. The present manuscript is primarily a methods paper to show the feasibility of the iDEP technique applied to insulin vesicles. Experiments are performed on cultured cells in low or high glucose, with the conclusion that there are several distinct subpopulations of insulin vesicles in both conditions, but that the distributions in the two conditions are different. As it is already known that glucose induces release of mature insulin vesicles and stimulates new vesicle biosynthesis and maturation, this finding is not necessarily new, but is intended as a proof of principle experiment to show that the technique works. This is a promising new technology based on solid theory that has the possibility to transform the study of insulin vesicle subpopulations, itself an emerging field. The technique development is a major strength of the paper. Also, cellular fractionation and iDEP experiments are performed well, and it is clear that the distribution of vesicle populations is different in the low and high glucose conditions. However, more work is needed to characterize the vesicle populations being separated, leaving open the possibility that the separated populations are not only insulin vesicles, but might consist of other compartments as well. It is also unclear whether the populations might represent immature and mature vesicles, distinct pools of mature vesicles such as the readily releasable pool and the reserve pool, or vesicles of different age. Without a better characterization of these populations, it is not possible to assess how well the iDEP technique is doing what is claimed.

Major comments:

(1) There is no attempt to relate the separated populations of vesicles to known subpopulations of insulin vesicles such as immature and mature vesicles, or the more recently characterized Syt9 and Syt7 vesicle subpopulations that differ in protein and lipid composition (Kreutzberger et al. 2020). Given that it is unclear exactly what populations of vesicles will be immunolabeled (see point #2 below), it is also possible that some of the "subpopulations" are other compartments being separated in addition to insulin vesicles. It will be important to examine other markers on these separated populations or to perform EM to show that they look like insulin vesicles.

(2) An antibody to synaptotagmin V is used to immunolabel vesicles, but there has been confusion between synaptotagmins V and IX in the literature and it isn't clear what exactly is being recognized by this antibody (this reviewer actually thinks it is Syt 9). If it is indeed recognizing Syt 9, it might already be labeling a restricted population of insulin vesicles (Kreutzberger et al. 2020). The specificity of this antibody should be clarified. Furthermore, Figure 2 is not convincing at showing that this synaptotagmin antibody specifically labels insulin vesicles nor is there convincing colocalization of this synaptotagmin antibody with insulin vesicles. In the image shown, several cells show very weak or no staining of both insulin and the synaptotagmin. The highlighted cell appears to show insulin mainly in a perinuclear structure (probably the Golgi) rather than in mature vesicles (which should be punctate), and insulin is not particularly well-colocalized with the synaptotagmin. Other cells in the image appear to have even less colocalization of insulin and synaptotagmin, and there is no quantification of colocalization. It seems possible that this antibody is recognizing other compartments in the cell, which would change the interpretation of the populations measured in the iDEP experiments. It would also be good to perform synaptotagmin staining under glucose-stimulating conditions, in case this alters the localization.

(3) The EKMr values of the vesicle populations between the low and high glucose conditions don't seem to precisely match. It is unclear if this just a technical limitation in comparing between experiments or instead suggests that glucose stimulation does not just change the proportion of vesicles in the subpopulations (i.e. the relative fluorescent intensities measured), but rather the nature of the subpopulations (i.e. they have distinct biophysical characteristics). This again gets to the issue of what these vesicle subpopulations represent. If glucose stimulation is simply converting immature to mature vesicles, one might expect it to change the proportion of vesicles, but not the biophysical properties of each subpopulation.

(4) The title of the paper promises "isolation" of insulin vesicles, but the manuscript only presents separation and no isolation of the separated populations. Isolation of the separated populations is important to be able to better define what these populations are (see point #1 above). Isolation is also critical if this is to be a valuable technique in the future. Yet the paper is unclear on whether it is actually technically feasible to isolate the populations separated by iDEP. In line 367, it states "this method provides a mechanism for the isolation and concentration of fractions which show the largest difference between the two population patterns for further bioanalysis (imaging, proteomics, lipidomics, etc.)." However, in line 361 it says "developing the capability to port the collected individual boluses will enable downstream analyses such as mass spectrometry or electron microscopy," suggesting that true isolation of these populations is not yet feasible. This should be clarified.

Reviewer #1 (Public Review):

Summary:

The manuscript describes a model based on 5-state cellular automata of development of an infection. The model is motivated and qualitatively justified by time-resolved measurements of expression levels of viral, interferon-producing, and antiviral genes. The model is set up in such a way that the crucial difference in outcomes (infection spreading vs. confinement) depends on the initial fraction of special virus-sensing cells. Those cells (denoted as 'type a') cannot be infected and do not support the propagation of infection, but rather inhibit it in a somewhat autocatalytic way. Presumably, such feedback makes the transition between two outcomes very sharp: a minor variation in concentration of 'a' cells results in qualitative change from one outcome to another. As in any percolation-like system, the transition between propagation and inhibition of infection goes through a critical state with all its attributes, including a power-law distribution of the cluster size (corresponding to the fraction of infected cells) with a fairly universal exponent and a cutoff at the upper limit of this distribution.

Strengths:

The proposed model suggests a well-justified explanation for the frequently observed yet puzzling diversity of outcomes of viral infections such as COVID.

Weaknesses:

None.

Reviewer #1 (Public Review):

This paper can be seen as an extension of a recent study by two of the same authors [1]. In the previous paper, the authors considered two variants of the Moran process, labelled Model A and Model B, and examined differences between the evolutionary dynamics of these two models. They further described the site frequency spectra, expected allele counts, and expected singleton counts of these models, building on analytical results from prior studies, and used numerical simulations to investigate the models' evolutionary dynamics. Finally, they compared the site frequency spectra of the two models (using numerical simulations) to spectra derived from a small breast cancer data set (two sets of three samples).

In the new paper, the authors consider the same two Moran process variants (Model A and Model B) and some related branching processes. As before, they compare the site frequency spectra and various summary statistics of these models, but here they present only numerical simulations (except that some prior analytical results are summarized in Appendix A, which are never referred to in the main text and seem unconnected to the study). They then compare the site frequency spectra of these models (again using numerical simulations) to those derived from the same breast cancer samples as before and thus infer some evolutionary parameters.

The first main conclusion is that the critical branching process and the Moran process models behave similarly and generate similar site frequency spectra. This finding is unsurprising (indeed, the authors acknowledge that the result "has been expected"). For a reasonably large population size, the population size in the critical branching process has been shown to vary relatively little over time and the model is thus essentially a continuous time Moran process (see, for example, Equation 8.55 in ref 2). Nor is it surprising that the authors see stronger similarities when they select only the subset of branching process replicates in which the final population size is particularly close to the initial population size (this is because, in these replicates, the population size likely varies even less than usual).

The second main conclusion is that, although "the mutational SFS alone is not adequate" to quantify the strength of selection, "All fitted values for the selective disadvantage of passenger mutations are nonzero, supporting the view that they exert deleterious selection during tumorigenesis". Although the question of whether mildly deleterious mutations play an important role in cancer evolution is of considerable interest, it's debatable whether the results presented here help resolve the issue.

Many prominent researchers have called into question whether cancer evolutionary parameters can be reliably inferred from site frequency spectra (e.g., [3-7]), even using sophisticated statistical methods. The statistical approach used here (though not named as such in the paper) is a crude kind of approximate Bayesian computation. To improve the accuracy of the results, it would have been better to have set reasonably vague priors for the uncertain mutation rates, rather than fixing them arbitrarily. It would also have been better to have chosen a likelihood function explicitly based on an analysis of the sampling and error distributions, rather than just summing the absolute logged deviations. It is well known that "Checking the model is crucial to statistical analysis" and "A good Bayesian analysis, therefore, should include at least some check of the adequacy of the fit of the model to the data and the plausibility of the model for the purposes for which the model will be used" [8]. The authors' failure to describe any attempt to validate or check their model, using simulated data or otherwise, casts doubt on the reliability of their inferences.

Putting aside the potential biassing effects of sampling error, measurement error, and the limitations of the authors' statistical method, it is well established that both population growth and spatial structure profoundly alter the shape of site frequency spectra in ways that can mimic the effects of selection (e.g. [9-11]). Indeed, Figures 3, 4 and 5 show that the critical and super-critical branching processes generate markedly different site frequency spectra. It follows that if the population dynamics and spatial structure of the mathematical model used for inference don't match those of the biological process that produced the data then any inferred evolutionary parameter values will be unreliable. Breast cancer has two indisputable ecological features that shape its evolutionary dynamics: the cell population expands by many orders of magnitude from a single cell, and the population is spatially structured. In the authors' mathematical model, the population size is initially 100 cells and either remains constant or varies little, and there is no spatial structure. These profound mismatches between model and data cast further doubt on what is supposed to be the paper's most important biological finding.

In this paper the authors offer no justification for their decision to model breast cancer as a non-growing, non-spatial cell population. Nor do they engage with the extensive recent literature on the challenges of inferring evolutionary parameters from cancer site frequency spectra (they cite none of the many relevant papers listed at https://www.sottorivalab.org/neutral-evolution.html). Their 2022 paper [1] claims that, "it sometimes makes sense to consider cancer growth in the framework of constant-population models. Our models correspond to the situation in which a constant population of N "healthy" stem cells is gradually replaced by a growing clone of transformed cells with increasing fitness." No evidence was presented to support this hypothesis regarding breast cancer progression. On the other hand, a wealth of evidence supports the consensus view that, in breast cancer and other human solid tumours, the number of cells with unlimited proliferative potential is several orders of magnitude greater than 100 and grows over time (e.g. [12]).

Analytic expressions for the site frequency spectra with neutral mutations are already known. It is well known that the site frequency spectrum of an exponentially growing population has a tail following a power law S_k ~ k^(-2) [13, 14]. Similarly, it is known that for the critical branching process or the Moran process, the site frequency spectrum at equilibrium is S_k ~ k^(-1) [13, 15]. Especially noteworthy yet uncited studies that use those results about site frequency spectra to make inferences based on sequencing data include ref 16, in which selection is inferred, and ref 17, in which evolutionary parameters of constant populations (healthy cell populations) are inferred.

Although the paper is well written, the figures are ineffective in communicating the results. As others have put it, "A figure is meant to express an idea or introduce some facts or a result that would be too long (or nearly impossible) to explain only with words" and "If your figure is able to convey a striking message at first glance, chances are increased that your article will draw more attention from the community" [18]. On the contrary, Figures 3, 4, 5 and 6 are bewilderingly complicated, crowded, and repetitive. These figures comprise no fewer than fifty-six plots, each containing numerous curves or histograms, spread across four pages. To compare the results of different scenarios, the reader is presumably expected to put these figures side by side and try to spot the differences, hampered by inconsistent axis ranges, absence of axis labels, absence of titles, absence of legends, and unreliable captions ("cyan" seems to refer to pale blue, and "orange" to something closer to red). For example, the only notable difference between Figures 3 and 4 is in the shape of a single green curve in panel I. In the main text of a published paper, one would expect fewer, more carefully curated figures drawing attention to salient features, so that the reader can infer the main results with minimal effort. The rest can be put in supplementary figures.

In summary, this paper adds somewhat to our understanding of some standard mathematical models; whether it tells us anything new about cancer is open to debate.

References<br /> (1) Kurpas, Monika K., and Marek Kimmel. "Modes of selection in tumors as reflected by two mathematical models and site frequency spectra." Frontiers in Ecology and Evolution 10 (2022): 889438.<br /> (2) Bailey, Norman TJ. The elements of stochastic processes with applications to the natural sciences. John Wiley & Sons, 1964.<br /> (3) Tarabichi, Maxime, et al. "Neutral tumor evolution?." Nature Genetics 50.12 (2018): 1630-1633.<br /> (4) McDonald, Thomas O., Shaon Chakrabarti, and Franziska Michor. "Currently available bulk sequencing data do not necessarily support a model of neutral tumor evolution." Nature Genetics 50.12 (2018): 1620-1623.<br /> (5) Balaparya, Abdul, and Subhajyoti De. "Revisiting signatures of neutral tumor evolution in the light of complexity of cancer genomic data." Nature Genetics 50.12 (2018): 1626-1628.<br /> (6) Noorbakhsh, Javad, and Jeffrey H. Chuang. "Uncertainties in tumor allele frequencies limit power to infer evolutionary pressures." Nature Genetics 49.9 (2017): 1288-1289.<br /> (7) Bozic, Ivana, Chay Paterson, and Bartlomiej Waclaw. "On measuring selection in cancer from subclonal mutation frequencies." PLoS Computational Biology 15.9 (2019): e1007368.<br /> (8) Neher, Richard A., and Oskar Hallatschek. "Genealogies of rapidly adapting populations." Proceedings of the National Academy of Sciences 110.2 (2013): 437-442.<br /> (9) Gelman, Andrew, et al. Bayesian data analysis (Third Edition). Chapman and Hall/CRC, 2014.<br /> (10) Fusco, Diana, et al. "Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments." Nature Communications 7.1 (2016): 12760.<br /> (11) Noble, Robert, et al. "Spatial structure governs the mode of tumour evolution." Nature Ecology & Evolution 6.2 (2022): 207-217.<br /> (12) Lawson, Devon A., et al. "Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells." Nature 526.7571 (2015): 131-135.<br /> (13) Gunnarsson, Einar B., Leder, Kevin, and Foo Jasmine. "Exact site frequency spectra of neutrally evolving tumors: A transition between power laws reveals a signature of cell viability" Theoretical Population Biology 142 (2021) 67-90<br /> (14) Durrett, Richard "Branching Process Models of Cancer" Springer (2015)<br /> (15) Durrett, Richard "Probability Models for DNA Sequence Evolution" Springer Science & Business media (2008)<br /> (16) Williams, Mark J. et al. "Quantification of subclonal selection in cancer from bulk sequencing data." Nature Genetics 50 (6). 895-903 (2018)<br /> (17) Moeller, Marius E. et al. "Measures of genetic diversification in somatic tissues at bulk and single-cell resolution" eLife (2024) 12:RP89780<br /> (18) Rougier, Nicolas P., Michael Droettboom, and Philip E. Bourne. "Ten simple rules for better figures." PLoS Computational Biology 10.9 (2014): e1003833.

Aligning a Montgomery Ward (Brother) Signature 511 by Bill M.

Reviewer #2 (Public Review):

Summary:

The goals of this study were to develop a genetic approach that would specifically and comprehensively target axo-axonic cells (AACs) throughout the brain and then to describe the patterns and characteristics of the targeted AACs in multiple, selected brain regions. The investigators have been successful in providing the most complete description of the regional distribution of putative (pAACs) throughout the brain to date. The supporting evidence is convincing, and the findings should serve as a guide for more detailed studies of AACs within each brain region and lead to new insights into their connectivity and functional organization of this important group of GABAergic interneurons.

Strengths:

The study has numerous strengths. A major strength is the development of a unique intersectional genetic strategy that uses cell lineage (Nkx2.1) and molecular (Unc5b or Pthlh) markers to identify AACs specifically and, apparently, nearly completely throughout the mouse brain. While AACs have been described previously in the cerebral cortex, hippocampus and amygdala, there has been no specific genetic marker that selectively identifies all AACs in these regions.

Importantly, the current genetic strategy labels pAACs in additional brain regions, including the claustrum-insular complex, extended amygdala, and several olfactory centers in which AACs have not been previously recognized. In general, the findings provide support for the specificity of the methods for targeting AACs and include several examples of labeling near markers of axon initial segments, providing validation of their AAC identity.

The descriptions and numerous low magnification images of the brain provide a roadmap for subsequent, detailed studies of AACs in numerous brain regions. The overview and summaries of the findings in the Abstract, Introduction and Discussion are particularly clear and helpful in placing the extensive regional descriptions of AACs in context.

Weaknesses:

Considering the unique and striking characteristics of AACs, it would have been ideal to include a clear, high resolution confocal image of an AAC from the Unc5b;Nkx2.1 mouse that would display the beauty of these cells with their numerous cartridges of axon terminals, emanating from a single AAC. While several cells are illustrated, the processes are often obscured by other labeling or the background created by the blue Dapi labeling. A high-resolution image of an isolated cell would not only support the identity of the cells as AACs but also demonstrate the potential advantages of their labeling for more detailed anatomical and neurophysiological studies. High magnification views of the axon terminals adjacent to AnkG-labeled axon initial segments are included and provide strong support for the identity of the cells. However, they cannot convey the extensiveness and patterns of the axonal arborizations of these cells.

The intersectional genetic methods included use of the lineage marker Nkx2.1 with either Unc5b or Pthlh as the molecular marker. As described, the mice with intersectional targeting of Nkx2.1 and Unc5b appear to show the most specific brain-wide labeling for AACs, and the majority of the descriptions are from these mice. The targeting with Nkx2.1 and Pthlh is less convincing and there appears to be a disconnect between the descriptions and the images. While the descriptions emphasize that the labeling is very similar in the two types of mice, the images suggest distinct differences, including labeling of non AACs in striatum and layer 4 of the cortex in the Pthlh;Nkx2.1 mouse, as described in the manuscript. In addition, the Pthlh;Nkx2.1 mouse has higher cell targeting in some regions and fewer labeled cells in others. Perhaps it would be more accurate to present the Pthlh;Nkx2.1 mouse as differing from the Unc5b;Nkx2.1 mouse, but useful for AAC labeling in select regions and under some conditions, such as following tamoxifen administration at specific ages. As currently presented, the inclusion of the Pthlh;Nkx2.1 detracts from the otherwise convincing argument that the Unc5b;Nkx2.1 mouse provides a specific and comprehensive way to identify AACs.

Reviewer #1 (Public Review):

Summary:

In this study, Rosenblum et al introduce a novel and automatic way of calculating sleep cycles from human EEG. Previous results have shown that the slope of the non-oscillatory component of the power spectrum (called the aperiodic or fractal component) changes with the sleep stage. Building on this, the authors present an algorithm that extracts the continuous-time fluctuations in the fractal slope and propose that peaks in this variable can be used to identify sleep cycle limits. Cycles defined in this way are termed "fractal cycles". The main focus of the article is a comparison of fractal and classical, manually defined sleep cycles in numerous datasets.

Strengths:

The manuscript amply illustrates through examples the strong overlap between fractal and classical cycle identification. Accordingly, a high percentage (81%) can be matched one-to-one between methods and sleep cycle duration is well correlated (around R = 0.5). Moreover, the methods track certain global changes in sleep structure in different populations: shorter cycles in children and longer cycles in patients medicated with REM-suppressing anti-depressants. Finally, a major strength of the results is that they show similar agreement between fractal and classical sleep cycle length in 5 different data sets, showing that it is robust to changes in recording settings and methods.

These results suggest that the fractal cycle methodology could provide a valuable new method to study sleep architecture and avoid the time-consuming steps of manual cycle identification. Moreover, it has the potential to be applied to animal studies which rarely deal with sleep cycle structure.

Weaknesses:

The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method. This raises the question as to whether differences are due to one method being more reliable than another or whether they are also identifying different underlying biological differences. It is not clear for example whether the agreement between the two methods is better or worse than between two human scorers, which generally serve as a gold standard to validate novel methods. The authors provide some insight into differences between the methods that could account for differences in results. However, given that the fractal method is automatic it would be important to clearly identify criteria for recordings in which it will produce similar results to the classical method.

Reviewer #1 (Public Review):

Summary:

This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with amplitude comparable to aspartate, and cells can quickly adapt (and generally over-adapt). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.

Strengths:

The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH. The updated manuscript controls for the impact of pH on the fluorescent protein brightness that can bias the measured FRET signal. After correction the response amplitude and sharpness (hill coefficient) are comparable to conventional chemoattractants (e.g. aspartate), indicating the general mechanisms underlying the response may be similar. The authors suggest that the biphasic response of Tar mutants may be due to pH influencing the activity of other enzymes (CheA, CheR or CheB), which will be an interesting direction for future study.

Weaknesses:

The measured response may be biased by adaptation, especially for weak potassium signals. For other attractant stimuli, the response typically shows a low plateau before it recovers (adapts). In the case of potassium, the FRET signal does not have an obvious plateau following the stimuli of small potassium concentrations, perhaps due to the faster adaptation compared to other chemoattractants. It is possible cells have already partially adapted when the response reaches its minimum, so the measured response may be a slight underestimate of the true response. Mutants without adaptation enzymes appear to be sensitive to potassium only at much larger concentrations, where the pH significantly disrupts the FRET signal; more accurate measurements would require the development of new mutants and/or measurement techniques.

Note added after the second revision: The authors made a reasonable argument regarding the effects of adaptation, which were estimated to be small.

Reviewer #1 (Public Review):

In this paper, the authors evaluate the utility of brain-age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain-age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.

Comments on revised version:

I thank the authors for the revision of the manuscript and for being more explicit about the inherent conceptual limitations of Brain Age / Brain Cognition. I have no further comments.

Reviewer #1 (Public Review):

In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3. They demonstrate that circHIPK3 interacts with an RNA binding protein (IGF2BP2), sequestering it away from its target mRNAs. This interaction is shown to regulates the expression of hundreds of genes that share a specific sequence motif (11-mer motif) in their untranslated regions (3'-UTR), identical to one present in circHIPK3 where IGF2BP2 binds. The study further focuses on the specific case of STAT3 gene, whose mRNA product is found to be downregulated upon circHIPK3 depletion. This suggests that circHIPK3 sequesters IGF2BP2, preventing it from binding to and destabilizing STAT3 mRNA. The study presents evidence supporting this mechanism and discusses its potential role in tumor cell progression. These findings contribute to the growing complexity of understanding cancer regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.

Strengths:

The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of studies. They provide both genome-wide analysis and a specific case (STAT3) which is relevant for cancer progression. Overall, the authors have significantly improved their manuscript in their revised version.

Weaknesses:

There are seemingly contradictory effects of circHIPK3 and STAT3 depletion in cancer progression. However, the authors have addressed these issues in their revised manuscript, incorporating potential reasons that might explain such complexity.

Reviewer #1 (Public Review):

This paper describes RNA-sensing guide RNAs for controlled activation of CRISPR modification. This works by having an extended guide RNA with a sequence that folds back onto the targeting sequence such that the guide RNA cannot hybridise to its genomic target. The CRISPR is "activated" by the introduction of another RNA, referred to as a trigger, that competes with this "back folding" to make the guide RNA available for genome targeting. The authors first confirm the efficacy of the approach using several RNA triggers and a GFP reporter that is activated by dCas9 fused to transcriptional activators. A major potential application of this technique is the activation of CRISPR in response to endogenous biomarkers. As these will typically be longer than the first generation triggers employed by the authors they test some extended triggers, which also work though not always to the same extent. They then introduce MODesign which may enable the design of bespoke or improved triggers. After that, they determine that the mode of activation by the RNA trigger involves cleavage of the RNA complexes. Finally, they test the potential for their system to work in a developmental setting - specifically zebrafish embryos. There is some encouraging evidence, though the effects appear more subtle than those originally obtained in cell culture.

Overall, the potential of a CRISPR system that can be activated upon sensing an RNA is high and there are a myriad of opportunities and applications for it. This paper represents a reasonable starting point having developed such a system in principle.<br /> The weakness of the study is that it does not demonstrate that the system can be used in a completely natural setting. This would require an endogenous transcript as the RNA trigger with a clear readout. The authors now acknowledge this limitation in their revised manuscript. Future studies and experiments should focus on these aspects in order for the system to be employed to its full and intended potential.

Reviewer #1 (Public Review):

In the presence of predators, animals display attenuated foraging responses and increased defensive behaviors that serve to protect them from potential predatory attacks. Previous studies have shown that the basolateral nucleus of the amygdala (BLA) and the periaqueductal gray matter (PAG) are necessary for the acquisition and expression of conditioned fear responses. However, it remains unclear how BLA and PAG neurons respond to predatory threats when animals are foraging for food. To address this question, Kim and colleagues conducted in vivo electrophysiological recordings from BLA and PAG neurons and assessed approach-avoidance responses while rats search for food in the presence of a robotic predator.

The authors observed that rats exhibited a significant increase in the latency to obtain the food pellets and a reduction in the pellet success rate when the predator robot was activated. A subpopulation of PAG neurons showing increased firing rate in response to the robot activation didn't change their activity in response to food pellet retrieval during the pre- or post-robot sessions. Optogenetic stimulation of PAG neurons increased the latency to procure the food pellet in a frequency- and intensity-dependent manner, similar to what was observed during the robot test. Combining optogenetics with single-unit recordings, the authors demonstrated that photoactivation of PAG neurons increased the firing rate of 10% of BLA cells. A subsequent behavioral test in 3 of these same rats demonstrated that BLA neurons responsive to PAG stimulation displayed higher firing rates to the robot than BLA neurons nonresponsive to PAG stimulation. Next, because the PAG does not project monosynaptically to the BLA, the authors used a combination of retrograde and anterograde neural tracing to identify possible regions that could convey robot-related information from PAG to the BLA. They observed that neurons in specific areas of the paraventricular nucleus of the thalamus (PVT) that are innervated by PAG fibers contained neurons that were retrogradely labeled by the injection of CTB in the BLA. In addition, PVT neurons showed increased expression of the neural activity marker cFos after the robot test, suggesting that PVT may be a mediator of PAG signals to the BLA.

Overall, the idea that the PAG interacts with the BLA via the midline thalamus during a predator vs. foraging test is new and quite interesting. The authors have used appropriated tools to address their questions.

In this revised version of the manuscript, the authors have made important modifications in the text, inserted new data analyses, and incorporated additional references, as recommended by the reviewers. These modifications have significantly improved the quality of the manuscript.

Reviewer #1 (Public Review):

Summary:

This work describes a new protein factor required for filamentous phage assembly. The protein PSB15 binds to the packaging signal of the ssDNA, Trx and cardiolipin. A mechanism how the phage DNA is targeted to the assembly site in the bacterial inner membrane is discussed.

Strengths:

The work describes a clever way to detect factors required for phage propagation by looking at the plaque size of pseudorevertants that arise after infection of a phage with a directed mutation in the packaging signal. This led to the detection of a phage protein expressed from ORF9, the PSB15.

The authors convincingly show that PSB15 is expressed in infected cells and can complement a phage with a mutated orf9.

Weaknesses:

Given the fact that the phage LF-UK is not well explored, many open questions should be mentioned in the introduction. For the study, it is important to know if the phageLF-UK has a mimick or homolog of gV and gXI, and if not, whether PSB15 could take their role.

I am not convinced of the proposition of their term "checkpoint". The truth is that the authors do not know the real purpose of PSB15. I do not see an advantage for a checkpoint that only adds an additional step to enter the phage assembly site. There must be a biochemical reason for the action of PSB15. Looking at Figure 7, the step from "Release" to "Loading" is just adding many unknowns, e.g. how to transfer the DNA, how to dispose of PSB15 and Trx? Also, in the previous step are three question marks that do not add any solid information.

The in vivo study of subcellular localization is very questionable. Why is there a single fluorescent dot if there are thousands of PSB15 molecules expressed in the cell? I have my doubts that the conclusions the authors make here are correct and meaningful. The movies do not add anything significant.

Joint Public Review:

Summary

This manuscript explores the transcriptomic identities of olfactory ensheathing cells (OECs), glial cells that support life-long axonal growth in olfactory neurons, as they relate to spinal cord injury repair. The authors show that transplantation of cultured, immunopurified rodent OECs at a spinal cord injury site can promote injury-bridging axonal regrowth. They then characterize these OECs using single-cell RNA sequencing, identifying five subtypes and proposing functional roles that include regeneration, wound healing, and cell-cell communication. They identify one progenitor OEC subpopulation and also report several other functionally relevant findings, notably, that OEC marker genes contain mixtures of other glial cell type markers (such as for Schwann cells and astrocytes), and that these cultured OECs produce and secrete Reelin, a regrowth-promoting protein that has been disputed as a gene product of OECs.

This manuscript offers an extensive, cell-level characterization of OECs, supporting their potential therapeutic value for spinal cord injury and suggesting potential underlying repair mechanisms. The authors use various approaches to validate their findings, providing interesting images that show the overlap between sprouting axons and transplanted OECs, and showing that OEC marker genes identified using single-cell RNA sequencing are present in vivo, in both olfactory bulb tissue and spinal cord after OEC transplantation.

Despite the breadth of information presented, however, further quantification of results and explanation of experimental approaches would be needed to support some of the authors' claims. Additionally, a more thorough discussion is needed to contextualize their findings relative to previous work.

(1) Important quantification is lacking for the data presented. For example, multiple figures include immunohistochemistry or immunocytochemistry data (Figures 1, 5, 6), but they are presented without accompanying measures like fractions of cells labeled or comparisons against controls. As a result, for axons projecting via OEC bridges in Figure 1, it is unclear how common these bridges are in the presence or absence of OECs. For Figure 6., it is unclear whether cells having an alternative OEC morphology coincide with progenitor OEC subtype marker genes to a statistically significant degree. Similar quantification is missing in other types of data such as Western blot images (Fig. 9) and OEC marker gene data (for which p-values are not reported; Table S2).

The addition of quantitative measures and, where appropriate, statistical comparisons with p-values or other significance measures, would be important for supporting the authors' claims and more rigorously conveying the results.

(2) Some aspects of the experimental design that are relevant to the interpretation of the results are not explained. For example, OECs appear to be collected from only female rats, but the potential implications of this factor are not discussed.

Additionally, it is unclear from the manuscript to what degree immunopurified cells are OECs as opposed to other cell types. The antibody used to retain OECs, nerve growth factor receptor p75 (Ngfr-p75), can also be expressed by non-OEC olfactory bulb cell types including astrocytes [1-3]. The possible inclusion of Ngfr-p75-positive but non-OEC cell types in the OEC culture is not sufficiently addressed. Such non-OEC cell types are also not distinguished in the analysis of single-cell RNA sequencing data (only microglia, fibroblasts, and OECs are identified; Figure 2). Thus, it is currently unclear whether results related to the OEC subtype may have been impacted by these experimental factors.

(3) The introduction, while well written, does not discuss studies showing no significant effect of OEC implantation after spinal cord injury. The discussion also fails to sufficiently acknowledge this variability in the efficacy of OEC implantation. This omission amplifies bias in the text, suggesting that OECs have significant effects that are not fully reflected in the literature. The introduction would need to be expanded to properly address the nuance suggested by the literature regarding the benefits of OECs after spinal cord injury. Additionally, in the discussion, relating the current study to previous work would help clarify how varying observations may relate to experimental or biological factors.

(a) Cragnolini, A.B. et al., Glia, (2009), doi: 10.1002/glia.20857.<br /> (b) Vickland H. et al., Brain Res., (1991), doi: 10.1016/0006-8993(91)91659-O.<br /> (c) Ung K. et al., Nat Commun., (2021), doi: 10.1038/s41467-021-25444-3.

Reviewer #1 (Public Review):

Summary:

It seems as if the main point of the paper is about the new data related to rat fish although your title is describing it as extant cartilaginous fishes and you bounce around between the little skate and ratfish. So here's an opportunity for you to adjust the title to emphasize ratfish is given the fact that leader you describe how this is your significant new data contribution. Either way, the organization of the paper can be adjusted so that the reader can follow along the same order for all sections so that it's very clear for comparative purposes of new data and what they mean. My opinion is that I want to read, for each subheading in the results, about the the ratfish first because this is your most interesting novel data. Then I want to know any confirmation about morphology in little skate. And then I want to know about any gaps you fill with the cat shark. (It is ok if you keep the order of "skate, ratfish, then shark, but I think it undersells the new data).

Strengths:

The imagery and new data availability for ratfish are valuable and may help to determine new phylogenetically informative characters for understanding the evolution of cartilaginous fishes. You also allude to the fossil record.

Opportunities:

I am concerned about the statement of ratfish paedomorphism because stage 32 and 33 were not statistically significantly different from one another (figure and prior sentences). So, these ratfish TMDs overlap the range of both 32 and 33. I think you need more specimens and stages to state this definitely based on TMD. What else leads you to think these are paedomorphic? Right now they are different, but it's unclear why. You need more outgroups.

Your headings for the results subsection and figures are nice snapshots of your interpretations of the results and I think they would be better repurposed in your abstract, which needs more depth.

Historical literature is more abundant than what you've listed. Your first sentence describes a long fascination and only goes back to 1990. But there are authors that have had this fascination for centuries and so I think you'll benefit from looking back. Especially because several of them have looked into histology and development of these fishes.

I agree that in the past 15 years or so a lot more work has been done because it can be done using newer technologies and I don't think your list is exhaustive. You need to expand this list and history which will help with your ultimate comparative analysis without you needed to sample too many new data yourself.

I'd like to see modifications to figure 7 so that you can add more continuity between the characters, illustrated in figure 7 and the body of the text. Generally Holocephalans are the outgroup to elasmobranchs - right now they are presented as sister taxa with no ability to indicate derivation. Why isn't the catshark included in this diagram?

In the last paragraph of the introduction, you say that "the data argue" and I admit, I am confused. Whose data? Is this a prediction or results or summary of other people's work? Either way, could be clarified to emphasize the contribution you are about to present.

Reviewer #1 (Public Review):

Summary:

The main observation that the sperm from CRISP proteins 1 and 3 KO lines are post-fertilization less developmentally competent is convincing. However, the molecular characterization of the mechanism that leads to these defects and the temporal appearance of the defects requires additional studies.

Strengths:

The generation of these double mutant mice is valuable for the field. Moreover, the fact that the double mutant line of Crisp 1 and 3 is phenotypically different from the Crisp 1 and 4 line suggests different functions of these epididymis proteins. The methods used to demonstrate that developmental defects are largely due to post-fertilization defects are also a considerable strength. The initial characterization of these sperm has altered intracellular Ca2+ levels, and increased rates of DNA fragmentation are valuable.

Weaknesses:

The study is mechanistically incomplete because there is no direct demonstration that the absence of these proteins alters the epididymal environment and fluid, wherein during the passage through the epididymis the sperm become affected. Also, a direct demonstration of how the proteins in question cause or lead to DNA damage and increased Ca2+ requires further characterization.

Reviewer #1 (Public Review):

Summary:

Using a cross-modal sensory selection task in head-fixed mice, the authors attempted to characterize how different rules reconfigured representations of sensory stimuli and behavioral reports in sensory (S1, S2) and premotor cortical areas (medial motor cortex or MM, and ALM). They used silicon probe recordings during behavior, a combination of single-cell and population-level analyses of neural data, and optogenetic inhibition during the task.

Strengths:

A major strength of the manuscript was the clarity of the writing and motivation for experiments and analyses. The behavioral paradigm is somewhat simple but well-designed and well-controlled. The neural analyses were sophisticated, clearly presented, and generally supported the authors' interpretations. The statistics are clearly reported and easy to interpret. In general, my view is that the authors achieved their aims. They found that different rules affected preparatory activity in premotor areas, but not sensory areas, consistent with dynamical systems perspectives in the field that hold that initial conditions are important for determining trial-based dynamics.

I think this is a well-performed, well-written and interesting study that shows differences in rule representations in sensory and premotor areas, and finds that rules reconfigure preparatory activity in motor cortex to support flexible behavior.

Reviewer #1 (Public Review):

Summary:

The classical pro/antisaccade task has become a valuable diagnostic tool in neurology and psychiatry (Antoniades et al., 2013, Vision Res). Although it is well-established that antisaccades require substantially longer latencies than prosaccades, the exact attentional mechanisms underlying these differences are not yet fully elucidated. This study investigates the separate influences of exogenous and endogenous attention on saccade generation. These two mechanisms are often confounded in classical pro/antisaccade tasks. In the current study, the authors build on their previous work using an urgent choice task (Salinas et al., 2019, eLife) to time-resolve the influences of exogenous and endogenous factors on saccade execution. The key contribution of the current study is to show that, when controlling for exogenous capture, antisaccades continue to require longer processing times. This longer processing time may be explained by a coupling between endogenous attention and saccade motor plans.

Strengths:

In the classical pro/antisaccade task the direction of exogenous capture (caused by the presentation of the cue) is typically congruent with the direction of prosaccades and incongruent with antisaccades. A key strength of the current study is the introduction of different experimental conditions that control for the effects of exogenous capture on saccade generation. In particular, Experiments 3 and 4 provide strong evidence for two independent (exogenous and endogenous) mechanisms that guide saccadic choices, acting at different times. Differences in timing for pro and antisaccades during the endogenous phase were consistent and independent of whether the exogenous capture biased early saccades toward the correct prosaccade direction or toward the correct antisaccade directions.

As in previous studies by the same group (Salinas et al., 2019, eLife; Goldstein et al., 2023, eLife), the detailed analysis of the time course of goal-directed saccades allowed the authors to determine the exact, additional time of 30 ms that is necessary to generate a correct antisaccade versus prosaccade.

Overall, the manuscript is very well written, and the data are presented clearly.

Weaknesses:

The main research question could be defined more clearly. In the abstract and at some points throughout the manuscript, the authors indicate that the main purpose of the study was to assess whether the allocation of endogenous attention requires saccade planning [e.g., ll.3-5 or ll.247-248]. While the data show a coupling between endogenous attention and saccades, they do not point to a specific direction of this coupling (i.e., whether endogenous attention is necessary to successfully execute a saccade plan or whether a saccade plan necessarily accompanies endogenous attention).

Some of the analyses were performed only on subgroups of the participants. The reporting of these subgroup analyses is transparent and data from all participants are reported in the supplementary figures. Still, these subgroup analyses may make the data appear more consistent, compared to when data is considered across all participants. For instance, the exogenous capture in Experiments 1 and 2 appears much weaker in Figure 2 (subgroup) than Figure S3 (all participants). Moreover, because different subgroups were used for different analyses, it is often difficult to follow and evaluate the results. For instance, the tachometric curves in Figure 2 (see also Figure 3 and 4) show no motor bias towards the cue (i.e., performance was at ~50% for rPTs <75 ms). I assume that the subsequent analyses of the motor bias were based on a very different subgroup. In fact, based on Figure S2, it seems that the motor bias was predominantly seen in the unreliable participants. Therefore, I often found the figures that were based on data across all participants (Figures 7 and S3) more informative to evaluate the overall pattern of results.

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #1 (Public Review):

Olszyński and colleagues present data showing variability from canonical "aversive calls", typically described as long 22 kHz calls rodents emit in aversive situations. Similarly long but higher-frequency (44 kHz) calls are presented as a distinct call type, including analyses both of their acoustic properties and animals' responses to hearing playback of these calls. While this work adds an intriguing and important reminder, namely that animal behavior is often more variable and complex than perhaps we would like it to be, there is some caution warranted in the interpretation of these data.

The exclusive use of males is a major concern lacking adequate justification and should be disclosed in the title and abstract to ensure readers are aware of this limitation. With several reported sex differences in rat vocal behaviors this means caution should be exercised when generalizing from these findings. The occurrence of an estrus cycle in typical female rats is not justification for their exclusion. Note also that male rodents experience great variability in hormonal states as well, distinguishing between individuals and within individuals across time. The study of endocrinological influences on behavior can be separated from the study of said behavior itself, across all sexes. Similarly, concerns about needing to increase the number of animals when including all sexes are usually unwarranted (see Shansky [2019] and Phillips et al. [2023]).

Regarding the analysis where calls were sorted using DBSCAN based on peak frequency and duration, my comment on the originally reviewed version stands. It seems that the calls are sorted by an (unbiased) algorithm into categories based on their frequency and duration, and because 44kHz calls differ by definition on frequency and duration the fact that the algorithm sorts them as a distinct category is not evidence that they are "new calls [that] form a separate, distinct group". I appreciate that the authors have softened their language regarding the novelty and distinctness of these calls, but the manuscript contains several instances where claims of novelty and specificity (e.g. the subtitle on line 193) is emphasized beyond what the data justifies.

The behavioral response to call playback is intriguing, although again more in line with the hypothesis that these are not a distinct type of call but merely represent expected variation in vocalization parameters. Across the board animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls. This does raise interesting questions about how, ethologically, animals may interpret such variation and integrate this interpretation in their responses. However, the categorical approach employed here does not address these questions fully.

I appreciate the amendment in discussing the idea of arousal being the key determinant for the increased emission of 44kHz, and the addition of other factors. Some of the items in this list, such as annoyance/anger and disgust/boredom, don't really seem to fit the data. I'm not sure I find the idea that rats become annoyed or disgusted during fear conditioning to be a particularly compelling argument. As such the list appears to be a collection of emotion-related words, with unclear potential associations with the 44kHz calls.

Later in the Discussion the authors argue that the 44kHz aversive calls signal an increased intensity of a negative valence emotional state. It is not clear how the presented arguments actually support this. For example, what does the elongation of fear conditioning to 10 trials have to do with increased negative emotionality? Is there data supporting this relationship between duration and emotion, outside anthropomorphism? Each of the 6 arguments presented seems quite distant from being able to support this conclusion.

In sum, rather than describing the 44kHz long calls as a new call type, it may be more accurate to say that sometimes aversive calls can occur at frequencies above 22 kHz. Individual and situational variability in vocalization parameters seems to be expected, much more so than all members of a species strictly adhering to extremely non-variable behavioral outputs.

[Editors' note: The reviewer agrees that the additional analysis has ruled out the possibility that the calls are due to fatigue.]

Reviewer #1 (Public Review):

Summary:

The authors assess the accuracy of short variant calling (SNPs and indels) in bacterial genomes using Oxford Nanopore reads generated on R10.4 flow cells from a very similar genome (99.5% ANI), examining the impact of variant caller choice (three traditional variant callers: bcftools, freebayes, and longshot, and three deep learning based variant callers: clair3, deep variant, and nano caller), base calling model (fast, hac and sup) and read depth (using both simplex and duplex reads).

Strengths:

Given the stated goal (analysis of variant calling for reads drawn from genomes very similar to the reference), the analysis is largely complete and results are compelling. The authors make the code and data used in their analysis available for re-use using current best practices (a computational workflow and data archived in INSDC databases or Zenodo as appropriate).

Weaknesses:

While the medaka variant caller is now deprecated for diploid calling, it is still widely used for haploid variant calling and should at least be mentioned (even if the mention is only to explain its exclusion from the analysis).

Appraisal:

The experiments the authors engaged in are well structured and the results are convincing. I expect that these results will be incorporated into "best practice" bacterial variant calling workflows in the future.

Reviewer #1 (Public Review):

Summary:

Winged seeds or ovules from the Devonian are crucial to understanding the origin and early evolutionary history of wind dispersal strategy. Based on exceptionally well-preserved fossil specimens, the present manuscript documented a new fossil plant taxon (new genus and new species) from the Famennian Series of Upper Devonian in eastern China and demonstrated that three-winged seeds are more adapted to wind dispersal than one-, two- and four-winged seeds by using mathematical analysis.

Strengths:

The manuscript is well organised and well presented, with superb illustrations. The methods used in the manuscript are appropriate.

Weaknesses:

I would only like to suggest moving the "Mathematical analysis of wind dispersal of ovules with 1-4 wings" section from the supplementary information to the main text, leaving the supplementary figures as supplementary materials.

Reviewer #1 (Public Review):

Summary:

By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to identifying a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

Strengths:

In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

Weaknesses:

The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as species-specific gamete recognition. Additionally, using a TPM >1 threshold for protein-coding transcripts may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis.

Reviewer #1 (Public Review):

Summary:

The manuscript by Anbarcia et al. re-evaluates the function of the enigmatic Rete Ovarii (RO), a structure that forms in close association with the mammalian ovary. The RO has generally been considered a functionless structure in the adult ovary. This manuscript follows up on a previous study from the lab that analyzed ovarian morphogenesis using high-resolution microscopy (McKey et al., 2022). The present study adds finer details to RO development and possible function by (1) identifying new markers for OR sub-regions (e.g. GFR1a labels the connecting rete) suggesting that the sub-regions are functionally distinct, (2) showing that the OR sub-regions are connected by a luminal system that allows transport of material from the extra-ovarian rete (EOR) to the inter-ovarian rete (IOG), (3) identifies proteins that are secreted into the OR lumen and that may regulate ovarian homeostasis, and finally, (4) better defines how the vasculature, nervous, and immune system integrates with the OR.

Strengths:

The data is beautifully presented and convincing. They show that the RO is composed of three distinct domains that have unique gene expression signatures and thus likely are functionally distinct.

Weaknesses:

It is not always clear what the novel findings are that this manuscript is presenting. It appears to be largely similar to the analysis done by McKey et al. (2022) but with more time points and molecular markers. The novelty of the present study's findings needs to be better articulated.

Joint Public Review:

Summary:

Idiopathic scoliosis (IS) is a common spinal deformity. Various studies have linked genes to IS, but underlying mechanisms are unclear such that we still lack understanding of the causes of IS. The current manuscript analyzes IS patient populations and identifies EPHA4 as a novel associated gene, finding three rare variants in EPHA4 from three patients (one disrupting splicing and two missense variants) as well as a large deletion (encompassing EPHA4) in a Waardenburg syndrome patient with scoliosis. EPHA4 is a member of the Eph receptor family. Drawing on data from zebrafish experiments, the authors argue that EPHA4 loss of function disrupts the central pattern generator (CPG) function necessary for motor coordination.

Strengths:

The main strength of this manuscript is the human genetic data, which provides convincing evidence linking EPHA4 variants to IS. The loss of function experiments in zebrafish strongly support the conclusion that EPHA4 variants that reduce function lead to IS.

Weaknesses:

The conclusion that disruption of CPG function causes spinal curves in the zebrafish model is not well supported. The authors' final model is that a disrupted CPG leads to asymmetric mechanical loading on the spine and, over time, the development of curves. This is a reasonable idea, but currently not strongly backed up by data in the manuscript. Potentially, the impaired larval movements simply coincide with, but do not cause, juvenile-onset scoliosis. Support for the authors' conclusion would require independent methods of disrupting CPG function and determining if this is accompanied by spine curvature. At a minimum, the language of the manuscript could be toned down, with the CPG defects put forward as a potential explanation for scoliosis in the discussion rather than as something this manuscript has "shown". An additional weakness of the manuscript is that the zebrafish genetic tools are not sufficiently validated to provide full confidence in the data and conclusions.

Reviewer #1 (Public Review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

Weaknesses:

The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.

Reviewer #1 (Public Review):

Summary:

The study seeks to establish accurate computational models to explore the role of hydrodynamic interactions on energy savings and spatial patterns in fish schools. Specifically, the authors consider a system of (one degree-of-freedom) flapping airfoils that passively position themselves with respect to the streamwise direction, while oscillating at the same frequency and amplitude, with a given phase lag and at a constant cross-stream distance. By parametrically varying the phase lag and the cross-stream distance, they systematically explore the stability and energy costs of emergent configurations. Computational findings are leveraged to distill insights into universal relationships and clarify the role of the wake of the leading foil.

Strengths:

(1) The use of multiple computational models (computational fluid dynamics, CFD, for full Navier-Stokes equations and computationally efficient inviscid vortex sheet, VS, model) offers an extra degree of reliability of the observed findings and backing to the use of simplified models for future research in more complex settings.

(2) The systematic assessment of the stability and energy savings in multiple configurations of pairs and larger ensembles of flapping foils is an important addition to the literature.

(3) The discovery of a linear phase-distance relationship in the formation attained by pairs of flapping foils is a significant contribution, which helps compare different experimental observations in the literature.

(4) The observation of a critical size effect for in-line formations of larger, above which cohesion and energetic benefits are lost at once, is a new discovery in the field.

Weaknesses:

(1) The extent to which observations on one-degree-of-freedom flapping foils could translate to real fish schools is presently unclear so some of the conclusions on live fish schools are likely to be overstated and would benefit from some more biological framing.

(2) The analysis of non-reciprocal coupling is not as novel as the rest of the study and potentially not as convincing due to the chosen linear metric of interaction (that is, the flow agreement).

Overall, this is a rigorous effort on a critical topic: findings of the research can offer important insight into the hydrodynamics of fish schooling, stimulating interdisciplinary research at the interface of computational fluid mechanics and biology.

Reviewer #1 (Public Review):

Summary:

This is an interesting study by Xu et al showing the effects of infection with the Treponema pallidum virus (which causes syphilis disease) on neuronal development using iPSC-derived human brain organoids as a model and single-cell RNA sequencing. This work provides an important insight into the impact of the virus on human development, bridging the gap between the phenomena observed in studies using animal models as well as non-invasive human studies showing developmental abnormalities in fetuses infected with the virus in utero through maternal vertical transmission.

Using single-cell RNAseq in combination with qPCR and immunofluorescence techniques, the authors show that T. pallidum infected organoids are smaller in size, in particular during later growth stages, contain a larger number of undifferentiated neuronal lineage cells, and exhibit decreased numbers of specific neuronal subcluster, which the authors have identified as undifferentiated hindbrain neurons.

The study is an important first step in understanding how T. pallidum affects human neuronal development and provides important insight into the potential mechanisms that underlie the neurodevelopmental abnormalities observed in infected human fetuses.

Strengths:

(1) The study is well written, and the data quality is good for the most part.

(2) The study provides an important first step in utilizing human brain organoids to study the impact of T. pallidum infection on neuronal development.

(3) The study's conclusions may provide important insight to other researchers focused on studying how viral infections impact neuronal development.

Reviewer #1 (Public Review):

The revised manuscript "Diffusive lensing as a mechanism of intracellular transport and compartmentalization" is very similar to the original manuscript. The main difference between the revised and the original manuscript is that the authors have removed the reference to viscosity gradient and instead talk of diffusivity gradient. With this change the manuscript the analysis and claims in the manuscript are much more aligned. The manuscript, as the original version, explores the role of spatially varying diffusion constant in three scenarios:

(i) Spatial localization of non-particles<br /> (ii) Clustering in presence of inter-particle interactions<br /> (iii) Moment analysis for non-interacting particles in space with discrete patches of inhomogeneous diffusivity.

Since the manuscript has not changed much the strengths and weaknesses, in my opinion, remain similar to that of the original manuscript.

Strengths: The implications of a heterogeneous environment on phase separation and reaction kinetics in cells are under-explored. This makes the general theme of this manuscript relevant and interesting.

Weaknesses: The central part of the paper "diffusive lensing", i.e., particles localizing in the region of low diffusion constant is not new. Some of the papers authors cite already show that. The parts on phase separation and frap analysis that could provide new results are not rigorous enough for a theory paper.

I reiterate some of my comments from the original version that are valid for the revised version as well.

My main criticism was not to say that some convention should be used or some not. But instead, the main point was to say that just because there is spatial diffusion constant that does not mean there will be a spatial gradient of particles. From the authors response to my comments, it is clear that they understand the subtilties around it and are aware of the relevant papers. However, a reader not familiar with this discussion may work under the impression that if there if there is a spatialy varying diffusion constant in cell there will be an accumulation of particles in the region of low diffusivity but that may not always be the case. Moreover, localisation of particles in the region of low diffusivity has been reported in many different context. Some of the papers that the author cite already show that. For example, in Rupprecht et al. 2018 non-isothermal interpretation is applied to the dynamics of objects inside cells.

Given that the central result is not new. The paper could still be of general interest to the biophysics community if the follow up sections (ii) Clustering in presence of inter-particle interactions and (iii) Moment analysis for non-interacting particles in space with discrete patches of inhomogeneous diffusivity were analysed rigorously.

Joint Public Review:

This manuscript by Yue et al. aims to understand the molecular mechanisms underlying the better reproductive outcomes of Tibetans at high altitude by characterizing the transcriptome and histology of full-term placenta of Tibetans and compare them to those Han Chinese at high elevations.

The approach is innovative, and the data collected are valuable for testing hypotheses regarding the contribution of the placenta to better reproductive success of populations that adapted to hypoxia. The authors identified hundreds of differentially expressed genes (DEGs) between Tibetans and Han, including the EPAS1 gene that harbors the strongest signals of genetic adaptation. The authors also found that such differential expression is more prevalent and pronounced in the placentas of male fetuses than those of female fetuses, which is particularly interesting, as it echoes with the more severe reduction in birth weight of male neonates at high elevation observed by the same group of researchers (He et al., 2022).

Comments on latest version:

The revised manuscript has incorporated the suggested changes and weakened conclusions regarding natural selection. Limitations of the study are also clearly stated in the Discussion section.

Reviewer #1 (Public Review):

This study is one in a series of excellent papers by the Forstmann group focusing on the ability of fMRI to reliably detect activity in small subcortical nuclei - in this case, specifically those purportedly involved in the hyper- and indirect inhibitory basal ganglia pathways. I have been very fond of this work for a long time, beginning with the demonstration of De Hollander, Forstmann et al. (HBM 2017) of the fact that 3T fMRI imaging (as well as many 7T imaging sequences) do not afford sufficient signal to noise ratio to reliably image these small subcortical nuclei. This work has done a lot to reshape my view of seminal past studies of subcortical activity during inhibitory control, including some that have several thousand citations.

Comments on revised version:

This is my second review of this article, now entitled "Multi-study fMRI outlooks on subcortical BOLD responses in the stop-signal paradigm" by Isherwood and colleagues.

The authors have been very responsive to the initial round of reviews.

I still think it would be helpful to see a combined investigation of the available 7T data, just to really drive the point home that even with the best parameters and a multi-study sample size, fMRI cannot detect any increases in BOLD activity on successful stop compared to go trials. However, I agree with the authors that these "sub samples still lack the temporal resolution seemingly required for looking at the processes in the SST."

As such, I don't have any more feedback.

Reviewer #1 (Public Review):

In this work, the authors investigate an important question - under what circumstances should a recurrent neural network optimised to produce motor control signals receive preparatory input before the initiation of a movement, even though it is possible to use inputs to drive activity just-in-time for movement?

This question is important because many studies across animal models have show that preparatory activity is widespread in neural populations close to motor output (e.g. motor cortex / M1), but it isn't clear under what circumstances this preparation is advantageous for performance, especially since preparation could cause unwanted motor output during a delay.

They show that networks optimised under reasonable constraints (speed, accuracy, lack of pre-movement) will use input to seed the state of the network before movement, and that these inputs reduce the need for ongoing input during the movement. By examining many different parameters in simplified models they identify a strong connection between the structure of the network and the amount of preparation that is optimal for control - namely, that preparation has the most value when nullspaces are highly observable relative to the readout dimension and when the controllability of readout dimensions is low. They conclude by showing that their model predictions are consistent with the observation in monkey motor cortex that even when a sequence of two movements is known in advance, preparatory activity only arises shortly before movement initiation.

Overall, this study provides valuable theoretical insight into the role of preparation in neural populations that generate motor output, and by treating input to motor cortex as a signal that is optimised directly this work is able to sidestep many of the problematic questions relating to estimating the potential inputs to motor cortex.

Reviewer #1 (Public Review):

Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure.

Reviewer #1 (Public Review):

Summary:

People with Parkinson's disease often experience a variety of nonmotor symptoms, the biological bases of which remain poorly understood. Johansson et al began to study potential roles of the dorsal raphe nucleus (DRN) degeneration in the pathophysiology of neuropsychiatric symptoms in PD.

Strengths:

Boi et al validated a transgenic reporter mouse line that can reliably label dopaminergic neurons in the DRN. This brain region shows severe neurodegeneration and has been proposed to contribute to the manifestation of neuropsychiatric symptoms in PD. Using this mouse line (and others), Boi and colleagues characterized electrophysiological and morphological phenotypes of dopaminergic and serotoninergic neurons in the raphe nucleus. This study involved very careful topographical registration of recorded neurons to brain slices for post hoc immunohistochemical validation of cell identity, making it an elegant and thorough piece of work.

Of relevance to PD pathophysiology, the authors evaluated the physiological and morphological changes of DRN serotoninergic and dopaminergic neurons after a partial loss of nigrostriatal dopamine neurons, which serves as a mouse model of early parkinsonian pathology. Moreover, the authors identified a series of physiological and morphological changes of subtypes of DRN neurons that depend on nigral dopaminergic neurodegeneration, LC noradrenergic neurodegeneration, or both. Indeed this work highlights the importance of LC noradrenergic degeneration in PD pathophysiology.

Overall, this is a well-designed study with high significance to the Parkinson's research field.

Reviewer #2 (Public Review):

Summary:

Tian et al. aimed to assess differences in biological motion (BM) perception between children with and without ADHD, as well as relationships to indices of social functioning and possible predictors of BM perception (including demographics, reasoning ability and inattention). In their study, children with ADHD showed poorer performance relative to typically developing children in three tasks measuring local, global, and general BM perception. The authors further observed that across the whole sample, performance in all three BM tasks was negatively correlated with scores on the social responsiveness scale (SRS), whereas within groups a significant relationship to SRS scores was only observed in the ADHD group and for the local BM task. Local and global BM perception showed a dissociation in that global BM processing was predicted by age, while local BM perception was not. Finally, general (local & global combined) BM processing was predicted by age and global BM processing, while reasoning ability mediated the effect of inattention on BM processing.

Strengths:

Overall, the manuscript is presented in a clear fashion and methods and materials are presented with sufficient detail so the study could be reproduced by independent researchers. The study uses an innovative, albeit not novel, paradigm to investigate two independent processes underlying BM perception. The results are novel and have the potential to have wide-reaching impact on multiple fields.

Weaknesses:

The manuscript has improved in clarity and conceptual and methodological considerations in response to the last review. However, the reported results still provide incomplete support for the claims the authors make in the paper.

In relation to other reviewers' earlier comments, the model notation used is still not consistent and model results are reported incompletely, which make it difficult to gain a full picture of the data and how they support the authors' secondary claims. For instance, across the models in the supplementary materials, ß coefficients are only reported selectively which makes it difficult to assess the model as a whole. Furthermore, different terms (task 1, task 2 vs. BM-Local, BM-global) are used to refer to the same levels of a variable, and it is unclear which levels of a dummy variable correspond to which task, making it overall very difficult to comprehend the modelling procedure.

Reviewer #1 (Public Review):

Summary:

The authors use innovative CRISPRi method to uncover regulators of cell density and volume in neutrophils. The results show that cells require NHE activity during chemoattractant-driven cell migration. Before migration occurs, cells also undergo a rapid cell volume increase. These results indicate that water flux, driven by ion channels, appears to play a central role in neutrophil migration. The paper is very well written and clear. The revised version has addressed all of my questions.

Reviewer #1 (Public Review):

This study presents a novel application of the inverted encoding (i.e., decoding) approach to detect the correlates of crossmodal integration in the human EEG (electrophysiological) signal. The method is successfully applied to data from a group of 41 participants, performing a spatial localization task on auditory, visual, and audio-visual events. The analyses clearly show a behavioural superiority for audio-visual localization. Like previous studies, the results when using traditional univariate ERP analyses were inconclusive, showing once more the need for alternative, more sophisticated approaches. Instead, the principal approach of this study, harnessing the multivariate nature of the signal, captured clear signs of super-additive responses, considered by many as the hallmark of multisensory integration. Unfortunately, the manuscript lacks many important details in the descriptions of the methodology and analytical pipeline. Although some of these details can eventually be retrieved from the scripts that accompany this paper, the main text should be self-contained and sufficient to gain a clear understanding of what was done. (A list of some of these is included in the comments to the authors). Nevertheless, I believe the main weakness of this work is that the positive results obtained and reported in the results section are conditioned upon eye movements. When artifacts due to eye movements are removed, then the outcomes are no longer significant.

Therefore, whether the authors finally achieved the aims and showed that this method of analysis is truly a reliable way to assess crossmodal integration, does not stand on firm ground. The worst-case scenario is that the results are entirely accounted for by patterns of eye movements in the different conditions. In the best-case scenario, the method might truly work, but further experiments (and/or analyses) would be required to confirm the claims in a conclusive fashion.

If finally successful, this approach could bring important advances in the many fields where multisensory integration has been shown to play a role, by providing a way to bring much-needed coherence across levels of analysis, from behaviour to single-cell electrophysiology. To achieve this, one would have to make sure that the pattern of super-additive effects, the standard self-imposed by the authors as a proxy for multisensory integration, shows up reliably regardless of eye movement or artifact corrections. One first step toward this goal would be, perhaps, to facilitate the understanding of results in context by reporting both the uncorrected and corrected analyses in the main results section. Second, one could try to support the argument given in the discussion, pointing out the origin of the super-additive effects in posterior electrode sites, by also modelling frontal electrode clusters and showing they aren't informative as to the effect of interest.

Joint Public Review:

Mahapatra and Takahashi report on the physiological consequences of pharmacologically blocking either clathrin and dynamin function during compensatory endocytosis or of the cortical actin scaffold both in the calyx of Held synapse and hippocampal boutons in acute slice preparations.

Although many aspects of these pharmacological interventions have been studied in detail during the past decades, this is a comprehensive and comparative study, which reveals some interesting differences between a fast synapse (Calyx of Held) tuned to reliably transmit at several 100 Hz and a more slow hippocampal CA1 synapse. In particular the authors find that acute disturbance of the synaptic actin network leads to a marked frequency-dependent enhancement of synaptic depression in the Calyx, but not in the hippocampal synapse. This striking difference between both preparations is the most interesting finding.

Comments on latest version:

The authors have done a great job revising the paper and only minor revisions are suggested to the Discussion of the paper.

Two quite relevant and recent papers should be cited and briefly discussed because they relate directly to Pitstop2 effects and actin-myosin-scaffold proteins in the calyx of Held synapse.

One is: Paksoy A et al, (2022) "Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo." Front. Synaptic Neurosci. 14:1056308. doi: 10.3389/fnsyn.2022.1056308. This paper shows with EM that changes caused by PitStop2 perturbation of "clathrin function suggest that clathrin plays a role in SV recycling from both, the plasma membrane and large endosomes, under physiological activity patterns, in vivo."

Second: A role for actin-myosin and MLCK in short-term plasticity has been shown by Srinivasan G., et al. (2008) "The Pool of Fast Releasing Vesicles Is Augmented by Myosin Light Chain Kinase Inhibition at the Calyx of Held Synapse." J Neurophysiol 99: 1810-1824, 2008. The data here suggests that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release but not the Pr of the synapse. In other words, MLCK inhibition augments super-priming of vesicles at the calyx of Held synapse.

Reviewer #1 (Public Review):

Summary:

The authors aim to test the sensory recruitment theory of visual memory, which assumes that visual sensory areas are recruited for working memory and that these sensory areas represent visual memories in a similar fashion to how perceptual inputs are represented. To test the overlap between working memory (WM) and perception, the authors use coarse stimulus (aperture) biases that are known to account for (some) orientation decoding in visual cortex (i.e., stimulus energy is higher for parts of an image where a grating orientation is perpendicular to an aperture edge, and stimulus energy drives decoding). Specifically, the authors show gratings (with a given "carrier" orientation) behind two different apertures: One is a radial modulator (with maximal energy aligned with the carrier orientation) and the other an angular modulator (with maximal energy orthogonal to the carrier orientation). When subject detect contrast changes in these stimuli (the perceptual task), orientation decoding only works when training and testing within each modulator, but not across modulators, showing the impact of stimulus energy on decoding performance. Instead, when subjects remember the orientation over a 12s delay, orientation decoding works irrespective of the modulator used. The authors conclude that representations during WM are therefore not "sensory-like", given that they are immune to aperture biases. This invalidates the sensory recruitment hypothesis, or at least the part assuming that when sensory areas that are recruited during WM, they are recruited in a manner that resembles how these areas are used during perception.

Strengths:

Duan and Curtis very convincingly show that aperture effects that are present during perception, do not appear to be present during the working memory delay. Especially when the debate about "why can we decode orientations from human visual cortex" was in full swing, many may have quietly assumed this to be true (e.g., "the memory delay has no stimuli, and ergo no stimulus aperture effects"), but it is definitely not self-evident and nobody ever thought to test it directly until now. In addition to the clear absence of aperture effects during the delay, Duan and Curtis also show that when stimulus energy aligns with the carrier orientation, cross-generalization between perception and memory does work (which could explain why perception-to-memory cross decoding also works). All in all, this is a clever manipulation, and I'm glad someone did it, and did it well.

Weaknesses:

There seems to be a major possible confound that prohibits strong conclusions about "abstractions" into "line-like" representation, which is spatial attention. What if subjects simply attend the end points of the carrier grating, or attend to the edge of the screen where the carrier orientation "intersects" in order to do the task? This may also result in reconstructions that have higher bold at areas close to the stimulus/screen edges along the carrier orientation. The question then would be if this is truly an "abstracted representation", or if subjects are merely using spatial attention to do the task.

Alternatively (and this reaches back to the "fine vs coarse" debate), another argument could be that during memory, what we are decoding is indeed fine-scale inhomogenous sampling of orientation preferences across many voxels. This is clearly not the most convincing argument, as the spatial reconstructions (e.g., Figure 3A and C) show higher BOLD for voxels with receptive fields that are aligned to the remembered orientation (which is in itself a form of coarse scale bias), but could still play a role.

To conclude that the spatial reconstruction from the data indeed comes from a line-like representation, you'd need to generate modeled reconstructions of all possible stimuli and representations. Yes, Figure 4 shows that a line results in a modeled spatial map that resembles the WM data, but many other stimuli might too, and some may better match the data. For example, the alternative hypothesis (attention to grating endpoints) may very well lead to a very comparable model output to the one from a line. But testing this would not suffice, as there may be an inherent inverse problem (with multiple stimuli that can lead to the same visual field model).

The main conclusion, and title of the paper, that visual working memories are abstractions of percepts, is therefore not supported. Subjects could be using spatial attention, for example. Furthermore, even if it is true that gratings are abstracted into lines, this form of abstraction would not generalize to any non-spatial feature (e.g., color cannot become a line, contrast cannot become a line, etc.), which means it has limited explanatory power.

Additional context:

The working memory and perception tasks are rather different. In this case, the perception task does not require the subject to process the carrier orientation (which is largely occluded, and possibly not that obvious without paying attention to it), but attention is paid to contrast. In this scenario, stimulus energy may dominate the signal. In the WM task, subjects have to work out what orientation is shown to do the task. Given that the sensory stimulus in both tasks is brief (1.5s during memory encoding, and 2.5s total in the perceptual task), it would be interesting to look at decoding (and reconstructions) for the WM stimulus epoch. If abstraction (into a line) happens in working memory, then this perceptual part of the task should still be susceptible to aperture biases. It allows the authors to show that it is indeed during memory (and not merely the task or attentional state of the subject) that abstraction occurs.

What's also interesting is what happens in the passive perceptual condition, and the fact that spatial reconstructions for areas beyond V1 and V2 (i.e., V3, V3AB, and IPS0-1) align with (implied) grating endpoints, even when an angular modulator is used (Figure 3C). Are these areas also "abstracting" the stimulus (in a line-like format)?

Review after revision:

(1) It's nice of the authors to simulate how a dot stimulus affects the image computable model, but this does not entirely address my concern about attention to endpoints. The assumption that attention can be used in the same manner as a physical stimulus to calculate stimulus energy is questionable. (also, why would a dot at 15º lead to high stimulus energy tangential to that orientation?). This simulation also does not at all address my concern about model mimicry (many possible inputs can lead to a line-like output).

(2) It's also nice that the authors agree that much more work needs to be done, and these results may not generalize to all forms of memory. Given this agreement, and until that "more work" is done, I strongly believe we should refrain from making hyperbolic claims that might preemptively imply all visual working memories are abstractions of percepts. Time (and much more work) will likely show things to be much more subtle and complex.

The work presented in this paper is cool, but it uses a specific case: spatial stimuli (gratings) with the task to remember orientation. This limits possible conclusions for several reasons (1) These results are specific to EVC, as visual maps are a prerequisite meaning that these results will not hold up in other, non-retinotopic areas. (2) The fact that subjects are "focusing" along the main stimulus axis (attention or not) can simply be a strategy employed by the majority of (but not all) subjects - a strategy that may not be necessary to do the task, and therefore not a canonical method of Abstraction. It may be a "shared preferred strategy" or something. (3) If subjects had to (for example) remember contrast, and not orientation, results may have been entirely different (I would hypothesize there is no line-like abstraction in this case). Vice versa, if the perceptual task would have been on orientation (instead of contrast), the authors admit that "participants would reformat the grating into a line-like representation to make the judgments" (quote from author's response under "Additional context"). Thus, the results may be entirely about the task/ cognitive state, and not about how perceptual information is abstracted into memory.

Instead of unveiling *the* working memory Abstraction, this work (very nicely) shows a specific instance of possible abstraction. A more correct (but admittedly, less "sexy") conclusion may be "Visual working memories of orientation can be abstracted into a line in early visual cortex". As it stands, the authors still do not acknowledge any of the alternatives that myself (see above) and the other reviewers have put forth, nor do they acknowledge recent work by Chunharas et al. (2023, BioRxiv), that directly applies principles of efficient coding to address the exact same question of working memory abstraction. The link between a "line-like" representation and efficient coding implied by the authors (in their response) is merely tentative to me, but it would be great if the authors could explain this further.

These were, and remain, the major weaknesses in the original submission, that in my view have not been adequately addressed by the authors, as many overly broad conclusions about abstractions are currently still present in the manuscript (in for example the title).

Reviewer #1 (Public Review):

In this study, Gonzalez Alam et al. report a series of functional MRI results about the neural processing from the visual cortex to high-order regions in the default-mode network (DMN), compiling evidence from task-based functional MRI, resting-state connectivity, and diffusion-weighted imaging. Their participants were first trained to learn the association between objects and rooms/buildings in a virtual reality experiment; after the training was completed, in the task-based MRI experiment, participants viewed the objects from the earlier training session and judged if the objects were in the semantic category (semantic task) or if they were previously shown in the same spatial context (spatial context task). Based on the task data, the authors utilised resting-state data from their previous studies, visual localiser data also from previous studies, as well as structural connectivity data from the Human Connectome Project, to perform various seed-based connectivity analysis. They found that the semantic task causes more activation of various regions involved in object perception while the spatial context task causes more activation in various regions for place perception, respectively. They further showed that those object perception regions are more connected with the frontotemporal subnetwork of the DMN while those place perception regions are more connected with the medial-temporal subnetwork of the DMN. Based on these results, the authors argue that there are two main pathways connecting the visual system to high-level regions in the DMN, one linking object perception regions (e.g., LOC) leading to semantic regions (e.g., IFG, pMTG), the other linking place perception regions (e.g., parahippocampal gyri) to the entorhinal cortex and hippocampus.

Below I provide my takes on (1) the significance of the findings and the strength of evidence, (2) my guidance for readers regarding how to interpret the data, as well as several caveats that apply to their results, and finally (3) my suggestions for the authors.

(1) Significance of the results and strength of the evidence

I would like to praise the authors for, first of all, trying to associate visual processing with high-order regions in the DMN. While many vision scientists focus specifically on the macroscale organisation of the visual cortex, relatively few efforts are made to unravel how neural processing in the visual system goes on to engage representations in regions higher up in the hierarchy (a nice precedent study that looks at this issue is by Konkle and Caramazza, 2017). We all know that visual processing goes beyond the visual cortex, potentially further into the DMN, but there's no direct evidence. So, in this regard, the authors made a nice try to look at this issue.

Having said this, the authors' characterisation of the organisation of the visual cortex (object perception/semantics vs. place perception/spatial contexts) does not go beyond what has been known for many decades by vision neuroscience. Specifically, over the past two decades, numerous proposals have been put forward to explain the macroscale organisation of the visual system, particularly the ventrolateral occipitotemporal cortex. A lateral-medial division has been reliably found in numerous studies. For example, some researchers found that the visual cortex is organised along the separation of foveal vision (lateral) vs. peripheral vision (medial), while others found that it is structured according to faces (lateral) vs. places (medial). Such a bipartite division is also found in animate (lateral) vs. inanimate (medial), small objects (lateral) vs. big objects (medial), as well as various cytoarchitectonic and connectomic differences between the medial side and the lateral side of the visual cortex. Some more recent studies even demonstrate a tripartite division (small objects, animals, big objects; see Konkle and Caramazza, 2013). So, in terms of their characterisation of the visual cortex, I think Gonzalez Alam et al. do not add any novel evidence to what the community of neuroscience has already known.

However, the authors' effort to link visual processing with various regions of the DMN is certainly novel, and their attempt to gather converging evidence with different methodologies is commendable. The authors are able to show that, in an independent sample of resting-state data, object-related regions are more connected with semantic regions in the DMN while place-related regions are more connected with navigation-related regions in the DMN, respectively. Such patterns reveal a consistent spatial overlap with their Kanwisher-type face/house localiser data and also concur with the HCP white-matter tractography data. Overall, I think the two pathways explanation that the authors seek to argue is backed by converging evidence. The lack of travelling wave type of analysis to show the spatiotemporal dynamics across the cortex from the visual cortex to high-level regions is disappointing though because I was expecting this type of analysis would provide the most convincing evidence of a 'pathway' going from one point to another. Dynamic caudal modelling or Granger causality may also buttress the authors' claim of pathway because many readers, like me, would feel that there is not enough evidence to convincingly prove the existence of a 'pathway'.

(2) Guidance to the readers about interpretation of the data

The organisation of the visual cortex and the organisation of the DMN historically have been studied in parallel with little crosstalk between different communities of researchers. Thus, the work by Gonzalez Alam et al. has made a nice attempt to look at how visual processing goes beyond the realm of the visual cortex and continues into different subregions of the DMN.

While the authors of this study have utilised multiple methods to obtain converging evidence, there are several important caveats in the interpretation of their results:

(1) While the authors choose to use the term 'pathway' to call the inter-dependence between a set of visual regions and default-mode regions, their results have not convincingly demonstrated a definitive route of neural processing or travelling. Instead, the findings reveal a set of DMN regions are functionally more connected with object-related regions compared to place-related regions. The results are very much dependent on masking and thresholding, and the patterns can change drastically if different masks or thresholds are used.

(2) Ideally, if the authors could demonstrate the dynamics between the visual cortex and DMN in the primary task data, it would be very convincing evidence for characterising the journey from the visual cortex to DMN. Instead, the current connectivity results are derived from a separate set of resting state data. While the advantage of the authors' approach is that they are able to verify certain visual regions are more connected with certain DMN regions even under a task-free situation, it falls short of explaining how these regions dynamically interact to convert vision into semantic/spatial decision.

(3) There are several results that are difficult to interpret, such as their psychophysiological interactions (PPI), representational similarity analysis, and gradient analysis. For example, typically for PPI analysis, researchers interrogate the whole brain to look for PPI connectivity. Their use of targeted ROI is unusual, and their use of spatially extensive clusters that encompass fairly large cortical zones in both occipital and temporal lobes as the PPI seeds is also an unusual approach. As for the gradient analysis, the argument that the semantic task is higher on Gradient 1 than the spatial task based on the statistics of p-value = 0.027 is not a very convincing claim (unhelpfully, the figure on the top just shows quite a few blue 'spatial dots' on the hetero-modal end which can make readers wonder if the spatial context task is really closer to the unimodal end or it is simply the authors' statistical luck that they get a p-value under 0.05). While it is statistically significant, it is weak evidence (and it is not pertinent to the main points the authors try to make).

(3) My suggestion for the authors

There are several conceptual-level suggestions that I would like to offer to the authors:

(1) If the pathway explanation is the key argument that you wish to convey to the readers, an effective connectivity type of analysis, such as Granger causality or dynamic caudal modelling, would be helpful in revealing there is a starting point and end point in the pathway as well as revealing the directionality of neural processing. While both of these methods have their issues (e.g., Granger causality is not suitable for haemodynamic data, DCM's selection of seeds is susceptible to bias, etc), they can help you get started to test if the path during task performance does exist. Alternatively, travelling wave type of analysis (such as the results by Raut et al. 2021 published in Science Advances) can also be useful to support your claims of the pathway.

(2) I think the thresholding for resting state data needs to be explained - by the look of Figure 2E and 3E, it looks like whole-brain un-thresholded results, and then you went on to compute the conjunction between these un-thresholded maps with network templates of the visual system and DMN. This does not seem statistically acceptable, and I wonder if the conjunction that you found would disappear and reappear if you used different thresholds. Thus, for example, if the left IFG cluster (which you have shown to be connected with the visual object regions) would disappear when you apply a conventional threshold, this means that you need to seriously consider the robustness of the pathway that you seek to claim... it may be just a wild goose that you are chasing.

(3) There are several analyses that are hard to interpret and you can consider only reporting them in the supplementary materials, such as the PPI results and representational similarity analysis, as none of these are convincing. These analyses do not seem to add much value to make your argument more convincing and may elicit more methodological critiques, such as statistical issues, the set-up of your representational theory matrix, and so on.

Reviewer #1 (Public Review):

The study by Chikermane and colleagues investigates the functional, structural, and dopaminergic network substrates of cortical beta oscillations (13-30 Hz). The major strength of the work lies in the methodology taken by the authors, namely a multimodal lesion network mapping. First, using invasive electrophysiological recordings from healthy cortical territories of epileptic patients they identify regions with the highest beta power. Next, they leverage open-access MRI data and PET atlases and use the identified high-beta regions as seeds to find (1) the whole-brain functional and structural maps of regions that form the putative underlying network of high-beta regions and (2) the spatial distribution of dopaminergic receptors that show correlation with nodal connectivity of the identified networks. These steps are achieved by generating aggregate functional, structural, and dopaminergic network maps using lead-DBS toolbox, and by contrasting the results with those obtained from high-alpha regions.

The main findings are:<br /> (1) Beta power is strongest across frontal, cingulate, and insular regions in invasive electrophysiological data, and these regions map onto a shared functional and structural network.<br /> (2) The shared functional and structural networks show significant positive correlations with dopamine receptors across the cortex and basal ganglia (which is not the case for alpha, where correlations are found with GABA).

Nevertheless, a few clarifications regarding the choice of high-power electrodes and distributions of functional connectivity maps (i.e., strength and sign across cortex and sub-cortex) can help with understanding the results.

Reviewer #1 (Public Review):

Vision is a highly active process. Humans move their eyes 3-4 times per second to sample information with high visual acuity from our environment, and where eye movements are directed is critical to our understanding of active vision. Here, the authors propose that the cost of making a saccade contributes critically to saccade selection (i.e., whether and where to move the eyes). The authors build on their own recent work that the effort (as measured by pupil size) that comes with planning and generating an eye movement varies with saccade direction. To do this, the authors first measured pupil size for different saccade directions for each participant. They then correlated the variations in pupil size obtained in the mapping task with the saccade decision in a free-choice task. The authors observed a striking correlation: pupil size in the mapping task predicted the decision of where to move the eyes in the free choice task. In this study, the authors provide a number of additional insightful analyses (e.g., based on saccade curvature, and saccade latency) and experiments that further support their claim that the decision to move the eyes is influenced by the effort to move the eyes in a particular direction. One experiment showed that the same influence of assumed saccade costs on saccade selection is observed during visual search in natural scenes. Moreover, increasing the cognitive load by adding an auditory counting task reduced the number of saccades, and in particular reduced the costly saccades. In sum, these experiments form a nice package that convincingly establishes the association between pupil size and saccade selection.

In my opinion, the causal structure underlying the observed results is not so clear. While the relationship between pupil size and saccade selection is compelling, it is not clear that saccade-related effort (i.e., the cost of a saccade) really drives saccade selection. Given the correlational nature of this relationship, there are other alternatives that could explain the finding. For example, saccade latency and the variance in landing positions also vary across saccade directions. This can be interpreted for instance that there are variations in oculomotor noise across saccade directions, and maybe the oculomotor system seeks to minimize that noise in a free-choice task. In fact, given such a correlational result, many other alternative mechanisms are possible. While I think the authors' approach of systematically exploring what we can learn about saccade selection using pupil size is interesting, it would be important to know what exactly pupil size can add that was not previously known by simply analyzing saccade latency. For example, saccade latency anisotropies across saccade directions are well known, and the authors also show here that saccade costs are related to saccade latency. An important question would be to compare how pupil size and saccade latency uniquely contribute to saccade selection. That is, the authors could apply the exact same logic to their analysis by first determining how saccade latencies (or variations in saccade landing positions; see Greenwood et al., 2017 PNAS) vary across saccade directions and how this saccade latency map explains saccade selection in subsequent tasks. Is it more advantageous to use one or the other saccade metric, and how well does a saccade latency map correlate with a pupil size map?

In addition to eye-movement-related anisotropies across the visual field, there are of course many studies reporting visual field anisotropies (see Himmelberg, Winawer & Carrasco, 2023, Trends in Neuroscience for a review). It would be interesting to understand how the authors think about visual field anisotropies in the context of their own study. Do they think that their results are (in)dependent on such visual field variations (see Greenwood et al., 2017, PNAS; Ohl, Kroell, & Rolfs, 2024, JEP:Gen for a similar discussion)?

Finally, the authors conclude that their results "suggests that the eye-movement system and other cognitive operations consume similar resources that are flexibly allocated among each other as cognitive demand changes. The authors should speculate what these similar resources could mean? What are the specific operations of the auditory task that overlap in terms of resources with the eye movement system?

Reviewer #1 (Public Review):

In the manuscript "A microglia clonal inflammatory disorder in Alzheimer's Disease", Vicario et al. provide a compelling study elucidating a potential contribution of somatic mutations within the microglia population of the CNS that accelerates microglia activation and disease-associated gene signatures in Alzheimer's disease. Here they especially identified an "enrichment" of pathological SNVs in microglia, but not the peripheral blood, that are associated with clonal proliferative disorders and neurological diseases in a subset of patients with AD. Convincingly, they identified P-SNVs in microglia of AD patients located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in human microglia-like cells in vitro.

Overall, this study provides clear and detailed evidence from an AD patient cohort pointing to a potential contribution of microglia-specific somatic mutations to disease onset and/or progression in a subset of patients with Alzheimer's disease.

Strengths:<br /> As outlined above, the study identified P-SNVs in microglia of AD patients associated with clonal proliferative disorders, but also gave an in-depth analysis of re-occurring P-SNVs located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in HEK cells, BV2 cells, MAC cells, and human microglia-like cells in vitro.

Great care was taken here to validate their hypotheses at each step, as well as to identify the limitations of the possible conclusions. For example, they highlight that the pathway proposed to be affected may be an explanation for a subset of AD patients, and emphasize that it is yet unclear whether this accumulation of pathological SNVs is a cause or consequence of disease progression

The study clearly supports an enrichment of P-SNVs in several genes associated with clonal proliferative disorders in microglia and nicely separates this from SNVs associated with clonal hematopoiesis in the peripheral blood found in AD patients and controls.

The authors further acknowledged that several age-matched control patients were diagnosed with cancer or tumor-associated diseases and carefully dissected the occurring SNVs in these patients are not associated with the P-SNVs identified in the microglial compartment of the AD cohort.

Weaknesses:

Even though the study is overall very convincing, several points could help to connect the seen somatic variants in microglia more with a potential role in disease progression. The connection of P-SNVs in the genes chosen from neurological disorders was not further highlighted by the authors.

The authors show in snRNA-seq data that a disease-associated microglia state seems to be enriched in patients with somatic variants in the CBL ring domain, however, this analysis could be deepened. For example, how this knowledge may translate to patient benefits when the relevant cell populations appear concentrated in a single patient sample (Figure 5; AD52) is unclear; increasing the analyzed patient pool for Figure 5 and showcasing the presence of this microglia state of interest in a few more patients with driving mutations for CBL or other MAPK pathway associated mutations would lend their hypotheses further credibility.

A potential connection between P-SNVs in microglia and disease pathology and symptoms was not further explored by the authors.

A recent preprint (Huang et al., 2024) connected the occurrence of somatic variants in genes associated with clonal hematopoiesis in microglia in a large cohort of AD patients, this study is not further discussed or compared to the data in this manuscript.

Reviewer #1 (Public Review):

Summary:

The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

Strengths:

(1) 7T high-resolution MRS is used.

(2) This study combines the behavioral tests, MRS, and fMRI.

Weaknesses:

(1) In the intro, it seems to me that the multiple-demand (MD) regions are the key in this study. However, I didn't see any results associated with the MD regions. Did I miss something??

(2) How was the sample size determined? Is it sufficient??

(3) In Schallmo elife 2018, there was no correlation between GABA concentration and SI. How can we justify the different results different here?

(4) Basically this study contains the data of SI, BDT, GABA in MT+ and V1, Glu in MT+ and V1-all 6 measurements. There should be 6x5/2 = 15 pairwise correlations. However, not all of these results are included in Figure 1 and supplementary 1-3. I understand that it is not necessary to include all figures. But I suggest reporting all values in one Table.

(5) In Melnick (2013), the IQ scores were measured by the full set of WAIS-III, including all subtests. However, this study only used the visual spatial domain of gF. I wonder why only the visuo-spatial subtest was used not the full WAIS-III?

(6) In the functional connectivity part, there is no explanation as to why only the left MT+ was set to the seed region. What is the problem with the right MT+?

(7) In Melnick (2013), the authors also reported the correlation between IQ and absolute duration thresholds of small and large stimuli. Please include these analyses as well.

Reviewer #1 (Public Review):

Summary:

This is a fine paper that serves the purpose to show that the use of light sheet imaging may be used to provide whole brain imaging of axonal projections. The data provided suggest that at this point the technique provides lower resolution than with other techniques. Nonetheless, the technique does provide useful, if not novel, information about particular brain systems.

Strengths:

The manuscript is well written. In the introduction a clear description of the functional organization of the barrel cortex is provided provides the context for applying the use of specific Cre-driver lines to map the projections of the main cortical projection types using whole brain neuroanatomical tracing techniques. The results provided are also well written, with sufficient detail describing the specifics of how techniques were used to obtain relevant data. Appropriate controls were done, including the identification of whisker fields for viral injections and determination of the laminar pattern of Cre expression. The mapping of the data provides a good way to visualize low resolution patterns of projections.

Weaknesses:

(1) The results provided are, as stated in the discussion, "largely in agreement with previously reported studies of the major projection targets". However it must be stated that the study does not "extend current knowledge through the high sensitivity for detecting sparse axons, the high specificity of labeling of genetically defined classes of neurons and the brain wide analysis for assigning axons to detailed brain regions" which have all been published in numerous other studies. ( the allen connectivity project and related papers, along with others). If anything the labeling of axons obtained with light sheet imaging in this study does not provide as detailed mapping obtained with other techniques. Some detail is provided of how the raw images are processed to resolve labeled axons, but the images shown in the figures do not demonstrate how well individual axons may be resolved, of particular interest would be to see labeling in terminal areas such as other cortical areas, striatum and thalamus. As presented the light sheet imaging appears to be rather low resolution compared to the many studies that have used viral tracing to look at cortical projections from genetically identified cortical neurons.<br /> (2) Amongst the limitations of this study is the inability to resolve axons of passage and terminal fields. This has been done in other studies with viral constructs labeling synaptophysin. This should be mentioned.<br /> (3) There is no quantitative analysis of differences between the genetically defined neurons projecting to the striatum, what is the relative area innervated by, density of terminals, other measures.<br /> (4) Figure 5 is an example of the type of large sets of data that can be generated with whole brain mapping and registration to the Allen CCF that provides information of questionable value. Ordering the 50 plus structures by the density of labeling does not provide much in terms of relative input to different types of areas. There are multiple subregions for different functional types ( ie, different visual areas and different motor subregions are scattered not grouped together. Makes it difficult to understand any organizing principles.<br /> (5) The GENSAT Cre driver lines used must have the specific line name used, not just the gene name as the GENSAT BAC-Cre lines had multiple lines for each gene and often with very different expression patterns. Rbp4_KL100, Tlx3_PL56, Sim1_KJ18, Ntsr1_ GN220.

Reviewer #1 (Public Review):

Abbasi et al. assess in this MEG study the directed connectivity of both cortical and subcortical regions during continuous speech production and perception. The authors observed bidirectional connectivity patterns between speech-related cortical areas as well as subcortical areas in production and perception. Interestingly, they found in speaking low-frequency connectivity from subcortical (the right cerebellum) to cortical (left superior temporal) areas, while connectivity from the cortical to subcortical areas was in the high frequencies. In listening a similar cortico-subcortical connectivity pattern was observed for the low frequencies, but the reversed connectivity in the higher frequencies was absent.

The work by Abbasi and colleagues addresses a relevant, novel topic, namely understanding the brain dynamics between speaking and listening. This is important because traditionally production and perception of speech and language are investigated in a modality-specific manner. To have a more complete understanding of the neurobiology underlying these different speech behaviors, it is key to also understand their similarities and differences. Furthermore, to do so, the authors utilize state-of-the-art directed connectivity analyses on MEG measurements, providing a quite detailed profile of cortical and subcortical interactions for the production and perception of speech. Importantly, and perhaps most interesting in my opinion, is that the authors find evidence for frequency-specific directed connectivity, which is (partially) different between speaking and listening. This could suggest that both speech behaviors rely (to some extent) on similar cortico-cortical and cortico-subcortical networks, but different frequency-specific dynamics.

These elements mentioned above (investigation of both production and perception, both cortico-cortical and cortico-subcortical connectivity is considered, and observing frequency-specific connectivity profiles within and between speech behaviors), make for important novel contributions to the field. Notwithstanding these strengths, I find that they are especially centered on methodology and functional anatomical description, but that precise theoretical contributions for neurobiological and cognitive models of speech are less transparent. This is in part because the study compares speech production and perception in general, but no psychophysical or psycholinguistic manipulations are considered. I also have some critical questions about the design which may pose some confounds in interpreting the data, especially with regard to comparing production and perception.

(1) While the cortico-cortical and cortico-subcortical connectivity profiles highlighted in this study and the depth of the analyses are impressive, what these data mean for models of speech processing remains on the surface. This is in part due, I believe, to the fact that the authors have decided to explore speaking and listening in general, without targeting specific manipulations that help elucidate which aspects of speech processing are relevant for the particular connectivity profiles they have uncovered. For example, the frequency-specific directed connectivity is it driven by low-level psychophysical attributes of the speech or by more cognitive linguistic properties? Does it relate to the monitoring of speech, timing information, and updating of sensory predictions? Without manipulations trying to target one or several of these components, as some of the referenced work has done (e.g., Floegel et al., 2020; Stockert et al., 2021; Todorović et al., 2023), it is difficult to draw concrete conclusions as to which representations and/or processes of speech are reflected by the connectivity profiles. An additional disadvantage of not having manipulations within each speech behavior is that it makes the comparison between listening and speaking harder. That is, speaking and listening have marked input-output differences which likely will dominate any comparison between them. These physically driven differences (or similarities for that matter; see below) can be strongly reduced by instead exploring the same manipulations/variables between speaking and listening. If possible (if not to consider for future work), it may be interesting to score psychophysical (e.g., acoustic properties) or psycholinguistic (e.g., lexical frequency) information of the speech and see whether and how the frequency-specific connectivity profiles are affected by it.

(2) Recent studies comparing the production and perception of language may be relevant to the current study and add some theoretical weight since their data and interpretations for the comparisons between production and perception fit quite well with the observations in the current work. These studies highlight that language processes between production and perception, specifically lexical and phonetic processing (Fairs et al., 2021), and syntactic processing (Giglio et al., 2024), may rely on the same neural representations, but are differentiated in their (temporal) dynamics upon those shared representations. This is relevant because it dispenses with the classical notion in neurobiological models of language where production and perception rely on (partially) dissociable networks (e.g., Price, 2010). Rather those data suggest shared networks where different language behaviors are dissociated in their dynamics. The speech results in this study nicely fit and extend those studies and their theoretical implications.

(3) The authors align the frequency-selective connectivity between the right cerebellum and left temporal speech areas with recent studies demonstrating a role for the right cerebellum for the internal modelling in speech production and monitoring (e.g., Stockert et al., 2021; Todorović et al., 2023). This link is indeed interesting, but it does seem relevant to point out that at a more specific scale, it does not concern the exact same regions between those studies and the current study. That is, in the current study the frequency-specific connectivity with temporal regions concerns lobule VI in the right cerebellum, while in the referenced work it concerns Crus I/II. The distinction seems relevant since Crus I/II has been linked to the internal modelling of more cognitive behavior, while lobule VI seems more motor-related and/or contextual-related (e.g., D'Mello et al., 2020; Runnqvist et al., 2021; Runnqvist, 2023).

(4) On the methodological side, my main concern is that for the listening condition, the authors have chosen to play back the speech produced by the participants in the production condition. Both the fixed order as well as hearing one's own speech as listening condition may produce confounds in data interpretation, especially with regard to the comparison between speech production and perception. Could order effects impact the observed connectivity profiles, and how would this impact the comparison between speaking and listening? In particular, I am thinking of repetition effects present in the listening condition as well as prediction, which will be much more elevated for the listening condition than the speaking condition. The fact that it also concerns their own voice furthermore adds to the possible predictability confound (e.g., Heinks-Maldonado et al., 2005). In addition, listening to one's speech which just before has been articulated may, potentially strategically even, enhance inner speech and "mouthing" in the participants, hereby thus engaging the production mechanism. Similarly, during production, the participants already hear their own voice (which serves as input in the subsequent listening condition). Taken together, both similarities or differences between speaking and listening connectivity may have been due to or influenced by these order effects, and the fact that the different speech behaviors are to some extent present in both conditions.

(5) The ability of the authors to analyze the spatiotemporal dynamics during continuous speech is a potentially important feat of this study, given that one of the reasons that speech production is much less investigated compared to perception concerns motor and movement artifacts due to articulation (e.g., Strijkers et al., 2010). Two questions did spring to mind when reading the authors' articulation artifact correction procedure: If I understood correctly, the approach comes from Abbasi et al. (2021) and is based on signal space projection (SSP) as used for eye movement corrections, which the authors successfully applied to speech production. However, in that study, it concerned the repeated production of three syllables, while here it concerns continuous speech of full words embedded in discourse. The articulation and muscular variance will be much higher in the current study compared to three syllables (or compared to eye movements which produce much more stable movement potentials compared to an entire discourse). Given this, I can imagine that corrections of the signal in the speaking condition were likely substantial and one may wonder (1) how much signal relevant to speech production behavior is lost?; (2) similar corrections are not necessary for perception, so how would this marked difference in signal processing affect the comparability between the modalities?

References:<br /> - Abbasi, O., Steingräber, N., & Gross, J. (2021). Correcting MEG artifacts caused by overt speech. Frontiers in Neuroscience, 15, 682419.<br /> - D'Mello, A. M., Gabrieli, J. D., & Nee, D. E. (2020). Evidence for hierarchical cognitive control in the human cerebellum. Current Biology, 30(10), 1881-1892.<br /> - Fairs, A., Michelas, A., Dufour, S., & Strijkers, K. (2021). The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. Cerebral Cortex Communications, 2(3), tgab040.<br /> - Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.<br /> - Giglio, L., Ostarek, M., Sharoh, D., & Hagoort, P. (2024). Diverging neural dynamics for syntactic structure building in naturalistic speaking and listening. Proceedings of the National Academy of Sciences, 121(11), e2310766121.<br /> - Heinks‐Maldonado, T. H., Mathalon, D. H., Gray, M., & Ford, J. M. (2005). Fine‐tuning of auditory cortex during speech production. Psychophysiology, 42(2), 180-190.<br /> - Price, C. J. (2010). The anatomy of language: a review of 100 fMRI studies published in 2009. Annals of the new York Academy of Sciences, 1191(1), 62-88.<br /> - Runnqvist, E., Chanoine, V., Strijkers, K., Pattamadilok, C., Bonnard, M., Nazarian, B., ... & Alario, F. X. (2021). Cerebellar and cortical correlates of internal and external speech error monitoring. Cerebral Cortex Communications, 2(2), tgab038.<br /> - Runnqvist, E. (2023). Self-monitoring: The neurocognitive basis of error monitoring in language production. In Language production (pp. 168-190). Routledge.<br /> - Stockert, A., Schwartze, M., Poeppel, D., Anwander, A., & Kotz, S. A. (2021). Temporo-cerebellar connectivity underlies timing constraints in audition. Elife, 10, e67303.<br /> - Strijkers, K., Costa, A., & Thierry, G. (2010). Tracking lexical access in speech production: electrophysiological correlates of word frequency and cognate effects. Cerebral cortex, 20(4), 912-928.<br /> - Todorović, S., Anton, J. L., Sein, J., Nazarian, B., Chanoine, V., Rauchbauer, B., ... & Runnqvist, E. (2023). Cortico-cerebellar monitoring of speech sequence production. Neurobiology of Language, 1-21.

Reviewer #1 (Public Review):

Summary:

This paper reports the finding that less fat accumulates in C. elegans that are feeding on Comamonas aquatica DA1877 (DA) vs the standard lab diet of Escherichia coli OP50 (OP50). While these bacteria are likely to be different in many ways, the authors found that fat accumulation phenotype depends on the vitamin B12 content of the bacterial diet and the involvement of B12 in the methionine cycle, affecting SAMS-1 and phosphatidylcholine (PC) synthesis. They report that low PC levels activate SREBP-1 (SBP-1 in C. elegans) and that an important target of SBP-1 is the delta 9 desaturase FAT-7. Finally, they describe a role for ASM-3, an acid sphingomyelinase, in influencing PC synthesis and fat accumulation in the worm.

Strengths:

This is a comprehensive story about how a dietary change affects fat accumulation in C. elegans. Their experimental evidence is convincing. The most novel aspect of this paper is that the coelomecyte expression of asm-3 contributes to PC/TAG homeostasis in C. elegans, which most likely occurs through the production of phosphocholine by the enzymatic breakdown of sphingomyelin by ASM-3. The phosphocholine will provide precursors for phosphatidylcholine (PC) synthesis, contributing to the PC synthesis pathway.

Weaknesses:

In the way the story is presented, the authors tend to imply that they discovered the pathways of B12, PC, SBP-1, and FAT-7, ignoring some important studies describing the relationship between PC synthesis and TAG accumulation in both the mammalian lipid metabolism field (liver) as well as in C. elegans. Many previous studies with similar results are not cited appropriately. Thus, the pathways reported in the paper are not new, and in this sense, the work is mostly confirmatory.

Reviewer #1 (Public Review):

Li et al. report here on the expression of a G-protein subunit Gng13 in ectopic tuft cells that develop after severe pulmonary injury in mice. By deleting this gene in ectopic tuft cells as they arise, the authors observed worsened lung injury and greater inflammation after influenza infection, as well as a decrease in the overall number of ectopic tuft cells. This was in stark contrast to deletion of Trpm5, a cation channel generally thought to be required for all functional gustatory signaling in tuft cells, where no phenotype is observed. Strengths here include a thorough assessment of lung injury via a number of different techniques. Weaknesses are notable: Confusingly, these findings are at odds with reports from other groups demonstrating no obvious phenotype upon influenza infection in mice lacking the transcription factor Pou2f3, which is essential for all tuft cell specification and development. The authors speculate that heterogeneity within nascent tuft cell populations, specifically the presence of pro- and anti-inflammatory tuft cells, may explain this difference, but they do not provide any data to support this idea.

Notes on revision: The authors provided responses to some of my critiques. I think the central discrepancy between the lack of a phenotype in Pou2f3 and Trpm5 KO mice compared to the stronger phenotype in the Chat-Cre / Gng13 KO mice remains unresolved and will require future work to provide a clear model. This may or may not ultimately involve tuft cell heterogeneity.

Reviewer #1 (Public Review):

Summary:

The revised manuscript is much improved. As stated previously, it is on an interesting and important topic and provides many new potentially important findings. The manuscript contains a large amount of high-quality data. In the revised manuscript, the authors have done a nice job addressing the concerns raised in the previous review. They have refined their conclusions and the evidence provided supports conclusions drawn. Likewise, the writing and low of the manuscript is much improved.

Strengths:

The manuscript contains a large amount of high-quality data that is used to draw interesting and important conclusions.

Weaknesses:

There are still some issues with grammar and word usage, but these should be easily corrected with some additional minor editing. Other than some minor editing, my only real question/concern is whether the data presented shows that SCC3 is directly involved in gene regulation. It may well be that changes in chromatin structure caused by mutations in SCC3 and the axial element protein containing genes examined indirectly affect transcript levels for the genes examined.

Reviewer #1 (Public Review):

Interactions known to be important for melanosome transport include exon F and the globular tail domain (GTD) of MyoVa with Mlph. Motivated by a discrepancy between in vitro and cell culture results regarding necessary interactions for MyoVa to be recruited to the melanosome, the authors used a series of pull-down and pelleting assays experiments to identify an additional interaction that occurs between exon G of MyoVa and Mlph. This interaction is independent of and synergistic with the interaction of Mlph with exon F. However, the interaction of the actin-binding domain of Mlph can occur either with exon G or with the actin filament, but not both simultaneously. These data lead to a modified recruitment model where both exon F and exon G enhance binding of Mlph to auto-inhibited MyoVa, and then via an unidentified switch (PKA?) the actin-binding domain of Mlph dissociates from MyoVa and interacts with the actin filament to enhance MyoVa processivity.

The only weakness noted is that the authors could have had a more complete story if they pursued whether PKA phosphorylation/dephosphorylation of Mlph is indeed the switch for the actin-binding domain of Mlph to interact with exon G versus the actin filament.

Reviewer #1 (Public Review):

Summary:

The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

Strengths:

The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

Weaknesses:

(1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

(2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

Reviewer #1 (Public Review):

The comments below are from my review of the first submission of this article. I would now like to thank the authors for their hard work in responding to my comments. I am happy with the changes they have made, in particular the inclusion of further experimental evidence in Figures 2 and 4. I have no further comments to make.

In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development timecourse. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.

I am convinced that the novel relationships reported here between miR-199a/b-5p and target genes FZD6, ITGA3 and CAV1 are likely to be genuine. It is important for researchers working on this system and related diseases to know all the miRNA/mRNA relationships but, as the authors have already published work studying the most dynamic miRNA (miR-140-5p) in this biological system I was not convinced that this study of the second miRNA in their list provided a conceptual advance on their previous work.

I was also concerned with the lack of reporting of details of the manipulation experiments. The authors state that they have over-expressed miR-199a-5p (Figure 2A) and knocked down miR-199b-5p (Figure 2B) but they should have reported their proof that these experiments had worked as predicted, e.g. showing the qRT-PCR change in miRNA expression. Similarly, I was concerned that one miRNA was over-expressed while the other was knocked down - why did the authors not attempt to manipulate both miRNAs in both directions? Were they unable to achieve a significant change in miRNA expression or did these experiments not confirm the results reported in the manuscript?

I had a number of issues with the way in which some of the data is presented. Table 1 only reported whether a specific pathway was significant or not for a given differential expression analysis but this concealed the extent of this enrichment or the level of statistical significance reported. Could it be redrawn to more similarly match the format of Figure 3A? The various shades of grey in Figure 2 and Figure 4 made it impossible to discriminate between treatments and therefore identify whether these data supported the conclusions made in the text. It also appeared that the same results were reported in Figure 3B and 3C and, indeed, Figure 3B was not referred to in the main text. Perhaps this figure could be made more concise by removing one of these two sets of panels?

Overall, while I think that this is an interesting and valuable paper, I think its findings are relatively limited to those interested in the role of miRNAs in this specific biomedical context.

Reviewer #1 (Public Review):

The inferior colliculus (IC) is the central auditory system's major hub. It integrates ascending brainstem signals to provide acoustic information to the auditory thalamus. The superficial layers of the IC ("shell" IC regions as defined in the current manuscript) also receive a massive descending projection from the auditory cortex. This auditory cortico-collicular pathway has long fascinated the hearing field, as it may provide a route to funnel "high-level" cortical signals and impart behavioral salience upon an otherwise behaviorally agnostic midbrain circuit.

Accordingly, IC neurons can respond differently to the same sound depending on whether animals engage in a behavioral task (Ryan and Miller 1977; Ryan et al., 1984; Slee & David, 2015; Saderi et al., 2021; De Franceschi & Barkat, 2021). Many studies also report a rich variety of non-auditory responses in the IC, far beyond the simple acoustic responses one expects to find in a "low-level" region (Sakurai, 1990; Metzger et al., 2006; Porter et al., 2007). A tacit assumption is that the behaviorally relevant activity of IC neurons is inherited from the auditory cortico-collicular pathway. However, this assumption has never been tested, owing to two main limitations of past studies:

(1) Prior studies could not confirm if data were obtained from IC neurons that receive monosynaptic input from the auditory cortex.

(2) Many studies have tested how auditory cortical inactivation impacts IC neuron activity; the consequence of cortical silencing is sometimes quite modest. However, all prior inactivation studies were conducted in anesthetized or passively listening animals. These conditions may not fully engage the auditory cortico-collicular pathway. Moreover, the extent of cortical inactivation in prior studies was sometimes ambiguous, which complicates interpreting modest or negative results.

Here, the authors' goal is to directly test if the auditory cortex is necessary for behaviorally relevant activity in IC neurons. They conclude that surprisingly, task relevant activity in cortico-recipient IC neuron persists in absence of auditory cortico-collicular transmission. To this end, a major strength of the paper is that the authors combine a sound-detection behavior with clever approaches that unambiguously overcome the limitations of past studies.

First the authors inject a transsynaptic virus into the auditory cortex, thereby expressing a genetically encoded calcium indicator in the auditory cortex's postsynaptic targets in the IC. This powerful approach enables 2-photon Ca2+ imaging from IC neurons that unambiguously receive monosynaptic input from auditory cortex. Thus, any effect of cortical silencing should be maximally observable in this neuronal population. Second, they abrogate auditory cortico-collicular transmission using lesions of auditory cortex. This "sledgehammer" approach is arguably the most direct test of whether cortico-recipient IC neurons will continue to encode task-relevant information in absence of descending feedback. Indeed, their method circumvents the known limitations of more modern optogenetic or chemogenetic silencing, e.g. variable efficacy.

The authors have revised their manuscript and adequately addressed the major concerns. Although more in depth analyses of these rich datasets are definitely possible, the current results nevertheless stand on their own. Indeed, the work serves as a beacon to move away from the idea that cortico-collicular projections function primarily to impart behavioral relevance upon auditory midbrain neurons. This knowledge inspires a search for alternative explanations as to the role of auditory cortico-collicular synapses in behavior.

Reviewer #1 (Public Review):

Summary:<br /> This is an excellent study by a superb investigator who discovered and is championing the field of migrasomes. This study contains a hidden "gem" - the induction of migrasomes by hypotonicity and how that happens. In summary, an outstanding fundamental phenomenon (migrasomes) en route to becoming transitionally highly significant.

Strengths:

Innovative approach at several levels. Migrasomes - discovered by Dr Yu's group - are an outstanding biological phenomenon of fundamental interest and now of potentially practical value.

Weaknesses:

I feel that the overemphasis on practical aspects (vaccine), however important, eclipses some of the fundamental aspects that may be just as important and actually more interesting. If this can be expanded, the study would be outstanding.

Reviewer #1 (Public Review):

Summary:

In this paper, Wang et al show that differentiated peridermal cells of the zebrafish epidermis extend cytoneme-like protrusions toward the less differentiated, intermediate layer below. They present evidence that expression of a dominant-negative cdc42, inhibits cytoneme formation and leads to elevated expression of a marker of undifferentiated keratinocytes, krtt1c19e, in the periderm layer. Data is presented suggesting the involvement of Delta-Notch signaling in keratinocyte differentiation. Finally, changes in expression of the inflammatory cytokine IL-17 and its receptors is shown to affect cytoneme number and periderm structure in a manner similar to Notch and cdc42 perturbations.

Strengths:

Overall, the idea that differentiated cells signal to underlying undifferentiated cells via membrane protrusions in skin keratinocytes is interesting and novel, and it is clear that periderm cells send out thin membrane protrusions that contain a Notch ligand. Further, perturbations that affect cytoneme number, Notch signaling, and IL-17 expression clearly lead to changes in periderm structure and gene expression.

Weaknesses:

More work is needed to determine whether the effects on keratinocyte differentiation are due to a loss of cytonemes themselves, or to broader effects of inhibiting cdc42. Moreover, more evidence is needed to support the claim that periderm cytonemes deliver Delta ligands to induce Notch signaling below. Without these aspects of the study being solidified, understanding how IL-17 affects these processes seems premature.

Reviewer #1 (Public Review):

Summary:

Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.

Strengths:

Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.

Weaknesses:

I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).

In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.

Reviewer #1 (Public Review):

Summary:

Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.

Strengths:

(1) Both human and mouse samples were examined.<br /> (2) The HA model was used.<br /> (3) genome-wide DNA methylation analysis was performed.

Weaknesses:

(1) In some experiments, the selection of the control groups was not ideal.<br /> (2) More details on analyzing methods and information on replicates need to be included.<br /> (3) Discussion can be improved by comparing findings to other relevant studies.<br /> (4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.

Reviewer #1 (Public Review):

Summary:

In this study the authors use an elegant set of single-molecule experiments to assess the transcriptional and post-transcriptional regulation of RecB. The question stems from a previous observation from the same lab, that RecB protein levels are low and not induced under DNA damage. The authors first show that recB transcript levels are low and have a short half-life. They further show that RecB levels are likely regulated via translational control. They provide evidence for low noise in RecB protein levels across cells and show that the translation of the mRNA increases under double-strand break conditions. Authors identify Hfq binding sites in the recBCD operon and show that Hfq regulates the levels of RecB protein without changing the mRNA levels. They suggest that RecB translation is directly controlled by Hfq binding to mRNA, as mutating one of the binding sites has a direct effect on RecB protein levels.

Strengths:

The implication of Hfq in regulation of RecB translation is important and suggests mechanisms of cellular response to DNA damage that are beyond the canonically studied mechanisms (such as transcriptional regulation by LexA). Data are clearly presented and the writing is direct and easy to follow. Overall, the study is well-designed and provides novel insights into the regulation of RecB, that is part of the complex required to process break ends.

Weaknesses:

Some key findings need additional support/ clarifications to strengthen the conclusions. These are suggested to the authors.

Reviewer #1 (Public Review):

I thank the authors for addressing almost all my comments on the previous version of this manuscript, which studies the representation by gender and name origin of authors from Nature and Springer Nature articles in Nature News.

The representation of author identities is an important step towards equality in science, and the authors found that women are underrepresented in news quotes and mentions with respect to the proportion of women authors.

The research is rigorously conducted. It presents relevant questions and compelling answers. The documentation of the data and methods is thoroughly done, and the authors provide the code and data for reproduction.

Reviewer #1 (Public Review):

The study offers intriguing insights, yet interpretations warrant caution, as the authors themselves acknowledged in their discussion of limitations.

The observed grid-like neural activity might not signify navigating a social landscape but rather a sensory feature space. The study's design had participants associate each face with a pair of bar lengths, with the purported 'navigation' being merely a response to the morphing of bar graph images. Crucially, the task did not necessitate any social cognitive processing to estimate grid-like activity. When making social decisions in a separate task, it is unclear whether participants were actually traversing a social space mentally or simply recalling the bar graphs linked to each face to calculate decision values. Notably, during the trust game, competence and trustworthiness did not equally influence decision-making (as illustrated by Equation 1), implying the possibility that the space represented may be more perceptual than social in nature.

The existence of a universal brain representation for faces within a social context is still debatable. Participants were not required to form a cognitive map of the six faces based on social traits; they could simply remember each face's trait values. While the study suggests that reaction times correlated with the perceived social distances between faces hint at the creation of internal representations, this phenomenon could occur without a true cognitive map of the face relationships. To convincingly argue for such internal representations in the brain, additional multivariate pattern analysis would be necessary to demonstrate that these are not merely the result of perceptual differences in the bar graphs associated with each face.

Reviewer #1 (Public Review):

Summary:

The study by He et al. investigates the relationship of an increased susceptibility of diabetes patients towards COVID-19. The paper raises the possibility that hyperglycemia-induced cathepsin L maturation could be one of the driving forces in this pathology, suggesting that an increased activity of CTSL leads to accelerated virus infection rates due to an elevated processing of the SARS-CoV-2 spike protein.

In a clinical case-control study, the team found that severity of corona infections was higher in diabetic patients, and their CTSL levels correlated well with the progression of the disease. They further showed an increase in CTSL activity in long term as well as acute hyperglycemia. SARS-CoV-2 increasingly infected cells that were cultured in serum from diabetic patients, the same was observed using high glucose medium. No effect was observed in the medium with increased concentrations of insulin. CTSL knockout abolished the glucose-dependent increase in infection.

Increased glucose levels did not correlate with an increase in CTSL transcription. Rather He et al. could show that high glucose levels led to CTSL translocation from the ER into the lysosome. It was the glucose-dependent processing of the protease to its active form which promoted infection.

Overall, it is a very complete study starting from a clinical observation and ending on the molecular mechanism. A strength is certainly the wide selection of experiments. The clinical study to investigate the effect of glucose on CTSL concentrations in healthy individuals sets the stage for experiments in cell culture, animal models and human tissue. The effect of CTSL knockout cell lines on glucose-induced SARS-CoV2 infection rates are convincing. Finally, the team used a combination of Western blots and confocal microscopy to identify the underlying molecular mechanisms.

The authors keep the diabetic condition at the center of their study and extend on previous knowledge of glucose-induced CTSL activation and their consequences for Covid19 infections. By doing so, they create a novel connection between CTSL involvement in SARS-CoV2 infections and diabetes. This enables novel, public awareness of the susceptibility of diabetes patients to the disease.

Reviewer #1 (Public Review):

Summary:

In this study, Liu et al. investigate the signaling pathway that triggers sliding motility in the bacterium B. subtilis in response to subinhibitory concentrations of the antibiotic chloramphenicol. The authors used a genetic approach to identify the master regulator CodY playing a regulatory role in this behavior. They used transcriptional and metabolomic profiling to delineate the spatiotemporal separation of the regulatory networks that define distinct metabolic states related to purine metabolism and pyruvate utilization, which are ultimately responsible for the induction of sliding in response to chloramphenicol. Many readers would be interested to read this work showing how extracellular signals modulate microbial physiology and metabolism.

Strengths:

This work presents numerous technical and conceptual strengths. In the opinion of this referee, the most significant conceptual strength of this work is to (once again) provide evidence that antibiotics are not merely produced by bacteria to eliminate competitors. Bacteria have evolved to respond to their presence and activate a range of physiological responses, which are poorly understood. Understanding these responses is critical to fully understand the evolutionary consequences associated with the use of antibiotics. From a technical standpoint, perhaps the most relevant aspect is the robust phenotypic assay developed by the authors to study sliding motility in the presence of chloramphenicol. This robustness enables genetic work using mutants and performing omics assays to characterize the response to chloramphenicol in detail. Additionally, two sets of results stood out and provided important value to this work. One is the comparison established between the sliding induced by chloramphenicol and the sliding generated in the ΔcodY mutant, to determine the genes and the metabolites (using transcriptomics and metabolomics) specifically associated with the response to chloramphenicol without being part of the general Cody-mediated induction of sliding. The second set of results led the authors to identify precise genes of bacterial metabolism (pdhA) responsible for the sliding phenotype in response to chloramphenicol, and conducted genetic experiments to demonstrate that the pdhA mutant does not respond to the presence of chloramphenicol.

Weaknesses:

This work has three main weaknesses, all related to transcriptomic and metabolomic analyses. Firstly, there is the challenge of understanding the essence of the omics results. This section presents an overwhelming array of genes involved in different metabolic pathways, without an obvious thread to tie these hits together. It is easy to get lost in this section. For instance, one cannot be certain if the hits from one particular metabolic pathway are significant enough to figure out to which degree is this pathway responsible for the sliding phenotype. This section contains a huge diversity of genes and pathways and needs to be streamlined. Related to this, the message of the omics experiments highlights a very close relationship between purine and pyruvate metabolism in sliding motility. However, it is unclear how these metabolic pathways may influence sliding or any other specific bacterial behavior. I do not mean to say that it is not possible, just that the connection/mechanism is missing. The third weakness concerns the omics results that sometimes are in conflict. The authors proposed that this may stem from a division of labor and the coexistence of different subpopulations with different metabolisms within the microbial community. While plausible, other possibilities are equally plausible and should be tested in a revised version of the work.

Reviewer #1 (Public Review):

By identifying a loss of function mutant of IQCH in infertile patient, Ruan et al. shows that IQCH is essential for spermiogenesis by generating a knockout mouse model of IQCH. Similar to infertile patient with mutant of IQCH, Iqch knockout mice are characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. Mechanistically, IQCH regulates the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis.

Although this manuscript contains a potentially interesting piece of work that delineates a mechanism of IQCH that associates with spermatogenesis, this reviewer feels that a number of issues require clarification and re-evaluation for a better understanding of the role of IQCH in spermatogenesis.

Line 251 - 253, "To elucidate the molecular mechanism by which IQCH regulates male fertility, we performed liquid chromatography tandem mass spectrometry (LC‒MS/MS) analysis using mouse sperm lysates and detected 288 interactors of IQCH (Figure 5-source data 1)."

The reviewer had already raised significant concerns regarding the text above, noting that "LC‒MS/MS analysis using mouse sperm lysates" would not identify interactors of IQCH. However, this issue was not addressed in the revised manuscript. In the Methods section detailing LC-MS/MS, the authors stated that it was conducted on "eluates obtained from IP". However, there was no explanation provided on how IP for LC-MS/MS was performed. Additionally, it was unclear whether LC-MS or LC-MS/MS was utilized. The primary concern is that if LC‒MS/MS was conducted for the IP of IQCH, IQCH itself should have been detected in the results; however, as indicated by Figure 5-source data 1, IQCH was not listed.

Reviewer #2 (Public Review):

The authors analysed functional MRI recordings of brain activity at rest, using state-of-the-art methods that reveal the diverse ways in which information can be integrated in the brain. In this way, they found brain areas that act as (synergistic) gateways for the 'global workspace', where conscious access to information or cognition would occur, and brain areas that serve as (redundant) broadcasters from the global workspace to the rest of the brain. The results are compelling and are consistent with the already assumed role of several networks and areas within the Global Neuronal Workspace framework. Thus, in a way, this work comes to stress the role of synergy and redundancy as complementary information processing modes, which fulfill different roles in the bigger context of information integration.

In addition, to prove that the identified high-order interactions are relevant to the phenomenon of consciousness, the same analysis was performed in subjects under anesthesia or with disorders of consciousness (DOC), showing that indeed the loss of consciousness is associated with a deficient integration of information within the gateway regions.

Reviewer #1 (Public Review):

In this study, the authors obtained multiple, novel and compelling datasets to better understand the relationship between histone H1 and RNA-directed DNA methylation in plants. Most of the authors' claims concerning H1 and RNA polymerase V (Pol V) are backed by convincing and independent lines of evidence. However, Pol V produces noncoding transcripts that act as scaffold RNAs, which AGO4-bound siRNAs recognize in plant chromatin to mediate RNA-directed DNA methylation. Detection of Pol V transcript products at the sites of Pol V redistribution in h1 mutants would significantly enhance the impact of this manuscript. Below I have listed several strengths and a weakness of the manuscript.

Strengths:

- The authors report high-quality NRPE1 ChIP-seq data, allowing them to directly test how and where Pol V occupancy depends on histone H1 function in Arabidopsis.<br /> - nrpe1 mutants generated via CRISPR/Cas9 in the h1 mutant background (nrpe1 h1.1-1 h1.2-1 triple mutants), allow the authors to study the role of Pol V in ectopic DNA methylation in H1-deficient plants.<br /> - Pol V recruitment via ZincFinger-DMS3 expression (a modified version of Pol V's DMS3 recruitment factor) sends Pol V to new genomic loci and thus provides the authors with an innovative dataset for understanding H1 function at these sites.

Weakness:

- The manuscript does not include detection or quantification of Pol V transcripts generated at ectopic sites in the h1 mutant background. Pol V encroachment into heterochromatin in the h1 mutant is indirectly shown by NRPE1-dependent methylation at such ectopic sites.

Previous studies have charted the relationship between H1 function and RNA-directed DNA methylation (RdDM) via analyses of Pol IV-dependent 24 nt siRNAs and factors that recruit Pol IV (Choi et al., 2021 and Papareddy et al., 2020). Harris and colleagues have extended this work and shown that histone H1 function also antagonizes Pol V occupancy in the context of constitutive heterochromatin. The authors thus provide important evidence to show that H1 limits the encroachment of both polymerases Pol IV and Pol V into plant heterochromatin.