Reviewer #1 (Public Review):

Summary:<br /> I really enjoyed this manuscript from Torsekar et al on "Contrasting responses to aridity by different-sized decomposers cause similar decomposition rates across a precipitation gradient". The authors aimed to examine how climate interacts with decomposers of different size categories to influence litter decomposition. They proposed a new hypothesis: "The opposing climatic dependencies of macrofauna and that of microorganisms and mesofauna should lead to similar overall decomposition rates across precipitation gradients".

This study emphasizes the importance as well as the contribution of different groups of organisms (micro, meso, macro, and whole community) across different seasons (summer with the following characteristics: hot with no precipitation, and winter with the following characteristics: cooler and wetter winter) along a precipitation gradient. The authors made use of 1050 litter baskets with different mesh sizes to capture decomposers contribution. They proposed a new hypothesis that was aiming to understand the "dryland decomposition conundrum". They combined their decomposition experiment with the sampling of decomposers by using pittfall traps across both experiment seasons. This study was carried out in Israel and based on a single litter species that is native to all seven sites. The authors found that microorganism contribution dominated in winter while macrofauna decomposition dominated the overall decomposition in summer. These seasonality differences combined with the differences in different decomposers groups fluctuation along precipitation resulted in similar overall decomposition rates across sites.<br /> I believe this manuscript has a potential to advance our knowledge on litter decomposition.

Strengths:<br /> Well design study with combination of different approaches (methods) and consideration of seasonality to generalize pattern.<br /> The study expands to current understanding of litter decomposition and interaction between factors affecting the process (here climate and decomposers).

Weaknesses:<br /> The study was only based on a single litter species.

Reviewer #2 (Public Review):

Summary: Torsekar et al. use a leaf litter decomposition experiment across seasons, and in an aridity gradient, to provide a careful test of the role of different-sized soil invertebrates in shaping the rates of leaf litter decomposition. The authors found that large-sized invertebrates are more active in the summer and small-sized invertebrates in the winter. The summed effects of all invets then translated into similar levels of decomposition across seasons. The system breaks down in hyper-arid sites.

Strengths: This is a well-written manuscript that provides a complete statistical analysis of a nice dataset. The authors provide a complete discussion of their results in the current literature.

Weaknesses: I have only three minor comments. Please standardize the color across ALL figures (use the same color always for the same thing, and be friendly to color-blind people). Fig 1 may benefit from separating the orange line (micro and meso) into two lines that reflect your experimental setup and results. I would mention the dryland decomposition conundrum earlier in the Introduction. And the manuscript is full of minor grammatical errors. Some careful reading and fixing of all these minor mistakes here and there would be needed.

Reviewer #1 (Public Review):

Summary:<br /> In this article, the authors investigate whether the connectivity of the hippocampus is altered in individuals with aphantasia ¬- people who have reduced mental imagery abilities and where some describe having no imagery, and others describe having vague and dim imagery. The study investigated this question using a fMRI paradigm, where 14 people with aphantasia and 14 controls were tested, and the researchers were particularly interested in the key regions of the hippocampus and the visual-perceptual cortices. Participants were interviewed using the Autobiographical Interview regarding their autobiographical memories (AMs), and internal and external details were scored. In addition, participants were queried on their perceived difficulty in recalling memories, imagining, and spatial navigation, and their confidence regarding autobiographical memories was also measured. Results showed that participants with aphantasia reported significantly fewer internal details (but not external details) compared to controls; that they had lower confidence in their AMs; and that they reported finding remembering and imagining in general more difficult than controls. Results from the fMRI section showed that people with aphantasia displayed decreased hippocampal and increased visual-perceptual cortex activation during AM retrieval compared to controls. In contrast, controls showed strong negative functional connectivity between the hippocampus and the visual cortex. Moreover, resting state connectivity between the hippocampus and visual cortex predicted better visualisation skills. The authors conclude that their study provides evidence for the important role of visual imagery in detail-rich vivid AM, and that this function is supported by the connectivity between the hippocampus and visual cortex. This study extends previous findings of reduced episodic memory details in people with aphantasia, and enables us to start theorising about the neural underpinnings of this finding.

The data provided good support for the conclusion that the authors draw, namely that there is a 'tight link between visual imagery and our ability to retrieve vivid and detail-rich personal past events'. However, as the authors also point out, the exact nature of this relationship is difficult to infer from this study alone, as the slow temporal resolution of fMRI cannot establish the directionality between the hippocampus and the visual-perceptual cortex. This is an exciting future avenue to explore.

Weaknesses:<br /> A weakness of the study is that some of the questions used are a bit vague, and no objective measure is used, which could have been more informative. For example, the spatial navigation question (reported as 'How difficult is it typically for you to orient you spatially?' - a question which is ungrammatical, but potentially reflects a typo in the manuscript) could have been more nuanced to tap into whether participants relied mostly on cognitive maps (likely supported by the hippocampus) or landmarks. It would also have been interesting to conduct a spatial navigation task, as participants do not necessarily have insight into their spatial navigation abilities (they could have been overconfident or underconfident in their abilities). Secondly, the question 'how difficult is it typically for you to use your imagination?' could also be more nuanced, as imagination is used in a variety of ways, and we only have reason to hypothesise that people with aphantasia might have difficulties in some cases (i.e. sensory imagination involving perceptual details). It is unlikely that people with aphantasia would have more difficulty than controls in using their imagination to imagine counterfactual situations and engage in counterfactual thought (de Brigard et al., 2013, https://doi.org/10.1016%2Fj.neuropsychologia.2013.01.015) due to its non-sensory nature, but the question used does not distinguish between these types of imagination. Again, this is a ripe area for future research. The general phrasing of 'how difficult is [x]' could also potentially bias participants towards more negative answers, something which ought to be controlled for in future research.

Strengths:<br /> A great strength of this study is that it introduces a fMRI paradigm in addition to the autobiographical interview, paralleling work done on episodic memory in cognitive science (e.g. Addis and Schacter, 2007, https://doi.org/10.1016%2Fj.neuropsychologia.2006.10.016 ), which has examined episodic and semantic memory in relation to imagination (future simulation) in non-aphantasic participants as well as clinical populations. Future work could build on this study, and for example use the recombination paradigm (Addis et al. 2009, 10.1016/j.neuropsychologia.2008.10.026 ), which would shed further light on the ability of people with aphantasia to both remember and imagine events. Future work could also build on the interesting findings regarding spatial navigation, which together with previous findings in aphantasia (e.g. Bainbridge et al., 2021, https://doi.org/10.1016/j.cortex.2020.11.014 ) strongly suggests that spatial abilities in people with aphantasia are unaffected. This can shed further light on the different neural pathways of spatial and object memory in general. In general, this study opens up a multitude of new avenues to explore and is likely to have a great impact on the field of aphantasia research.

Reviewer #2 (Public Review):

Summary:<br /> This study investigates to what extent neural processing of autobiographical memory retrieval is altered in people who are unable to generate mental images ('aphantasia'). Self-report as well as objective measures were used to establish that the aphantasia group indeed had lower imagery vividness than the control group. The aphantasia group also reported fewer sensory and emotional details of autobiographical memories. In terms of brain activity, compared to controls, aphantasics had a reduction in activity in the hippocampus and an increase in activity in the visual cortex during autobiographical memory retrieval. For controls, these two regions were also functionally connected during autobiographical memory retrieval, which did not seem to be the case for aphantasics. Finally, resting-state connectivity between the visual cortex and hippocampus was positively related to autobiographical vividness in the control group but negatively in the aphantasia group. The results are in line with the idea that aphantasia is caused by an increase in noise within the visual system combined with a decrease in top-down communication from the hippocampus.

Recent years have seen a lot of interest in the influence of aphantasia on other cognitive functions and one of the most consistent findings is deficits in autobiographical memory. This is one of the first studies to investigate the neural correlates underlying this difference, thereby substantially increasing our understanding of aphantasia and the relationship between mental imagery and autobiographical memory.

Strengths:<br /> One of the major strengths of this study is the use of both self-report as well as objective measures to quantify imagery ability. Furthermore, the fMRI analyses are hypothesis-driven and reveal unambiguous results, with alterations in hippocampal and visual cortex processing seeming to underlie the deficits in autobiographical memory.

Weaknesses:<br /> In terms of weaknesses, the control task, doing mathematical sums, also differs from the autobiographical memory task in aspects that are unrelated to imagery or memory, such as self-relevance and emotional salience, which makes it hard to conclude that the differences in activity are reflecting only the cognitive processes under investigation.

Overall, I believe that this is a timely and important contribution to the field and will inspire novel avenues for further investigation.

Reviewer #1 (Public Review):

Summary:

This manuscript from Park et al examines the molecular, anatomical and functional properties of a subset of wide-field amacrine cell (WAC) types in mouse retina. More than 60 mouse amacrine cell types have been identified by single-cell transcriptomic studies (Yan et al., 2020, PMID: 32457074), but the functions of most of these are unknown and WACs are particularly understudied. The authors use intersectional genetics to target a subset of mouse WACs that co-express Bhlhe22 and the kappa opioid receptor (referred to as B/K WACs). They used electrophysiological and anatomical approaches to determine how WACs contribute to neural computations in the retina.

Strengths:

Overall, the paper presents a technically impressive set of experiments that build strong evidence for the presence of at least 3 discrete WAC types in the B/K transgenic line. These cells vary with respect to their morphology, dendritic stratification, response polarity (On vs Off) and resting membrane potentials. All types have long, monostratified dendrites and appear to lack axons. Electrophysiological recordings establish that these WACs are non-spiking, while calcium imaging revealed orientation selectivity in dendritic segments with tuning that correlates strongly with dendritic orientation. The authors go on to use optogenetics to show that WACs provide strong GABA-A receptor mediated inhibitory input to OFF and ON alpha sustained RGCs. This connectivity is further substantiated, at least for the OFF sustained alpha RGCs, by connectomic analyses from serial block face EM volumes. The use of the APEX2 reporter system to label the B/K cells in one of the EM volumes is particularly nice, making identification of the B/K WACs unambiguous. The conclusions are largely well supported by the experimental data. The study provides novel insights into the structure and function of specific WACs that will provide a foundation for further studies investigating the role of these amacrine cells in retinal circuits.

Weaknesses:

A limitation of the study is that the B/K WAC types described here could not be aligned to specific transcriptomic identities. The authors show more than 15 GABA expressing ACs express Bhlhe22 in the transcriptomic dataset, but it is unclear which of these also express the kappa opioid receptor (Opkr1).

The optogenetic evidence suggests that WACs provide GABA-A receptor mediated inhibitory input to both the sustained OFF and ON alpha RGCs. However, at least in the examples shown, there appears to be a dramatic difference in the timecourse of the rising phase of the inhibitory inputs to these two cell types, with the inputs to the ON sustained alpha RGCs appearing slower than those in the OFF sustained and OFF transient alpha RGCs. This apparent temporal difference was accompanied by a relatively lower sensitivity to light stimulation for the ON sustained cells. The slow timecourse seems unexpected for a direct GABA-A mediated synaptic connections between the WACs and ON alpha sustained cells. Moreover, since the connectomic analyses do not examine inputs to ON RGC types, the direct synaptic connection between B/K WACs and On alpha RGC is less well substantiated.

Reviewer #2 (Public Review):

Summary:

An important frontier in research on the mammalian retina is to understand the role of inhibitory amacrine cells in visual processing. These cell types have been found to play roles in tuning the output of the retina to specific visual features like motion and orientation. These cell types are understudied for two main reasons. First, there are many types of them-over 60 types in the mouse--, and second, they are quite unconventional as far as neurons go, as they have dendrites but often lack axons. The manuscript "Molecular identification of wide-field amacrine cells in mouse retina that encode stimulus orientation" by Park et al. provides a characterization of two (or possibly more) cell types within the amacrine cell class. Specifically, they characterize types of widefield amacrine cells (WACs), which they have gained genetic access to using an intersectional transgenic mouse strategy (Bhlhe22 x KOR). The authors used a broad range of experiments to characterize these WACs' anatomical properties, their stimulus tuning, and their wiring within the retina to their postsynaptic partners. These experiments include anatomy, electrophysiology, calcium imaging, and electron microscopy.

Strengths:

Overall, the manuscript presents strong evidence that the Bhlhe22 x KOR WACs represent multiple WAC types in the retina and that these cell types are orientation tuned. The most exciting finding is that their orientation tuning is correlated with the physical orientations of the dendrites, which suggests that this anatomical feature supports the tuning in these cells.

Weaknesses:

(1) The one common thought about widefield amacrine cells (WACs) is that these are spiking cells, which allows them to transmit signals along their long dendrites. The authors state that "none of the recorded cells fired conventional action potentials (spikes)." (p.7) This is a surprising finding, which leads to an interesting question: how do these cells integrate information from their presynaptic partners to generate the orientation tuning observed without the ability to conduct over long distances? However, the authors have not fully ruled out that the cells do spike.<br /> For instance, one possibility is that spiking requires a specific stimulus and the authors did not play that stimulus during their recordings. Most somatic recordings did not result in very large depolarizations, and the cell could still be below threshold. Depolarizing the cell to attempt to evoke spikes directly could be used to explore this possibility. A second possibility is that the dendrites spike, but these spikes are attenuated at the soma. Direct injections of current into the cells to evoke such spikes could be used to observe whether dendritic spiking occurs. A third possibility is that some important machinery for spiking is being washed out by the whole cell recordings. Cell attached recordings could be used to assess whether spiking occurs in an intact cell. The authors may wish to address these possibilities experimentally, but at least should qualify their statement about spiking in these cells and discuss these possibilities.

(2) It was unclear in this paper how many cell types are present in the intersectional cross. I think the paper would be stronger if they clarified that. For instance, in Fig. 1B: the authors show Bhlhe22 expression in amacrine cells from a previous study. They should also show the expression of the other gene they used in their intersectional strategy, the Kappa Opioid receptor (Oprk1), which is available in the same dataset. Another piece of analysis that could help would be clearer quantification of the anatomical features of the cells. For instance, the cells shown in Fig. 2 A2 vs. B2 have clear differences in number of dendrites and the relative angles of the dendrites. The On cells appear to have more dendrites evenly spread around the soma, while the Off cells appear to have more clumping along a line. Is this the case for all the cells recorded, or just these examples? The authors should present some population-level quantification.

(3) In Fig. 4E, the preferred orientation of calcium responses and physical orientation of the dendrites appears to clump around specific orientations. The Methods don't mention if the retinas were aligned to the body axis during the dissection. Is this clumping real, or is this an artifact of the analysis? If there are specific preferred orientations to these WAC cell types, that would be important to discuss in the paper - for instance how this relates to the preferred direction in the direction selectivity system or how it might relate to the function of these cells for behavior.

Reviewer #3 (Public Review):

Summary:

Amacrine cells are a heterogeneous collection of retinal interneurons. Most are inhibitory, and like inhibitory neurons in other neural circuits, strongly shape retinal function. With a few exceptions, the role of amacrine cells in retinal signaling is poorly understood. This paper introduces an approach to study a set of wide-field amacrine cells that extend processes over large regions of the retina.

Strengths:

A substantial strength of the paper is the combination of genetic manipulations, electrophysiology, optogenetics and electron microscopy used to study these cells. As a result of that broad set of techniques, the results cover many properties of how the cells work and provide a nice overview. The paper is also (with a few exceptions below) clearly presented and the experiments look to be carefully executed with clean results.

Weaknesses:

My largest concern with the paper is that overall the results provided an initial view of an interesting set of issues about the function of these cells, but the interesting initial results are not pursued in more depth.

Spatial spread of signals in neurites<br /> An immediate question about axonless WACs is the extent of spread of signals along their processes, and hence whether they act as a collection of independent or semi-independent elements. This bears directly on interpretation of the responses to oriented stimuli for example. Did you do any experiments that might provide additional information about this issue? For example, if you stimulate one of the WACs peripherally do you see a strong modulation of the somatic voltage? Or in the imaging experiments, if you mask a region of the processes so that it is not receiving a stimulus, do you see responses "leak" into that occluded region from surrounding stimulated regions?

Orientation tuning and connectivity<br /> The most developed functional results in the paper relate to the sensitivity of the WAC processes to oriented stimuli. Interpretation of these results depends on a few factors. First is the spread of signals in the WAC processes - as noted above. Second is connectivity. The paper shows that the B/K WAC activity increases inhibitory input to Off-delta and On-alpha ganglion cells. These cells, as noted in the paper, are not orientation tuned. But the orientation tuned ganglion cells stratify in a similar location within the IPL, and hence are situated in an appropriate place to receive input from the B/K WACs. Did you focus exclusively on connections to the Off-delta and On-alpha cells (along with the Off-alpha) or did you look at any other ganglion cell types? This should at least get discussed in more detail.

In several places it is unclear whether the paper intends to be a methods paper or a basic research paper. One example is the last sentence of the abstract. If it is intended to be a basic research paper (which is my overall impression) then I suggest shifting the emphasis in some of those key locations towards results and away from methods.

Reviewer #1 (Public Review):

Summary:<br /> The thalamus is a central subcortical structure that receives anatomical connections from various cortical areas, each displaying a unique pattern. Previous studies have suggested that certain cortical areas may establish more extensive connections within the thalamus, influencing distributed information flow. Despite these suggestions, a quantitative understanding of cortical areas' anatomical connectivity patterns within the thalamus is lacking. In this study, the researchers addressed this gap by employing diffusion magnetic resonance imaging (dMRI) on a large cohort of healthy adults from the Human Connectome Project. Using brain-wide probabilistic tractography, a framework was developed to measure the spatial extent of anatomical connections within the thalamus for each cortical area. Additionally, the researchers integrated resting-state functional MRI, cortical myelin, and human neural gene expression data to investigate potential variations in anatomical connections along the cortical hierarchy. The results unveiled two distinct cortico-thalamic tractography motifs: 1) a sensorimotor cortical motif featuring focused thalamic connections to the posterolateral thalamus, facilitating fast, feed-forward information flow; and 2) an associative cortical motif characterized by diffuse thalamic connections targeting the anteromedial thalamus, associated with slower, feed-back information flow. These motifs exhibited consistency across human subjects and were corroborated in macaques, underscoring cross-species generalizability. In summary, the study illuminates differences in the spatial extent of anatomical connections within the thalamus for sensorimotor and association cortical areas, potentially contributing to functionally distinct cortico-thalamic information flow.

Strengths:<br /> * Quantitative Approach: The study employs diffusion magnetic resonance imaging (dMRI) and probabilistic tractography on a substantial sample size of 828 healthy adults, providing a robust quantitative analysis of anatomical connectivity patterns within the thalamus.

* Multi-Modal Integration: By incorporating resting-state functional MRI, cortical myelin, and human neural gene expression data, the study offers a comprehensive approach to understanding anatomical connections, enriching the interpretation of findings and enhancing the study's overall validity.

* Cross-Species Generalizability: The identification of consistent cortico-thalamic tractography motifs in both human subjects and macaques demonstrates the robustness and cross-species generalizability of the findings, strengthening the significance and broader applicability of the study.

* Supplementary Analyses: There are numerous, excellent examples of clear surrogates used to test the major claims of the paper. This is exemplary work.

Weaknesses:<br /> * Indirect Estimates of White Matter Connections: While dMRI is a valuable tool, it inherently provides indirect and inferred information about neural pathways. The accuracy and specificity of tractography can be influenced by various factors, including fiber crossing, partial volume effects, and algorithmic assumptions. A potential limitation in the accuracy of indirect estimates might affect the precision of spatial extent measurements, introducing uncertainty in the interpretation of cortico-thalamic connectivity patterns. Addressing the methodological limitations associated with indirect estimates and considering complementary approaches could strengthen the overall robustness of the findings.

Reviewer #2 (Public Review):

Summary:<br /> This paper by Howell and colleagues focuses on describing macro patterns of anatomical connections between cortical areas and the thalamus in the human brain. This research topic poses significant challenges due to the inability to apply the gold standard of mapping anatomical connections, and viral tracing, to humans. Moreover, when applied to animal models, viral tracing often has limited scope and resolution. As a result, the field has thus far lacked a comprehensive and validated description of thalamocortical anatomical connectivity in humans.

The paper focuses on an intriguing question: whether anatomical connections from the cortex to the thalamus exhibit a diffuse pattern, targeting multiple thalamic sub-regions, or a more focal pattern, selectively targeting specific thalamic subregions. This novel and significant question holds substantial implications for our understanding of thalamocortical information processing. The authors have developed a sophisticated and innovative quantitative metric to address this question. The study revealed two main patterns: a focal pattern originating from sensorimotor cortical regions to the posterior thalamus and a more diffuse pattern from associative cortical regions to the anterior-medial thalamus. These findings are then framed within the context of thalamocortical motifs involved in feedforward versus feedback processing.

While this paper has several strengths, including its significance and methodological sophistication, its extension to non-human primates, and other forms of data for testing hierarchy, there are important limitations. These limitations, discussed in more detail below, primarily concern tracking accuracy and the known limitations of using diffusion data to track thalamocortical connections in humans. These limitations may potentially introduce systematic biases into the results, weakening their support. Addressing these limitations through better validation is crucial, though some may remain unresolved due to the fundamental constraints of diffusion imaging.

Strengths:<br /> This research holds significant basic, clinical, and translational importance as it contributes to our understanding of how thalamocortical anatomical connectivity is organized. Its relevance spans cognitive, systems, and clinical neuroscientists in various subfields.

The central question addressed in this study, concerning whether cortico-thalamic projections are focal or diffuse, is both novel and previously unexplored to the best of my knowledge. It offers valuable insights into the potential capabilities of the thalamocortical system in terms of parallel or integrative processing.

The development of quantitative metrics to analyze anatomical connectivity is highly innovative and suitable for addressing the research questions at hand.

The findings are not only interesting but also robust, aligning with data from other sources that suggest a hierarchical organization in the brain.

Using PCA to integrate results across a range of thresholds is innovative.

The study's sophisticated integration of a diverse range of data and methods provides strong, converging support for its main findings, enhancing the overall credibility of the research.

Weaknesses:<br /> Structural thalamocortical connectivity was estimated from diffusion imaging data obtained from the HCP dataset. Consequently, the robustness and accuracy of the results depend on the suitability of this data for such a purpose. Conducting tractography on the cortical-thalamic system is recognized as a challenging endeavor for several reasons. First, diffusion directions lose their clearly defined principal orientations once they reach the deep thalamic nuclei, rendering the tracking of structures on the medial side, such as the medial dorsal (MD) and pulvinar nuclei difficult. Somewhat concerning is those are regions that authors found to show diffuse connectivity patterns. Second, the thalamic radiata diverge into several directions, and routes to the lateral surface often lack the clarity necessary for successful tracking. It is unclear if all cortical regions have similar levels of accuracy, and some of the lateral associative regions might have less accurate tracking, making them appear to be more diffuse, biasing the results.

While the methodology employed by the authors appears to be state-of-the-art, there exists uncertainty regarding its appropriateness for validation, given the well-documented issues of false positives and false negatives in probabilistic diffusion tractography, as discussed by Thomas et al. 2014 PNAS. Although replicating the results in both humans and non-human primates strengthens the study, a more compelling validation approach would involve demonstrating the method's ability to accurately trace known tracts from established tracing studies or, even better, employing phantom track data. Many of the control analyses the authors presented, such as track density, do not speak to accuracy.

Because the authors included data from all thresholds, it seems likely that false positive tracks were included in the results. The methodology described seems to unavoidably include anatomically implausible pathways in the spatial extent analyses.

If tracking the medial thalamus is indeed less accurate, characterized by higher false positives and false negatives, it could potentially lead to increased variability among individual subjects. In cases where results are averaged across subjects, as the authors have apparently done, this could inadvertently contribute to the emergence of the "diffuse" motif, as described in the context of the associative cortex. This presents a critical issue that requires a more thorough control analysis and validation process to ensure that the main results are not artifacts resulting from limitations in tractography.

The thresholding approach taken in the manuscript aimed to control for inter-areal differences in anatomical connection strength that could confound the ED estimates. Here I am not quite clear why inter-areal differences in anatomical connection strength have to be controlled. A global threshold applied on all thalamic voxels might kill some connections that are weak but do exist. Those weak pathways are less likely to survive at high thresholds. In the meantime, the mean ED is weighted, with more conservative thresholds having higher weights. That being said, isn't it possible that more robust pathways might contribute more to the mean ED than weaker pathways?

Reviewer #3 (Public Review):

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

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

Weaknesses:<br /> Potential weaknesses of the study are that it seems to largely integrate aspects of the thalamus that have been already described before. The differentiation between sensory and association systems across thalamic subregions is something that has been described before (see: Oldham and Ball, 2023; Zheng et al., 2023; Yang et al., 2020 Mueller, 2020; Behrens, 2003).

Appraisal:<br /> However, the aim of the study: 'to investigate the spatial extent of anatomical connectivity patterns within the thalamus in both humans and non-human primates and determine if such patterns differ between sensorimotor and association cortical areas' has been met.

Discussion:<br /> Overall, I think the study is a nice addition to the growing literature studying the anatomical connectivity between the thalamus and cortex and its functional implications.

Reviewer #1 (Public Review):

Summary:<br /> Wilmes and colleagues present a computational model of a cortical circuit for predictive processing which tackles the issue of how to learn predictions when different levels of uncertainty are present for the predicted sensory stimulus. When a predicted sensory outcome is highly variable, deviations from the average expected stimulus should evoke prediction errors that have less impact on updating the prediction of the mean stimulus. In the presented model, layer 2/3 pyramidal neurons represent either positive or negative prediction errors, SST neurons mediate the subtractive comparison between prediction and sensory input, and PV neurons represent the expected variance of sensory outcomes. PVs therefore can control the learning rate by divisively inhibiting prediction error neurons such that they are activated less, and exert less influence on updating predictions, under conditions of high uncertainty.

Strengths:<br /> The presented model is a very nice solution to altering the learning rate in a modality and context-specific way according to expected uncertainty and, importantly, the model makes clear, experimentally testable predictions for interneuron and pyramidal neuron activity. This is therefore an important piece of modelling work for those working on cortical and/or predictive processing and learning. The model is largely well-grounded in what we know of the cortical circuit.

Weaknesses:<br /> Currently, the model has not been challenged with experimental data, presumably because data from an adequate paradigm is not yet available. I therefore only have minor comments regarding the biological plausibility of the model:

Beyond the fact that some papers show SSTs mediate subtractive inhibition and PVs mediate divisive inhibition, the selection of interneuron types for the different roles could be argued further, given existing knowledge of their properties. For instance, is a high PV baseline firing rate, or broad sensory tuning that is often interpreted as a 'pooling' of pyramidal inputs, compatible with or predicted by the model?

On a related note, SSTs are thought to primarily target the apical dendrite, while PVs mediate perisomatic inhibition, so the different roles of the interneurons in the model make sense, particularly for negative PE neurons, where a top-down excitatory predicted mean is first subtractively compared with the sensory input, s, prior to division by the variance. However, sensory input is typically thought of as arising 'bottom-up', via layer 4, so the model may match the circuit anatomy less in the case of positive PE neurons, where the diagram shows 's' arising in a top-down manner. Do the authors have a justification for this choice?

In cortical circuits, assuming a 2:8 ratio of inhibitory to excitatory neurons, there are at least 10 pyramidal neurons to each SST and PV neuron. Pyramidal neurons are also typically much more selective about the type of sensory stimuli they respond to compared to these interneuron classes (e.g., Kerlin et al., 2012, Neuron). A nice feature of the proposed model is that the same interneurons can provide predictions of the mean and variance of the stimulus in a predictor-dependent manner. However, in a scenario where you have two types of sensory stimulus to predict (e.g., two different whiskers stimulated), with pyramidal neurons selective for prediction errors in one or the other, what does the model predict? Would you need specific SST and PV circuits for each type of predicted stimulus?

Reviewer #2 (Public Review):

Summary:<br /> This computational modeling study addresses the observation that variable observations are interpreted differently depending on how much uncertainty an agent expects from its environment. That is, the same mismatch between a stimulus and an expected stimulus would be less significant, and specifically would represent a smaller prediction error, in an environment with a high degree of variability than in one where observations have historically been similar to each other. The authors show that if two different classes of inhibitory interneurons, the PV and SST cells, (1) encode different aspects of a stimulus distribution and (2) act in different (divisive vs. subtractive) ways, and if (3) synaptic weights evolve in a way that causes the impact of certain inputs to balance the firing rates of the targets of those inputs, then pyramidal neurons in layer 2/3 of canonical cortical circuits can indeed encode uncertainty-modulated prediction errors. To achieve this result, SST neurons learn to represent the mean of a stimulus distribution and PV neurons its variance.

The impact of uncertainty on prediction errors is an understudied topic, and this study provides an intriguing and elegant new framework for how this impact could be achieved and what effects it could produce. The ideas here differ from past proposals about how neuronal firing represents uncertainty. The developed theory is accompanied by several predictions for future experimental testing, including the existence of different forms of coding by different subclasses of PV interneurons, which target different sets of SST interneurons (as well as pyramidal cells). The authors are able to point to some experimental observations that are at least consistent with their computational results. The simulations shown demonstrate that if we accept its assumptions, then the authors' theory works very well: SSTs learn to represent the mean of a stimulus distribution, PVs learn to estimate its variance, firing rates of other model neurons scale as they should, and the level of uncertainty automatically tunes the learning rate, so that variable observations are less impactful in a high uncertainty setting.

Strengths:<br /> The ideas in this work are novel and elegant, and they are instantiated in a progression of simulations that demonstrate the behavior of the circuit. The framework used by the authors is biologically plausible and matches some known biological data. The results attained, as well as the assumptions that go into the theory, provide several predictions for future experimental testing.

Weaknesses:<br /> Overall, I found this manuscript to be frustrating to read and to try to understand in detail, especially the Results section from the UPE/Figure 4 part to the end and parts of the Methods section. I don't think the main ideas are so complicated, and it should be possible to provide a much clearer presentation.

For me, one source of confusion is the comparison across Figure 1EF, Figure 2A, Figure 3A, Figure 4AB, and Figure 5A. All of these are meant to be schematics of the same circuit (although with an extra neuron in Figure 5), yet other than Figures 1EF and 4AB, no two are the same! There should be a clear, consistent schematic used, with identical labeling of input sources, neuron types, etc. across all of these panels.

The flow of the Results section overall is clear until the ``Calculation of the UPE in Layer 2/3 error neurons' and Figure 4, where I find that things become significantly more confusing. The mention of NMDA and calcium spikes comes out of the blue, and it's not clear to me how this fits into the authors' theory. Moreover: Why would this property of pyramidal cells cause the PV firing rate to increase as stated? The authors refer to one set of weights (from SSTs to UPE) needing to match two targets (weights from s to UPE and weights from mean representation to UPE); how can one set of weights match two targets? Why do the authors mention ``out-of-distribution detection' here when that property is not explored later in the paper? (see also below for other comments on Figure 4)

Coming back to one of the points in the previous paragraph: How realistic is this exact matching of weights, as well as the weight matching that the theory requires in terms of the weights from the SSTs to the PVs and the weights from the stimuli to the PVs? This point should receive significant elaboration in the discussion, with biological evidence provided. I would not advocate for the authors' uncertainty prediction theory, despite its elegant aspects, without some evidence that this weight matching occurs in the brain. Also, the authors point out on page 3 that unlike their theory, "...SSTs can also have divisive effects, and PVs can have subtractive effects, dependent on circuit and postsynaptic properties". This should be revisited in the Discussion, and the authors should explain why these effects are not problematic for their theory. In a similar vein, this work assumes the existence of two different populations of SST neurons with distinct UPE (pyramidal) targets. The Discussion doesn't say much about any evidence for this assumption, which should be more thoroughly discussed and justified.

Finally, I think this is a paper that would have been clearer if the equations had been interspersed within the results. Within the given format, I think the authors should include many more references to the Methods section, with specific equation numbers, where they are relevant throughout the Results section. The lack of clarity is certainly made worse by the current state of the Methods section, where there is far too much repetition and poor ordering of material throughout.

Reviewer #3 (Public Review):

Summary:<br /> The authors proposed a normative principle for how the brain's internal estimate of an observed sensory variable should be updated during each individual observation. In particular, they propose that the update size should be inversely proportional to the variance of the variable. They then proposed a microcircuit model of how such an update can be implemented, in particularly incorporating two types of interneurons and their subtractive and divisive inhibition onto pyramidal neurons. One type should represent the estimated mean while another represents the estimated variance. The authors used simulations to show that the model works as expected.

Strengths:<br /> The paper addresses two important issues: how uncertainty is represented and used in the brain, and the role of inhibitory neurons in neural computation. The proposed circuit and learning rules are simple enough to be plausible. They also work well for the designated purposes. The paper is also well-written and easy to follow.

Weaknesses:<br /> I have concerns with two aspects of this work.

(1) The optimality analysis leading to Eq (1) appears simplistic. The learning setting the authors describe (estimating the mean of a stationary Gaussian variable from a stream of observations) is a very basic problem in online learning/streaming algorithm literature. In this setting, the real "optimal" estimate is simply the arithmetic average of all samples seen so far. This can be implemented in an online manner with \hat{\mu}_{t} = \hat{\mu}_{t-1} +(s_t-\hat{\mu}_{t-1})/t. This is optimal in the sense that the estimator is always the maximum likelihood estimator given the samples seen up to time t. On the other hand, doing gradient descent only converges towards the MLE estimator after a large number of updates. Another critique is that while Eq (1) assumes an estimator of the mean (\hat{mu}), it assumes that the variance is already known. However, in the actual model, the variance also needs to be estimated, and a more sophisticated analysis thus needs to take into account the uncertainty of the variance estimate and so on. Finally, the idea that the update should be inverse to the variance is connected to the well-established idea in neuroscience that more evidence should be integrated over when uncertainty is high. For example, in models of two-alternative forced choices it is known to be optimal to have a longer reaction time when the evidence is noisier.

(2) While the incorporation of different inhibitory cell types into the model is appreciated, it appears to me that the computation performed by the circuit is not novel. Essentially the model implements a running average of the mean and a running average of the variance, and gates updates to the mean with the inverse variance estimate. I am not sure about how much new insight the proposed model adds to our understanding of cortical microcircuits.

Reviewer #1 (Public Review):

Summary:

Heer and Sheffield used 2 photon imaging to dissect the functional contributions of convergent dopamine and noradrenaline inputs to the dorsal hippocampus CA1 in head-restrained mice running down a virtual linear path. Mice were trained to collect water rewards at the end of the track and on test days, calcium activity was recorded from dopamine (DA) axons originating in the ventral tegmental area (VTA, n=7) and noradrenaline axons from the locus coeruleus (LC, n=87) under several conditions. When mice ran laps in a familiar environment, VTA DA axons exhibited ramping activity along the track that correlated with distance to reward and velocity to some extent, while LC input activity remained constant across the track, but correlated invariantly with velocity and time to motion onset. A subset of recordings taken when the reward was removed showed diminished ramping activity in VTA DA axons, but no changes in the LC axons, confirming that DA axon activity is locked to reward availability. When mice were subsequently introduced to a new environment, the ramping to reward activity in the DA axons disappeared, while LC axons showed a dramatic increase in activity lasting 90 s (6 laps) following the environment switch. In the final analysis, the authors sought to disentangle LC axon activity induced by novelty vs. behavioral changes induced by novelty by removing periods in which animals were immobile and established that the activity observed in the first 2 laps reflected novelty-induced signal in LC axons.

Strengths:

The results presented in this manuscript provide insights into the specific contributions of catecholaminergic input to the dorsal hippocampus CA1 during spatial navigation in a rewarded virtual environment, offering a detailed analysis of the resolution of single axons. The data analysis is thorough and possible confounding variables and data interpretation are carefully considered.

Weaknesses:

Aspects of the methodology, data analysis, and interpretation diminish the overall significance of the findings, as detailed below.

The LC axonal recordings are well-powered, but the DA axonal recordings are severely underpowered, with recordings taken from a mere 7 axons (compared to 87 LC axons). Additionally, 2 different calcium indicators with differential kinetics and sensitivity to calcium changes (GCaMP6S and GCaMP7b) were used (n=3, n=4 respectively) and the data pooled. This makes it very challenging to draw any valid conclusions from the data, particularly in the novelty experiment. The surprising lack of novelty-induced DA axon activity may be a false negative. Indeed, at least 1 axon (axon 2) appears to be showing a novelty-induced rise in activity in Figure 3C. Changes in activity in 4/7 axons are also referred to as a 'majority' occurrence in the manuscript, which again is not an accurate representation of the observed data.

The authors conducted analysis on recording data exclusively from periods of running in the novelty experiment to isolate the effects of novelty from novelty-induced changes in behavior. However, if the goal is to distinguish between changes in locus coeruleus (LC) axon activity induced by novelty and those induced by motion, analyzing LC axon activity during periods of immobility would enhance the robustness of the results.

The authors attribute the ramping activity of the DA axons to the encoding of the animals' position relative to reward. However, given the extensive data implicating the dorsal CA1 in timing, and the remarkable periodicity of the behavior, the fact that DA axons could be signalling temporal information should be considered.

The authors should explain and justify the use of a longer linear track (3m, as opposed to 2m in the DAT-cre mice) in the LC axon recording experiments.

Reviewer #2 (Public Review):

Summary:

The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

The main findings were as follows:

- In a familiar environment, the activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.<br /> - VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.<br /> - In contrast, the activity of LC axons ramped up before the initiation of movement on the Styrofoam wheel.<br /> - In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral, and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected the approach of a learned reward, and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

Strengths:

(1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that the activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

(2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

Weaknesses:

(1) The findings are correlational and do not allow strong conclusions on how VTA or LC inputs to dorsal CA1 affect cognition and behavior. However, as indicated above under Strengths, the findings will aid the interpretation of previous findings and help to generate new hypotheses as to how VTA or LC inputs to dorsal CA1 affect distinct cognitive and behavioral functions.

(2) Some aspects of the methodology would benefit from clarification.<br /> First, to help others to better scrutinize, evaluate, and potentially to reproduce the research, the authors may wish to check if their reporting follows the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the full and transparent reporting of research involving animals (https://arriveguidelines.org/). For example, I think it would be important to include a sample size justification (e.g., based on previous studies, considerations of statistical power, practical considerations, or a combination of these factors). The authors should also include the provenance of the mice. Moreover, although I am not an expert in 2-photon imaging, I think it would be useful to provide a clearer description of exclusion criteria for imaging data.<br /> Second, why were different linear tracks used for studies of VTA and LC axon activity (from line 362)? Could this potentially contribute to the partly distinct activity correlates that were found for VTA and LC axons?<br /> Third, the authors seem to have used two different criteria for defining immobility. Immobility was defined as moving at <5 cm/s for the behavioral analysis in Figure 3a, but as <0.2 cm/s for the imaging data analysis in Figure 4 (see legends to these figures and also see Methods, from line 447, line 469, line 498)? I do not understand why, and it would be good if the authors explained this.

(3) In the Results section (from line 182) the authors convincingly addressed the possibility that less time spent immobile in the novel environment may have contributed to the novelty-induced increase of LC axon activity in dorsal CA1 (Figure 4). In addition, initially (for the first 2-4 laps), the mice also ran more slowly in the novel environment (Figure 3aIII, top panel). Given that LC and VTA axon activity were both increasing with velocity (Figure 1F), reduced velocity in the novel environment may have reduced LC and VTA axon activity, but this possibility was not addressed. Reduced LC axon activity in the novel environment could have blunted the novelty-induced increase. More importantly, any potential novelty-induced increase in VTA axon activity could have been masked by decreases in VTA axon activity due to reduced velocity. The latter may help to explain the discrepancy between the present study and previous findings that VTA neuron firing was increased by novelty (see Discussion, from line 243). It may be useful for the authors to address these possibilities based on their data in the Results section, or to consider them in their Discussion.

(4) Sensory properties of the water reward, which the mice may be able to detect, could account for reward-related activity of VTA axons (instead of an expectation of reward). Do the authors have evidence that this is not the case? Occasional probe trials, intermixed with rewarded trials, could be used to test for this possibility.

Reviewer #3 (Public Review):

Summary:

Heer and Sheffield provide a well-written manuscript that clearly articulates the theoretical motivation to investigate specific catecholaminergic projections to dorsal CA1 of the hippocampus during a reward-based behavior. Using 2-photon calcium imaging in two groups of cre transgenic mice, the authors examine the activity of VTA-CA1 dopamine and LC-CA1 noradrenergic axons during reward seeking in a linear track virtual reality (VR) task. The authors provide a descriptive account of VTA and LC activities during walking, approach to reward, and environment change. Their results demonstrate LC-CA1 axons are activated by walking onset, modulated by walking velocity, and heighten their activity during environment change. In contrast, VTA-CA1 axons were most activated during the approach to reward locations. Together the authors provide a functional dissociation between these catecholamine projections to CA1. A major strength of their approach is the methodological rigor of 2-photon recording, data processing, and analysis approaches. These important systems neuroscience studies provide solid evidence that will contribute to the broader field of learning and memory. The conclusions of this manuscript are mostly well supported by the data, but some additional analysis and/or experiments may be required to fully support the author's conclusions.

Weaknesses:

(1) During teleportation between familiar to novel environments the authors report a decrease in the freezing ratio when combining the mice in the two experimental groups (Figure 3aiii). A major conclusion from the manuscript is the difference in VTA and LC activity following environment change, given VTA and LC activity were recorded in separate groups of mice, did the authors observe a similar significant reduction in freezing ratio when analyzing the behavior in LC and VTA groups separately?

(2) The authors satisfactorily apply control analyses to account for the unequal axon numbers recorded in the LC and VTA groups (e.g. Figure 1). However, given the heterogeneity of responses observed in Figures 3c, 4b and the relatively low number of VTA axons recorded (compared to LC), there are some possible limitations to the author's conclusions. A conclusion that LC-CA1 axons, as a general principle, heighten their activity during novel environment presentation, would require this activity profile to be observed in some of the axons recorded in most all LC-CA1 mice. Additionally, if the general conclusion is that VTA-CA1 axons ramp activity during the approach to reward, it would be expected that this activity profile was recorded in the axons of most all VTA-CA1 mice. Can the authors include an analysis to demonstrate that each LC-CA1 mouse contained axons that were activated during novel environments and that each VTA-CA1 mouse contained axons that ramped during the approach to reward?

(3) A primary claim is that LC axons projecting to CA1 become activated during novel VR environment presentation. However, the experimental design did not control for the presentation of a familiar environment. As I understand, the presentation order of environments was always familiar, then novel. For this reason, it is unknown whether LC axons are responding to novel environments or environmental change. Did the authors re-present the familiar environment after the novel environment while recording LC-CA1 activity?

Reviewer #1 (Public Review):

This is a short but important study. Basically, the authors show that α-synuclein overexpression's negative impact on synaptic vesicle recycling is mediated by its interaction with E-domain containing synapsins. This finding is highly relevant for synuclein function as well as for the pathophysiology of synucleinopathies. The data is clear, functional analysis is highly adequate.

Reviewer #1 (Public Review):

Neurons are not static-their activity patterns change as the result of learning, aging, and disease. Reliable tracking of activity from individual neurons across long time periods would enable studies of these important dynamics. For this reason, the authors' efforts to track electrophysiological activity across days without relying on matching neural receptive fields (which can change due to learning, aging, and disease) is very important.

By utilizing the tightly-spaced electrodes on Neuropixels probes, they are able to measure the physical distance and the waveform shape 'distance' between sorted units recorded on different days. To tune the matching algorithm and to validate the results, they used the visual receptive fields of neurons in the mouse visual cortex (which tend to change little over time) as ground truth. Their approach performs quite well, with a high proportion of neurons accurately matched across multiple weeks.

This suggests that the method may be useable in other cases where the receptive fields can't be used as ground truth to validate the tracking. This potential extendibility to tougher applications is where this approach holds the most promise. However, the study only looks at one brain area (visual cortex), in one species (mouse), using one type of spike sorter (Kilosort), and one type of behavioral prep (head-fixed). While the authors suggest methods to generalize their technique to other experimental conditions, no validation of those generalizations was done using data from different experimental conditions. Anyone using this method under different conditions would therefore need to perform such validation themselves.

Reviewer #2 (Public Review):

The manuscript presents a method for tracking neurons recorded with neuropixels across days, based on the matching of cells' spatial layouts and spike waveforms at the population level. The method is tested on neuropixel recordings of the visual cortex carried over 47 days, with the similarity in visual receptive fields used to verify the matches in cell identity.

This is an important tool as electrophysiological recordings have been notoriously limited in terms of tracking individual neuron's fate over time, unlike imaging approaches. The method is generally sound and properly tested but I think some clarifications would be helpful regarding the implementation of the method and some of the results.

(1) Page 6: I am not sure I understand the point of the imposed drift and how the value of 12µm is chosen.<br /> Is it that various values of imposed drift are tried, the EMDs computed to produce histograms as in Fig2c, values of rigid drifts estimated based on the histogram modes, and then the value associated with minimum cost selected? The corresponding manuscript section would need some clarification regarding this aspect.

(2) The EMD is based on the linear sum, with identical weight, of cell distance and waveform similarity measures. How performance is affected from using a different weighting of the 2 measures (for instance, using only cell distance and no waveform similarity)? It is common that spike waveforms associated to a given neuron appear different on different channels of silicon probes (i.e. the spike waveform changes depending the position of recording sites relative to the neuron), so I wonder if that feature is helping or potentially impeding the tracking.

(3) Fig.5: I assume the dots are representing time gaps for which cell tracking is estimated. The 3 different groups of colors correspond to the 3 mice used. For a given mouse, I would expect to always see 3 dots (for ref, putative and mixed) for a given tracking gap. However, for mouse AL036 for instance, at tracking duration of 8 days, a dot is visible for mixed but not for ref and putative. How come this is happening?

(4) Matched visual responses are measured by the sum of correlation of visual fingerprints, which are vectors of cells' average firing rate across visual stimuli, and correlation of PSTHs, which are implemented over all visual stimuli combined. I believe that some information is lost from combining all stimuli in the implementation of PSTHs (assuming that PSTHs show specificity to individual visual stimuli). The authors might consider, as alternative measure of matched visual responses, a correlation of the vector concatenations of all stimulus PSTHs. Such simpler measure would contain both visual fingerprint and PSTH information, and would not lose the information of PSTH specificity across visual stimuli.

2nd revision

(1) From reading the authors' response, I could understand several of the points I had previously missed. I still think that some part of the results are not straightforward to understand, the way it is written. Adding a few introductory sentences to the paragraphs (for instance the one related to my previous point #1) would really help the reader comprehend this important work.

(2) Following on my point #2, the w value used is 1500 and the recovery rate doesn't seems to reach a peak but rather a plateau for larger w values. From such large w value and the absence of a downward trend for increasing values, it would seem that only the 'waveform distance' matter and that the 'location distance' doesn't contribute much to the EMD distance. Is this correct?

Reviewer #1 (Public Review):

Summary: Nuclear depletion and cytoplasmic mislocalization/aggregation of the DNA and RNA binding protein TDP-43 are pathological hallmarks of multiple neurodegenerative diseases. Prior work has demonstrated that depletion of TDP-43 from the nucleus leads to alterations in transcription and splicing. Conversely, cytoplasmic mislocalization/aggregation can contribute to toxicity by impairing mRNA transport and translation as well as miRNA dysregulation. However, to date, models of TDP-43 proteinopathy rely on artificial knockdown- or overexpression-based systems to evaluate either nuclear loss or cytoplasmic gain of function events independently. Few model systems authentically reproduce both nuclear depletion and cytoplasmic miscloalization/aggreagtion events. In this manuscript, the authors generate novel iPSC-based reagents to manipulate the localization of endogenous TDP-43. This is a valuable resource for the field to study pathological consequences of TDP-43 proteinopathy in a more endogenous and authentic setting. However, in the current manuscript, there are a number of weaknesses that should be addressed to further validate the ability of this model to replicate human disease pathology and demonstrate utility for future studies.

Strengths: The primary strength of this paper is the development of a novel in vitro tool.

Weaknesses: There are a number of weaknesses detailed below that should be addressed to thoroughly validate these new reagents as more authentic models of TDP-43 proteinopathy and demonstrate their utility for future investigations.

(1) The authors should include images of their engineered TDP-43-GFP iPSC line to demonstrate TDP-43 localization without the addition of any nanobodies (perhaps immediately prior to addition of nanobodies). Additionally, it is unclear whether simply adding a GFP tag to endogenous TDP-43 impact its normal function (nuclear-cytoplasmic shuttling, regulation of transcription and splicing, mRNA transport etc).

(2) Can the authors explain why there is a significant discrepancy in time points selected for nanobody transduction and immunostaining or cell lysis throughout Figure 1 and 2? This makes interpretation and overall assessment of the model challenging.

(3) The authors should further characterize their TDP-43 puncta. TDP-43 immunostaining is typically punctate so it is unclear if the puncta observed are physiologic or pathologic based on the analyses carried out in the current version of this manuscript. Additionally, do these puncta co-localize with stress granule markers or RNA transport granule markers? Are these puncta phosphorylated (which may be more reminiscent of end-stage pathologic observations in humans)?

(4) The authors should include multiple time points in their evaluation of TDP-43 loss of function events and aggregation. Does loss of function get worse over time? Is there a time course by which RNA misprocessing events emerge or does everything happen all at once? Does aggregation get worse over time? Do these neurons die at any point as a result of TDP-43 proteinopathy?

(5) Can the authors please comment on whether or not their model is "tunable"? In real human disease, not every neuron displays complete nuclear depletion of TDP-43. Instead there is often a gradient of neurons with differing magnitudes of nuclear TDP-43 loss. Additionally, very few neurons (5-10%) harbor cytoplasmic TDP-43 aggregates at end-stage disease. These are all important considerations when developing a novel authentic and endogenous model of TDP-43 proteinopathy which the current manuscript fails to address.

Reviewer #2 (Public Review):

Summary:<br /> TDP-43 mislocalization occurs in nearly all of ALS, roughly half of FTD, and as a co-pathology in roughly half of AD cases. Both gain-of-function and loss-of-function mechanisms associated with this mislocalization likely contribute to disease pathogeneisis.

Here, the authors describe a new method to induce TDP-43 mislocalization in cellular models. They endogenously-tagged TDP-43 with a C-terminal GFP tag in human iPSCs. They then expressed an intrabody - fused with a nuclear export signal (NES) - that targeted GFP to the cytosol. Expression of this intrabody-NES in human iPSC-derived neurons induced nuclear depletion of homozygous TDP-43-GFP, caused its mislocalization to the cytosol, and at least in some cells appeared to cause cytosolic aggregates. This mislocalization was accompanied by induction of cryptic exons in well characterized transcripts known to be regulated by TDP-43, a hallmark of functional TDP-43 loss and consistent with pathological nuclear TDP-43 depletion. Interestingly, in heterozygous TDP-43-GFP neurons, expression of intrabody-NES appeared to also induce the mislocalization of untagged TDP-43 in roughly half of the neurons, suggesting that this system can also be used to study effects on untagged endogenous TDP-43 as well as TDP-43-GFP fusion protein.

Strengths:<br /> A clearer understanding of how TDP-43 mislocalization alters cellular function, as well as pathways that mitigate clearance of TDP-43 aggregates, is critical. But modeling TDP-43 mislocalization in disease-relevant cellular systems has proven to be challenging. High levels of overexpression of TDP-43 lacking an NES can drive endogenous TDP-43 mislocalization, but such overexpression has direct and artificial consequences on certain cellular features (e.g. altered exon skipping) not seen in diseased patients. Toxic small molecules such as MG132 and arsenite can induce TDP-43 mislocalization, but co-induce myriad additional cellular dysfunctions unrelated to TDP-43 or ALS. TDP-43 binding oligonucleotides can cause cytosolic mislocalization as well. Each system has pros and cons, and additional ways to induce TDP-43 mislocalization would be useful for the field. The method described in this manuscript could provide researchers with a powerful way to study the combined biology of cytosolic TDP-43 mislocalization and nuclear TDP-43 depletion, with additional temporal control that is lacking in current method. Indeed, the authors see some evidence of differences in RNA splicing caused by pure TDP-43 depletion versus their induced mislocalization model. Finally, their method may be especially useful in determining how TDP-43 aggregates are cleared by cells, potentially revealing new biological pathways that could be therapeutically targeted.

Weaknesses:<br /> The method and supporting data have limitations in its current form, outlined below, and in its current form the findings are rather preliminary.

• Tagging of TDP-43 with a bulky GFP tag may alter its normal physiological functions, for example phase separation properties and functions within complex ribonucleoprotein complexes. In addition, alternative isoforms of TDP-43 (e.g. "short" TDP-43, would not be GFP tagged and therefore these species would not be directly manipulatable or visualizable with the tools currently employed in the manuscript.<br /> • The data regarding potential mislocalization of endogenous TDP-43 in the heterozygous TDP-43-GFP lines is especially intriguing and important, yet very little characterization was done. Does untagged TDP-43 co-aggregate with the tagged TDP-43? Is localization of TDP-43 immunostaining the same as the GFP signal in these cells?<br /> • The experiments in which dox was used to induce the nanobody-NES, then dox withdrawn to study potential longer-lasting or self-perpetuating inductions of aggregation is potentially interesting. However, the nanobody was only measured at the RNA level. We know that protein half lives can be very long in neurons, and therefore residual nanobody could be present at these delayed time points. The key measurement to make would be at the protein level of the nanobody if any conclusions are be made from this experiment.<br /> • Potential differences in splicing and microRNAs between TDP-43 knockdown and TDP-43 mislocalization are potentially interesting. However, different patterns of dysregulated RNA splicing can occur at different levels of TDP-knockdown, thus it is difficult to asses whether the changes observed in this paper are due to mislocalization per se, or rather just reflect differences in nuclear TDP-43 abundance.

Reviewer #1 (Public Review):

Summary:<br /> Functionally important alternative isoforms are gold nuggets found in a swamp of errors produced by the splicing machinery.

The architecture of eukaryotic genomes, when compared with prokaryotes, is characterised by a preponderance of introns. These elements, which are still present within transcripts, are rapidly removed during the splicing of messenger RNA (mRNA), thus not contributing to the final protein. The extreme rarity of introns in prokaryotes, and the elimination of these introns from mRNAs before translation into protein, raises questions about the function of introns in genomes. One explanation comes from functional biology: introns are thought to be involved in post-transcriptional regulation and in the production of translational variants. The latter function is possible when the positions of the edges of the spliced intron vary. While some light has been shed on specific examples of the functional role of alternative splicing, to what extent are they representative of all introns in metazoans?

In this study, the hypothesis of a functional role for alternative splicing, and therefore to a certain extent for introns, is evaluated against another explanation coming from evolutionary biology: isoforms are above all errors of imprecision by the molecular machinery at work during splicing. This hypothesis is based on a principle established by Motoo Mikura, which has become central to population genetics, explaining that the evolutionary trajectory of a mutation with a given effect is intimately linked to the effective population size (Ne) where this mutation emerges. Thus, the probability of fixation of a weakly deleterious mutation increases when Ne decreases, and the probability of fixation of a weakly advantageous mutation increases when Ne increases. The genomes of populations with low Ne are therefore expected to accumulate more weakly deleterious mutations and fewer weakly advantageous mutations than populations with high Ne. In this framework, if splicing errors have only small effects on the fitness of individuals, then natural selection cannot increase the precision of the splicing machinery, allowing tolerance for the production of alternative isoforms.

In the past, the debate opposed one-off observations of effectively functional isoforms on the one hand, to global genomic quantities describing patterns without the possibility of interpreting them in detail. The authors here propose an elegant quantitative approach in line with the expected continuous variation in the effectiveness of selection, both between species and within genomes. The result describing the inter-specific pattern on a large scale confirms what was already known (there is a negative relationship between effective size and average alternative splicing rate). The essential novelty of this study lies in 1) the quantification, for each intron studied, of the relative abundance of each isoform, and 2) the analysis of a relationship between this abundance and the evolutionary constraints acting on these isoforms.

What is striking is the light shed on the general very low abundance of alternative isoforms. Depending on the species, 60% to 96% of cases of alternatively spliced introns lead to an isoform whose abundance is less than 5% of the total variants for a given intron.

In addition to the fact that 60%-96% of the total isoforms are more than 20 times less abundant than their majority form, this large proportion of alternative isoforms exhibit coding-phase shift at rates similar to what would be expected by chance, i.e. for a third of them, which reinforces the idea that there is no particular constraint on these isoforms.

The remaining 4%-40% of isoforms see their coding-phase shift rate decrease as their relative abundance increases. This result represents a major step forward in our understanding of alternative splicing and makes it possible to establish a quantitative model directly linking the relative abundance of an isoform with a putative functional role concerning only those isoforms produced in abundance. Only the (rare) isoforms which are abundantly produced are thought to be involved in a biological function.

Within the same genome, the authors show that only highly expressed genes, i.e. those that tend to be more constrained on average, are also the genes with the lowest alternative splicing rates on average.

The comparison between species in this study reveals that the smaller the effective size of a species, the more its genome produces isoforms that are low in abundance and low in constraint. Conversely, species with a large effective size relatively reduce rare isoforms, and increase stress on abundant isoforms.

To sum up:<br /> • the higher the effective size of a species, the fewer introns are spliced.<br /> • highly expressed genes are spliced less.<br /> • when splicing occurs, it is mainly to produce low-abundance isoforms.<br /> • low-abundance isoforms are also less constrained.

Taken together, these results reinforce a quantitative view of the evolution of alternative splicing as being mainly the product of imprecision in the splicing machinery, generating a great deal of molecular noise. Then, out of all this noise, a few functional gold nuggets can sometimes emerge. From the point of view of the reviewer, the evolutionary dynamics of genomes are depressing. The small effective population sizes are responsible for the accumulation of multiple slightly deleterious introns. Admittedly, metazoan genomes try to get rid of these introns during RNA maturation, but this mechanism is itself rendered imprecise by population sizes.

Strengths:<br /> • The authors simultaneously study the effects of effective population size, isoform abundance, and gene expression levels on the evolutionary constraints acting on isoforms. Within this framework, they clearly show that an isoform becomes functionally important only under certain rare conditions.<br /> • The authors rule out an effect putatively linked to variations in expression between different organs which could have biased comparisons between different species.

Weaknesses:<br /> • While the longevity of organisms as a measure of effective size seems to work overall, it may not be relevant for discriminating within a clade. For example, within Hymenoptera, we might expect them to have the same overall longevity, but that effective size would be influenced more by the degree of sociality: solitary bees/ants/wasps versus eusocial. I am therefore certain that the relationship shown in Figure 4D is currently not significant because the measure of effective size is not relevant for Hymenoptera. The article would have been even more convincing by contrasting the rates of alternative splicing between solitary versus social hymenopterans.<br /> • When functionalist biologists emphasise the role of the complexity of living things, I'm not sure they're thinking of the comparison between "drosophila" and "homo sapiens", but rather of a broader evolutionary scale. Which gives the impression of an exaggeration of the debate in the introduction.

Reviewer #2 (Public Review):

Summary:<br /> Two hypotheses could explain the observation that genes of more complex organisms tend to undergo more alternative splicing. On one hand, alternative splicing could be adaptive since it provides the functional diversity required for complexity. On the other hand, increased rates of alternative splicing could result through nonadaptive processes since more complex organisms tend to have smaller effective population sizes and are thus more prone to deleterious mutations resulting in more spurious splicing events (drift-barrier hypothesis). To evaluate the latter, B́enitiere et al. analyzed transcriptome sequencing data across 53 metazoan species. They show that proxies for effective population size and alternative splicing rates are negatively correlated. Furthermore, the authors find that rare, nonfunctional (and likely erroneous) isoforms occur more frequently in more complex species. Additionally, they show evidence that the strength of selection on splice sites increases with increasing effective population size and that the abundance of rare splice variants decreases with increased gene expression. All of these findings are consistent with the drift-barrier hypothesis.

This study conducts a comprehensive set of separate analyses that all converge on the same overall result and the manuscript is well organized. Furthermore, this study is useful in that it provides a modified null hypothesis that can be used for future tests of adaptive explanations for variation in alternative splicing.

Strengths:<br /> The major strength of this study lies in its complementary approach combining comparative and population genomics. Comparing evolutionary trends across phylogenetic diversity is a powerful way to test hypotheses about the origins of genome complexity. This approach alone reveals several convincing lines of evidence in support of the drift-barrier hypothesis. However, the authors also provide evidence from a population genetics perspective (using resequencing data for humans and fruit flies), making results even more convincing.

The authors are forward about the study's limitations and explain them in detail. They elaborate on possible confounding factors as well as the issues with data quality (e.g. proxies for Ne, inadequacies of short reads, heterogeneity in RNA-sequencing data).

Weaknesses:<br /> The authors primarily consider insects and mammals in their study. This only represents a small fraction of metazoan diversity. Sampling from a greater diversity of metazoan lineages would make these results and their relevance to broader metazoans substantially more convincing. Although the authors are careful about their tone, it is challenging to reconcile these results with trends across greater metazoans when the underlying dataset exhibits ascertainment bias and represents samples from only a few phylogenetic groups. Relatedly, some trends (such as Figure 1B-C) seem to be driven primarily by non-insect species, raising the question of whether some results may be primarily explained by specific phylogenetic groups (although the authors do correct for phylogeny in their statistics). How might results look if insects and mammals (or vertebrates) are considered independently?

Throughout the manuscript, the authors refer to infrequently spliced (mode <5%) introns as "minor introns" and frequently spliced (mode >95%) as "major introns". This is extremely confusing since "minor introns" typically represent introns spliced by the U12 spliceosome, whereas "major introns" are those spliced by the U2 spliceosome. Furthermore, it remains unclear whether the study only considers major introns or both major and minor introns. Minor introns typically have AT-AC splice sites whereas major introns usually have GT/GC-AG splice sites, although in rare cases the U2 can recognize AT-AC (see Wu and Krainer 1997 for example). The authors also note that some introns show noncanonical AT-AC splice sites while these are actually canonical splice sites for minor introns.

Reviewer #1 (Public Review):

Summary:<br /> Fang Huang et al found that RBM7 deficiency promotes metastasis by coordinating MFGE8 splicing switch and NF-kB pathway in breast cancer by utilizing clinical samples as well as cell and tail vein injection models.

Strengths:<br /> This study uncovers a previously uncharacterized role of MFGE8 splicing alteration in breast cancer metastasis, and provides evidence supporting RBM7 function in splicing regulation. These findings facilitate the mechanistic understanding of how splicing dysregulation contributes to metastasis in cancer, a direction that has increasingly drawn attention recently, and provides a potentially new prognostic and therapeutic target for breast cancer.

Weaknesses:<br /> This study can be strengthened in several aspects by additional experiments or at least by further discussions. First, how RBM7 regulates NF-kB, and how it coordinates splicing and canonical function as a component of NEXT complex should be clarified. Second, although the roles of MFGE8 splicing isoforms in cell migration and invasion have been demonstrated in transwell and wound healing assays, it would be more convincing to explore their roles in vivo such as the tail vein injection model. Third, the clinical significance would be considerably improved, if the therapeutic value of targeting MFGE8 splicing could be demonstrated.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, the authors reported the biological role of RBM7 deficiency in promoting metastasis of breast cancer. They further used a combination of genomic and molecular biology approaches to discover a novel role of RBM7 in controlling alternative splicing of many genes in cell migration and invasion, which is responsible for the RBM7 activity in suppressing metastasis. They conducted an in-depth mechanistic study on one of the main targets of RBM7, MFGE8, and established a regulatory pathway between RBM7, MFGE8-L/MFGE8-S splicing switch, and NF-κB signaling cascade. This link between RBM7 and cancer pathology was further supported by analysis of clinical data.

Strengths:<br /> Overall, this is a very comprehensive study with lots of data, and the evidence is consistent and convincing. Their main conclusion was supported by many lines of evidence, and the results in animal models are pretty impressive.

Weaknesses:<br /> However, there are some controls missing, and the data presentation needs to be improved. The writing of the manuscript needs some grammatical improvements because some of the wording might be confusing.

Specific comments:<br /> (1) Figure 2. The figure legend is missing for Figure 2C, which caused many mislabels in the rest of the panels. The labels in the main text are correct, but the authors should check the figure legend more carefully. Also in Figure 2C, it is not clear why the authors choose to examine the expression of this subset of genes. The authors only refer to them as "a series of metastasis-related genes", but it is not clear what criteria they used to select these genes for expression analysis.

(2) Line 218-220. The comparison of PSI changes in different types of AS events is misleading. Because these AS events are regulated in different mechanisms, they cannot draw the conclusion that "the presence of RBM7 may promote the usage of alternative splice sites". For example, the regulators of SE and IR may even be opposite, and thus they should discuss this in different contexts. If they want to conclude this point, they should specifically discuss the SE and A5SS rather than draw an overall conclusion.

(3) In the section starting at line 243, they first referred to the gene and isoforms as "EFG-E8" or "EFG-E8-L", but later used "EFGE8" and "EFGE8-L". Please be consistent here. In addition, it will be more informative if the authors add a diagram of the difference between two EFGE8 isoforms in terms of protein structure or domain configuration.

(4) Figure 7B and 7C. The figures need quantification of the inclusion of MFGE exon7 (PSI value) in addition to the RT-PCR gel. The difference seems to be small for some patients.

Minor comments:<br /> The writing in many places is a little odd or somewhat confusing, I am listing some examples, but the authors need to polish the whole manuscript more to improve the writing.

(1) Line 169-170, "...followed by profiling high-throughput transcriptome by RNA sequencing", should be "followed by high-throughput transcriptome profiling with RNA sequencing".

(2) Line 170, "displayed a wide of RBM7-regulated genes were enriched...", they should add a "that" after the "displayed" as the sentence is very long.

(3) Line 213, "PSI (percent splicing inclusion)" is not correct, PSI stands for "percent spliced in".

(4) Line 216-217, the sentence is long and fragmented, they should break it into two sentences.

(5) Line 224, the "tethering" should be changed to "recognizing". There is a subtle difference in the mechanistic implication between these two words.

(6) Line 250, should be changed to "..in the ratio of two MFGE8 isoforms".

Reviewer #1 (Public Review):

Summary:<br /> The study by Valles-Marti et al. was aimed at elucidating mechanisms of high-dose vitamin C (Ascorbate) sensitivity using proteomics of a large panel of cancer cell lines. The study is primarily based on correlating protein expression to vitamin C sensitivity based on IC50 from cell viability studies. As expected, cancer type-specific proteome patterns emerge and the authors conclude that some pan-cancer pathways, such as proliferation correlate with high sensitivity to VitC. In a subset of PDAC cells proteomics and phospho proteomics were also carried out following vitamin C treatment, albeit those studies did not identify significant changes in response to treatment.

Strengths:<br /> The premise for the work is of interest as high dose vitamin C is in clinical trials and thus studies investigating mechanisms of sensitivity and potential resistance mechanisms to this therapy are of interest to the field. The authors have collected large proteomic datasets on some of the most common cancer cells used and these data may be a useful resource for others when made publicly available. Although this is not necessarily novel, since proteomics data sets for some of the included cell lines are already available.

Weaknesses:<br /> The title suggests that the proteomics data presented "underscores high-dose vitamin C as a potent anti-cancer agent" However, while the proteomic data are extensive, it is my assessment that without further validation there are no clear pathways identified by the presented proteomics data that conclusively determine vitamin C sensitivity.

A major question arising from this work is how specific the proteomics data reflect sensitivity to vitamin C over general sensitivity to other cytotoxic agents. It would be of interest to compare the correlation of proteomic data and ascorbate sensitivity to the sensitivity of cell lines to other cytotoxic agents. (e.g. comparison to NCI-60 growth inhibition data). In other words, do the proteomic data that correlate with ascorbate sensitivity simply reflect susceptibility to other cytotoxic agents? The comments that vitamin C toxicity is not dependent on underlying histological or genetic subtypes of cancers ("one size fits all") suggest this.

The genetic backgrounds of tumor cells have not been taken into consideration in the analysis and how this may influence VitC susceptibility. An example that comes to mind is KEAP1/Nrf2 aberrations in lung cancer.

The study would be significantly strengthened if some of the proteins identified were further validated in eliciting low or high sensitivity to Vitamin C. Of particular interest are proteins that have functions related to known mechanisms of action of Vitamin C toxicity, such as iron homeostasis. Some of the metabolic-related protein changes are also of interest. For example, HCCS expression is mentioned several times as being associated with lower sensitivity to ascorbate. Providing experimental evidence that this protein is of significance to Vitamin C sensitivity and if this is due to its effects on iron and subsequent generation of ROS in response to VitC would be of significance.

Similarly, an interesting aspect of the findings is the authors' conclusion that proliferation is associated with Vitamin C sensitivity. The authors propose in their discussion that Vitamin C may be an attractive alternative to treat heavily pretreated and chemoresistant cancers. Thus it would be important to know which of the highly proliferative cell lines tested have a chemoresistance phenotype and are also more susceptible to Vitamin C toxicity. Perhaps partitioning the cells further into chemoresistant and sensitive cell lines to standard chemotherapy and then assessing which protein signatures are associated with Vitamin C sensitivity will allow for better elucidation of sensitivity mechanisms that are more relevant to using Vitamin C as an alternate therapy for chemoresistant tumors.

Following on from this, there is an interesting mechanistic question as to why more proliferative cells are more sensitive to vitamin C, and whether this is related to changes in metabolism and underlying changes in their steady-state levels of ROS. Further investigating this mechanistically based on the identified proteomic signatures could make the findings more significant.

Vitamin C can also generate H2O2 extracellularly in the presence of iron. Thus, Vitamin C toxicity could be affected by different abilities of the tumor cells to scavenge extracellular H2O2, such as different expression levels of extracellular antioxidant enzymes. Judging from the methods section, it does not appear that proteomic data include secreted proteins. Can the authors comment on how this may be a potential caveat?

In light of this, the strong effects of exogenous catalase addition on cell viability suggest that H2O2 may be produced by ascorbate in the media.

Similarly, can the authors comment on the cell culture conditions used to compare IC50s between cell lines, specifically if different media and FBS batches were used, as these have the potential to vary in metal/iron concentrations that might influence the pro-oxidant generation by high dose ascorbate in media. Specifically, have the authors looked into the iron content and how these different conditions may be contributing to intracellular H2O2 and extracellular H2O2 (AmplexRed) production in response to Vitamin C.

Other comments relate to methods:

How was ascorbate prepared? There is no mention of degassing of H2O and ensuring that H2O does not have mental impurities, which can lead to auto-oxidation.

The OxiSelect probe is based on DCFDA, which is an oxidant-sensitive probe that has been described to be fraught with artifacts. Thus it is advised to mention the caveats associated with the use of this probe (as outlined in PMCID: PMC3911769) and consider backing up these experiments with additional Oxidant probes.

Reviewer #2 (Public Review):

Summary:<br /> The authors generated proteome profiles of 51 cancer cell lines treated with pharmacologic ascorbate. The idea was to identify players responsible for the sensitivity or relative resistance to ascorbate to delineate mechanisms of action of this potentially transformative new treatment.

Strengths:<br /> The proteomic profiles themselves. The identification of MAPK and mTOR as overrepresented proteomic elements and close correlations between proliferation, cell cycle mediators, and sensitivity to ascorbate indicate that rapidly proliferating cancer may be more sensitive to ascorbate. Also, the finding that sensitivity to ascorbate is correlated to different pathways in different types of cancer is interesting. For instance, in some pancreatic and lung cancers sensitivity seemed to be related to iron handling while in breast DNA damage/repair seemed to be most involved.

Weaknesses:<br /> The study is quite descriptive. Although the proteomes indicate what pathways are more or less represented after ascorbate challenge there is little mechanistic information about their relevance to the sensitivity to ascorbate. Since activity is not assessed, proteins may be present in higher or lower abundance but not necessarily at the peak of their activity. Also, many statements are made as "known facts" but no references are provided.

Reviewer #3 (Public Review):

Summary:<br /> In the manuscript titled "Large pan-cancer cell screen coupled to (phospho-)proteomics underscores high-dose vitamin C (VitC) as a potent anti-cancer agent," the authors use a combination of proteomics and cell viability assays to understand the effect of Vitamin C on different solid tumor models in 51 different cancer cell lines. They found that many cancer cell lines are sensitive to high-dose Vitamin C, with IC50 values in the micromolar to millimolar range. Given that Vitamin C, when administered intravenously, can reach 20mM, this suggests that Vitamin C could provide some benefits to patients. The authors also generate and analyze bulk proteomic data for all 51 cell lines. They perform statistical analysis of these data to identify proteins that are up or downregulated in sensitive vs resistant cell lines in the same tumor and commonly across tumors. They then focus on PDAC cell lines and measure bulk and phosphoproteomics of PDAC cell lines 2, 4 and 24 hours after Vitamin C treatment.

Strengths: The strengths of the study are the rather large datasets accumulated on bulk proteomics of 51 different cancer cell lines. The IC50 values of these cell lines in response to Vitamin C is also useful.

Weaknesses:<br /> Though identifying targets to sensitize cancer cells to Vitamin C treatment is interesting, I felt the manuscript delved too much into listing off genes they found, with speculation on why the particular protein would be enriched in sensitive or resistant cell lines without testing any key claims experimentally.

Major Issues

(1) The overall premise of the study is that proteins that are enriched in Vitamin C-sensitive cell lines point to mechanisms of sensitivity and those enriched in Vitamin C resistant lines underlie mechanisms of resistance. Yet this is never directly tested. To show that the authors would need to knockdown/knockout a gene enriched in resistant lines and show this sensitizes cells to Vitamin C treatment or overexpress a protein associated with resistance and show that this leads to resistance in an otherwise sensitive cell line.

(2) One of the key strengths of this study is the large datasets generated, namely the proteomics data for 51 different cell lines. Yet the data is not included as a supplement or uploaded to a public repository.

Reviewer #1 (Public Review):

This manuscript proposes a complex incoherent model involving Ca2+ signaling in inflammasome activation. The experimental approaches used to study the calcium dynamics are highly problematic and the results shown are of very poor quality.

Major concerns:

(1) The analysis of lysosomal Ca2+release is being carried out after many hours of treatment. Such evidence is not meaningful to claim that PA activates Ca2+ efflux from lysosome and even if this phenomenon was robust, it is not doubtful that such kinetics are meaningful for the regulation of inflammasome activation. Furthermore, the evidence for lysosomal Ca2+ release is indirect and relies on a convoluted process that doesn't make any conceptual sense to me. In addition to these major shortcomings, the indirect evidence of perilysosomal Ca2+ elevation is also of very poor quality and from the standpoint of my expertise in calcium signaling, the data are incredulous. The use of GCaMP3-ML1, *transiently transfected* into BMDMs is highly problematic. The efficiency of transfection in BMDMs is always extremely low and overexpression of the sensor in a few rare cells can lead to erroneous observations. The overexpression also results in gross mislocalization of such membrane-bound sensors. The accumulation of GCaMP3-ML1 in the ER of these cells would prevent any credible measurements of perilysosomal Ca2+ signals. A meaningful investigation of this process in primary macrophages requires the generation of a mouse line wherein the sensor is expressed at low levels in myeloid cells, and shown to be localized almost exclusively in the lysosomal membrane. The mechanistic framework built around these major conceptual and technical flaws is not especially meaningful and since these are foundational results, I cannot take the main claims of this study seriously.

A few transfected cells may overexpress the protein through a strong promoter but this is not ideal. For reliable Ca2+ measurements, one needs low expression of the sensor in a substantially high percentage of cells. This can only be demonstrated by showing the time lapse of Ca2+ responses in the macrophages. More generally, I have nearly 2 decades of experience working with primary BMDMs and it is widely known that primary BMDMs are incredibly difficult to transfect - it is the nature of these cells. The claim that they get high efficiency of transfection is frankly too incredulous to take seriously.

(2) The cytosolic Ca2+ imaging shown in figure 1C doesn't make any sense. It looks like a snapshot of basal Ca2+ many hours after PA treatment - calcium elevations are highly dynamic. Snapshot measurements are not helpful and analyses of Calcium dynamics requires a recording over a certain timespan. Unfortunately, this technical approach has been used throughout the manuscript. Also, BAPTA-AM abrogates IL-1b secretion because IL-1b transcription is Ca2+ dependent - the result shown in figure 1D does not shed light on anything to do with inflammasome activation and it is misleading to suggest that.

(3) Trpm2-/- macrophages are known to be hyporesponsive to inflammatory stimuli - the reduced secretion of IL-1b by these macrophages is not novel. From a mechanistic perspective, this study does not add much to that observation and the proposed role of TRPM2 as a lysosomal Ca2+ release channel is not substantiated by good quality Ca2+ imaging data (see point 3 above). Furthermore, the study assumes that TRPM2 is a lysosomal ion channel. One paper reported TRPM2 in the lysosomes but this is a controversial claim, with no replication or further development in the last 14 years. This core assumption can be highly misleading to readers unfamiliar with TRPM2 biology and it is necessary to present credible evidence that TRPM2 is functional in the lysosomal membrane of macrophages. Ideally, this line of investigation should rest on robust demonstration of TRPM2 currents in patch-clamp electrophysiology of lysosomes. If this is not technically feasible for the authors, they should at least investigate TRPM2 localization on lysosomal membranes of macrophages.

In the revised manuscript, authors showed TRPM2 localization but these results are problematic. The authors provide no information on what TRPM2 antibody they used for this study and whether it has been validated by use of knockouts. The staining shows very high amounts of TRPM2 all across the cell - even more than LAMP2. In reality, TRPM2 expression in macrophages is very low. Are the authors overexpressing TRPM2? These data only add to my concerns about this manuscript.

(4) Apigenin and Quercetin are highly non-specific and their effects cannot be attributed to CD38 inhibition alone. Such conclusions need strong loss of function studies using genetic knockouts of CD38 - or at least siRNA knockdown. Importantly, if indeed TRPM2 is being activated downstream of CD38, this should be easily evident in whole cell patch clamp electrophysiology. TRPM2 currents can be resolved using this technique and authors have Trpm2-/- cells for proper controls. Authors attempted these experiments but the results are of very poor quality. If the TRPM2 current is being activated through ADPR generated by CD38 (in response to PA stimulation), then it is very odd that authors need to include 200 uM cADPR to see TRPM2 current (Fig. 3A). Oddly, even these data cast great doubt on the technical quality of the electrophysiology experiments. Even with such high concentrations of cADPr, the TRPM2 current is tiny and Trpm2-/- controls are missing. The current-voltage relationship is not shown, and I feel that the results are merely reporting leak currents seen in measurements with substandard seals. Also 20 uM ACA is not a selective inhibitor of TRPM2 - relying on ACA as the conclusive diagnostic is problematic.

(5) TRPM2 is expressed in many different cell lines. The broad metabolic differences observed by the authors in the Trpm2-/- mice cannot be attributed to macrophage-mediated inflammation. Such a conclusion requires the study of mice wherein Trpm2 is deleted selectively in macrophages or at least in the cells of the myeloid lineage.

(6) The ER-Lysosome Ca2+ refilling experiments rely on transient transfection of organelle-targeted sensors into BMDMs. See point #1 to understand why I find this approach to be highly problematic. Furthermore the data procured are also not convincing and lack critical controls (localization of sensors has not been demonstrated and their response to acute mobilization of Ca2+ has not been shown inspire any confidence in these results).

(7) Authors claim that SCOE is coupled to K+ efflux. But there is no credible evidence that SOCE is activated in PA stimulated macrophages. The data shown in Fig 4 supp 1 do not investigate SOCE in a reliable manner - the conclusion is again based on snapshot measurements and crude non-selective inhibitors. The correct way to evaluate SOCE is to record cytosolic Ca2+ elevations over a period of time in absence and presence of extracellular Ca2+. However, even such recordings can be unreliable since the phenomenon is being investigated hours after PA stimulation. So, the only definitive way to demonstrate that Orai channels are indeed active during this process is through patch clamp electrophysiology of PA stimulated cells.

Authors failed to respond to these concerns in a credible manner and simply tried to obfuscate the matters with extraneous arguments and wild claims. The revised manuscript was not a significant improvement. I have major concerns with this manuscript and let it be on record that this is very poor-quality science.

Reviewer #2 (Public Review):

In this manuscript by Kang et. al., the authors investigated the mechanisms of K+-efflux-coupled SOCE in NLRP3 inflammasome activation by LP（LPS+PA, and identified an essential role of TRPM2-mediated lysosomal Ca2+ release and subsequent IP3Rs-mediated ER Ca2+ release and store depletion in the process. K+ efflux is shown to be mediated by a Ca2+-activated K+ channel (KCa3.1). LP-induced cytosolic Ca2+ elevation also induced a delayed activation of ASK1 and JNK, leading to ASC oligomerization and NLRP3 inflammasome activation. Overall, this is an interesting and comprehensive study that has identified several novel molecular players in metabolic inflammation. The manuscript can benefit if the following concerns could be addressed.

(1) The expression of TRPM2 in the lysosomes of macrophages needs to more definitively established. For instance, the cADPR-induced TRPM2 currents should be abolished in the TRPM2 KO macrophages. Can you show the lysosomal expression of TRPM2, either with an antibody if available or with a fluorescently-tagged TRPM2 overexpression construct?

In the revised manuscript, the authors did not perform the KO control experiment to support that cADPR-induced currents were indeed mediated by TRPM2. Additonally, the co-localization analyses failed to convincingly establish the lysosomal perimeter membrane residence of TRPM2.

(2) Can you use your TRPM2 inhibitor ACA to pharmacologically phenocopy some results, e.g., about [Ca2+]ER, [Ca2+]LY, and [Ca2+]i from the TRPM2 knockout?

In the revised manuscript, most suggested experiments were not performed. In the only experiment that was conducted, Figure 3-figure supplement 1A, the effect of ACA was marginal.

(3) In Fig. S4A, bathing the cells in zero Ca2+ for three hours might not be ideal. Can you use a SOCE inhibitor, e.g, YM-58483, to make the point?

The specific suggested experiment was not performed.

(4) In Fig. 1A, you need a positive control, e.g., ionomycin, to show that the GPN response was selectively reduced upon LP treatment.

Results in a previous study cannot be used to substitute the missing control experiments in the current study.

Reviewer #2 (Public Review):

Summary: The study is titled "Leading an urban invasion: risk-sensitive learning is a winning strategy", and consists of three different parts. First, the authors analyse data on initial and reversal learning in Grackles confronted with a foraging task, derived from three populations labeled as "core", "middle" and "edge" in relation to the invasion front. The suggested difference between study populations does not surface, but the authors do find support for a difference between male and female individuals. Secondly, the authors confirm that the proposed mechanism can actually generate patterns such as observed in the Grackle data through agent-based forward simulations. In the third part, the authors present an evolutionary model, in which they show that learning strategies, as observed in male Grackles, do evolve in simplified urban conditions including different levels of environmental stability and environmental stochasticity.

Strengths: The manuscript's strength is that it combines real learning data collected across different populations of the Great-tailed grackle (Quiscalus mexicanus) with theoretical approaches to better understand the processes with which grackles learn and how such learning processes might be advantageous during range expansion and invasion. Furthermore, the authors also take sex into account revealing that males, the dispersing sex, show better reversal learning through higher reward-payoff sensitivity. I also find it refreshing to see that the authors took the time to preregister their study to improve transparency especially regarding data analysis.

Weakness: The small sample size of grackles across populations increases uncertainty as to parameter estimates and the conclusions drawn from these estimates.

After revision, the introduction is appropriate, and in the methods, the authors take great care in explaining the rational behind decisions as to the selection of analysis methods and parameters. I very much appreciate that the authors took such care in revising their paper, the quality of which has now greatly improved.

Reviewer #1 (Public Review):

Assessment:

The manuscript titled 'Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal 1 homeostasis' by Gaur et al discusses the role of Rab7 in the development of ulcerative colitis by regulating the lysosomal degradation of Clca1, a mucin protease. The manuscript presents interesting data, and provides a potential molecular mechanism for the pathological alterations observed in ulcerative colitis.

Strengths:

The manuscript used a multi-pronged approach and compares patient samples, mouse models of DSS and protocols that allow differentiation of goblet cells. They also use a nanogel-based delivery system for siRNAs, which is ideal for knockdown of specific genes in the gut.

Weaknesses:

The manuscript should also mention the limitations of the study.

Reviewer #2 (Public Review):

Summary:

In this work, the authors report a role for the well-studied GTPase Rab7 in gut homeostasis. The study combines cell culture experiments with mouse models and human ulcerative colitis patient tissues to propose a model where, Rab7 by delivering a key mucous component CLCA1 to lysosomes, regulates its secretion in the goblet cells. This is important for the maintenance of mucous permeability and gut microbiota composition. In the absence of Rab7, CLCA1 protein levels are higher in tissues as well as the mucus layer, corroborating with the anti-correlation of Rab7 (reduced) and CLCA1 (increased) from ulcerative colitis patients. The authors conclude that Rab7 maintains CLCA1 level by controlling its lysosomal degradation, thereby playing a vital role in mucous composition, colon integrity, and gut homeostasis.

Strengths:

The biggest strength of this manuscript is the combination of cell culture, mouse model, and human tissues. The experiments are largely well done and in most cases, the results support their conclusions. The authors go to substantial lengths to find a link, such as alteration in microbiota, or mucus proteomics.

Weaknesses:

There are also some weaknesses that need to be addressed. The association of Rab7 with UC in both mice and humans is clear, however, claims on the underlying mechanisms are less clear. Does Rab7 regulate specifically CLCA1 delivery to lysosomes, or is it an outcome of a generic trafficking defect? CLCA1 is a secretory protein, how does it get routed to lysosomes, i.e. through Golgi-derived vesicles, or by endocytosis of mucous components? Mechanistic details on how CLCA1 is routed to lysosomes will add substantial value.

Why does the level of Rab7 fluctuate during DSS treatment (Fig 1B)? Does the reduction seen in Rab7 levels (by WB) also reflect in reduced Rab7 endosome numbers? Are other late endosomal (and lysosomal) populations also reduced upon DSS treatment and UC? Is there a general defect in lysosomal function?

While it is clear that the pattern of Muc2 in WT and Rab7-/- cells are different, how this corroborates with the in vivo data on alterations in mucus layer permeability - as claimed - is not clear.

The use of an in vivo intestine-specific Rab7 silencing model is good. Why does Rab7 KD itself not capitulate aspects of DSS treatment, rather it seems to exacerbate it.

The use of mucous proteomics to identify mechanisms of Rab7-mediated phenotype is a good approach. The replicates in the proteomics dataset (Fig 6F) do not seem to match. Detailing of methodology used for analysis will help to overcome these doubts.

The work shows a role for a well-studied GTPase, Rab7, in gut homeostasis. This is an important finding and could provide scope and testable hypotheses for future studies aimed at understanding in detail the mechanisms involved.

Reviewer #1 (Public Review):

Summary:<br /> The authors compared four types of hiPSCs and four types of hESCs at the proteome level to elucidate the differences between hiPSCs and hESCs. Semi-quantitative calculations of protein copy numbers revealed increased protein content in iPSCs. Particularly in iPSCs, proteins related to mitochondrial and cytoplasmic were suggested to reflect the state of the original differentiated cells to some extent. However, the most important result of this study is the calculation of the protein copy numbers per cell, and the validity of this result is problematic. In addition, several experiments need to be improved, such as using cells of different genders (iPSC: female, ESC: male) in mitochondrial metabolism experiments.

Strengths:<br /> The focus on the number of copies of proteins is exciting and appreciated if the estimated calculation result is correct and biologically reproducible.

Weaknesses:<br /> The proteome results in this study were likely obtained by simply looking at differences between clones, and the proteome data need to be validated. First, there were only a few clones for comparison, and the gender and number of cells did not match between ESCs and iPSCs. Second, no data show the accuracy of the protein copy number per cell obtained by the proteome data.

Reviewer #2 (Public Review):

Summary:<br /> Pluripotent stem cells are powerful tools for understanding development, differentiation, and disease modeling. The capacity of stem cells to differentiate into various cell types holds great promise for therapeutic applications. However, ethical concerns restrict the use of human embryonic stem cells (hESCs). Consequently, induced human pluripotent stem cells (ihPSCs) offer an attractive alternative for modeling rare diseases, drug screening, and regenerative medicine. A comprehensive understanding of ihPSCs is crucial to establish their similarities and differences compared to hESCs. This work demonstrates systematic differences in the reprogramming of nuclear and non-nuclear proteomes in ihPSCs.

Strengths:<br /> The authors employed quantitative mass spectrometry to compare protein expression differences between independently derived ihPSC and hESC cell lines. Qualitatively, protein expression profiles in ihPSC and hESC were found to be very similar. However, when comparing protein concentration at a cellular level, it became evident that ihPSCs express higher levels of proteins in the cytoplasm, mitochondria, and plasma membrane, while the expression of nuclear proteins is similar between ihPSCs and hESCs. A higher expression of proteins in ihPSCs was verified by an independent approach, and flow cytometry confirmed that ihPSCs had larger cell sizes than hESCs. The differences in protein expression were reflected in functional distinctions. For instance, the higher expression of mitochondrial metabolic enzymes, glutamine transporters, and lipid biosynthesis enzymes in ihPSCs was associated with enhanced mitochondrial potential, increased ability to uptake glutamine, and increased ability to form lipid droplets.

Weaknesses:<br /> While this finding is intriguing and interesting, the study falls short of explaining the mechanistic reasons for the observed quantitative proteome differences. It remains unclear whether the increased expression of proteins in ihPSCs is due to enhanced transcription of the genes encoding this group of proteins or due to other reasons, for example, differences in mRNA translation efficiency. Another unresolved question pertains to how the cell type origin influences ihPSC proteomes. For instance, whether ihPSCs derived from fibroblasts, lymphocytes, and other cell types all exhibit differences in their cell size and increased expression of cytoplasmic and mitochondrial proteins. Analyzing ihPSCs derived from different cell types and by different investigators would be necessary to address these questions.

Reviewer #3 (Public Review):

Summary:<br /> In this study, Brenes and colleagues carried out proteomic analysis of several human induced pluripotent (hiPSC) and human embryonic stem cell (hESC) lines. The authors found quantitative differences in the expression of several groups of cytoplasmic and mitochondrial proteins. Overall, hiPSC expressed higher levels of proteins such as glutamine transporters, mitochondrial metabolism proteins, and proteins related to lipid synthesis. Based on the protein expression differences, the authors propose that hiPSC lines differ from hESC in their growth and metabolism.

Strengths:<br /> The number of generated hiPSC and hESC lines continues to grow, but potential differences between hiPSC and hESC lines remain to be quantified and explained. This study is a promising step forward in understanding of the differences between different hiPSC and hESC lines.

Weaknesses:<br /> It is unclear whether changes in protein levels relate to any phenotypic features of cell lines used. For example, the authors highlight that increased protein expression in hiPSC lines is consistent with the requirement to sustain high growth rates, but there is no data to demonstrate whether hiPSC lines used indeed have higher growth rates.

The authors claim that the cell cycle of the lines is unchanged. However, no details of the method for assessing the cell cycle were included so it is difficult to appreciate if this assessment was appropriately carried out and controlled for.

Details and characterisation of iPSC and ESC lines used in this study were overall lacking. The lines used are merely listed in methods, but no references are included for published lines, how lines were obtained, what passage they were used at, their karyotype status, etc. For details of basic characterisation, the authors should refer to the ISSC Standards for the use of human stem cells in research. In particular, the authors should consider whether any of the changes they see may be attributed to copy number variants in different lines.

The expression data for markers of undifferentiated state in Figure 1a would ideally be shown by immunocytochemistry or flow cytometry as it is impossible to tell whether cultures are heterogeneous for marker expression.

TEM analysis should ideally be quantified.

All figure legends should explicitly state what graphs are representing (e.g. average/mean; how many replicates (biological or technical), which lines)? Some data is included in Methods (e.g. glutamine uptake), but not for all of the data (e.g. TEM).

Validation experiments were performed typically on one or two cell lines, but the lines used were not consistent (e.g. wibj_2 versus H1 for respirometry and wibj_2, oaqd_3 versus SA121 and SA181 for glutamine uptake). Can the authors explain how the lines were chosen?

The authors should acknowledge the need for further functional validation of the results related to immunosuppressive proteins.

Differences in H1 histone abundance were highlighted. Can the authors speculate as to the meaning of these differences?

Reviewer #1 (Public Review):

Summary:<br /> In this study by Zhou, Wang, and colleagues, the authors utilize biventricular electromechanical simulations to illustrate how different degrees of ionic remodeling can contribute to different ECG morphologies that are observed in either acute or chronic post-myocardial infarction (MI) patients. Interestingly, the simulations show that abnormal ECG phenotypes - associated with a higher risk of sudden cardiac death - are predicted to have almost no correspondence with left ventricular ejection fraction, which is conventionally used as a risk factor for arrhythmia.

Strengths:<br /> The numerical simulations are state-of-the-art, integrating detailed electrophysiology and mechanical contraction predictions, which are often modeled separately. The simulation provides mechanistic interpretation, down to the level of single-cell ionic current remodeling, for different types of ECG morphologies observed in post-MI patients. Collectively, these results demonstrate compelling and significant evidence for the need to incorporate additional risk factors for assessing post-MI patients.

Weaknesses:<br /> The study is rigorous and well-performed. However, some aspects of the methodology could be clearer, and the authors could also address some aspects of the robustness of the results. Specifically, does variability in ionic currents inherent in different patients, or the location/size of the infarct and surrounding remodeled tissue impact the presentation of these ECG morphologies?

Reviewer #2 (Public Review):

Summary:<br /> The authors constructed multi-scale modeling and simulation methods to investigate the electrical and mechanical properties of acute and chronic myocardial infarction (MI). They simulated three acute MI conditions and two chronic MI conditions. They showed that these conditions gave rise to distinct ECG characteristics that have been seen in clinical settings. They showed that the post-MI remodeling reduced ejection fraction up to 10% due to weaker calcium current or SR calcium uptake, but the reduction of ejection fraction is not sensitive to remodeling of the repolarization heterogeneities.

Strengths:<br /> The major strength of this study is the construction of computer modeling that simulates both electrical behavior and mechanical behavior for post-MI remodeling. The links of different heterogeneities due to MI remodeling to different ECG characteristics provide some useful information for understanding complex clinical problems.

Weaknesses:<br /> The rationale (e.g., physiological or medical bases) for choosing the 3 acute MI and 2 chronic MI settings is not clear. Although the authors presented a huge number of simulation data, in particular in the supplemental materials, it is not clearly stated what novel findings or mechanistic insights this study gained beyond the current understanding of the problem.

Reviewer #1 (Public Review):

Herzog and colleagues investigated the interactions between working memory (WM) task condition (updating, maintenance) and BMI (body-mass-index), while considering selected dopaminergic genes (COMT, Taq1A, C957T, DARPP-32). Emerging evidence suggests that there might be a specific negative association with BMI in the updating but not maintenance condition, with potential bearings to reversal reward learning in obesity. The inclusion of multiple dopaminergic genes is a strength in the present study, considering the complexity of the interactions between tonic and phasic dopamine across the brain that may distinctly associate with the component processes of WM. Here, the finding was that BMI was negatively associated with WM performance regardless of the condition (updating, maintenance), but in models including moderation by either Taq1A or DARPP-32 (but not by COMT and C957T) an interaction by task condition was observed. Furthermore, a two-way interaction effect between BMI and genotype was observed exclusively in the updating condition. These findings are in line with the accounts by which striatal dopamine as reflected by Taq1A and DARPP-32 play an important role in working memory updating, while cortical dopamine as reflected by COMT is mainly associated with maintenance. The authors conclude that the genetic moderation reflects a compound negative effect of having high BMI and a risk allele in Taq1A or DARPP-32 to working memory updating specifically.

These data increment the accumulating evidence that the dopamine system may play an important role in obesity, but some of the claims in the present work are not entirely supported by the data and analysis presented. In particular, theoretical analysis of the extant evidence and formulation of the hypothesis remains elusive in terms of the potential mechanisms of updating/maintaining balance in obesity, and as such the interpretation of the present findings in the light of dopaminergic moderation warrants some caution. The result that Taq1A and DARPP-32 moderated the interaction between WM condition and BMI requires intricate post hoc analysis to understand the bearings to update. The authors found that Taq1A or DARPP-32 genotype moderated the negative association between BMI and WM exclusively in the update condition (significant two-way interaction effect), suggesting that the BMI-WM associations in other conditions were similar across genotypes. Importantly, visual inspection of the relationship between WM and BMI (Fig 4 & 5) suggests more prevalent positive effects of the putatively advantageous Taq1A-A1 and DARPP-32-AA genotypes to the overall negative relationship between WM and BMI in updating, but not in the other conditions. Given that an overall negative relationship was statistically supported across all conditions (model 1), a plausible interpretation would be that the updating condition stands out in terms of a positive moderation by putative advantageous genotypes, rather than compound negative consequences of BMI and genotype in updating. Critically, this interpretation stands in stark contrast with the interpretation put forth by the authors suggesting a specifically negative association between BMI and WM updating.

In conclusion, in its current form the title of the present work is ambivalent in terms of 1) the use of the term "impaired" in the context of cognitively normal individuals, 2) a BMI group difference specifically in the updating condition, and 3) the dopaminergic mechanisms based on observational data.

Reviewer #2 (Public Review):

Summary:<br /> The authors investigated if obesity is associated with elevated working memory deficits. Prior theorizing would suggest that individuals with a higher BMI would be worse at working memory updating, potentially due to impaired dopaminergic signaling in the striatum. However, the authors find that higher BMI was associated with worse working memory performance, irrespective of having to ignore or update new information. To further explore the putative dopaminergic mechanisms, participants are stratified according to genetic polymorphisms in COMT, Taq1A, DARPP, and C957T and the ratio of the amino acids phenylalanine and tyrosine, all implicated in dopamine-signaling. They find that especially for working memory updating, carriers of a risk allele of Taq1A and DARPP, but not of COMT and C957T, performed worse with increasing BMI. The detrimental effects of these polymorphisms on updating only surfaced for individuals with high but not low BMI.

Although the authors allude to potential imbalances in the striatal go/no-go dopamine pathways to explain these findings, the dopaminergic mechanisms of the effects remain speculative.

Strengths:<br /> Differentiating between working memory maintenance (ignoring) and updating is a powerful way to get a deeper insight into specific working memory deficits in individuals with obesity. This way of assessing working memory could potentially be applied to various populations at risk for cognitive or working memory deficits.

By pooling data from three studies, the authors reached a relatively large sample of 320 participants, which enables the assessment of more subtle effects on working memory, including the differentiation between updating and ignoring.

Working memory gating has long implicated striatal dopamine signaling. This paper shows that specific combinations of risk factors, a high BMI and carrying a risk allele, can contribute to very selective working memory impairments. More insight into how these risk factors interact can ultimately lead to more tailor-made treatments.

Weaknesses:<br /> The majority of participants seem to fall within the normal BMI range, whereas the interaction between BMI and genetic variations or amino acid ratio particularly surfaces at higher BMI. As genetic variations are usually associated with small effect sizes, the effective sample size, although large for a behavioral analysis only, might have been too small to detect meaningful effects of risk alleles of COMT and C957T.

The relationships between genetic variations, BMI, and specific disturbances in dopamine signaling are complex, as compensating mechanisms might be at play to mitigate any detrimental effects. The results would therefore benefit from more direct measures or manipulations of dopaminergic processes.

The introduction could benefit from a more elaborate description of the predicted effects: into which direction (better or worse updating) would the authors predict each effect to go and why? This is clearly explained for COMT, but not for e.g. DARPP-32.

Reviewer #1 (Public Review):

Drawing on insights from preceding studies, the researchers pinpointed mutations within the spag7 gene that correlate with metabolic aberrations in mice. The precise function of spag7 has not been fully described yet, thereby the primary objective of this investigation is to unravel its pivotal role in the development of obesity and metabolic disease in mice. First, they generated a mice model lacking spag7 and observed that KO mice exhibited diminished birth size, which subsequently progressed to manifest obesity and impaired glucose tolerance upon reaching adulthood. This behaviour was primarily attributed to a reduction in energy expenditure. In fact, KO animals demonstrated compromised exercise endurance and muscle functionality, stemming from a deterioration in mitochondrial activity. Intriguingly, none of these effects was observed when using a tamoxifen-induced KO mouse model, implying that Spag7's influence is predominantly confined to the embryonic developmental phase. Explorations within placental tissue unveiled that mice afflicted by Spag7 deficiency experienced placental insufficiency, likely due to aberrant development of the placental junctional zone, a phenomenon that could impede optimal nutrient conveyance to the developing fetus. Overall, the authors assert that Spag7 emerges as a crucial determinant orchestrating accurate embryogenesis and subsequent energy balance in the later stages of life.

The study boasts several noteworthy strengths. Notably, it employs a combination of animal models and a thorough analysis of metabolic and exercise parameters, underscoring a meticulous approach. Furthermore, the investigation encompasses a comprehensive evaluation of fetal loss across distinct pregnancy stages, alongside a transcriptomic analysis of skeletal muscle, thereby imparting substantial value. Upon addressing the previously mentioned aspects, the study is poised to exert a substantial influence on the field, its significance reverberating significantly. The methodologies and data presented undoubtedly hold the potential to facilitate the community's deeper understanding of the ramifications stemming from disruptions during pregnancy, shedding light on their enduring impact on the metabolic well-being of subsequent generations.

Reviewer #1 (Public Review):

Summary:

Plant roots grow following the gravity vector. Changes in the direction of gravity can be sensed in the root tip by specialized cells that hold starch granules. These starch granules act as levels. Movement and settling of the granules at the bottom of these specialized cells initiates an imbalanced distribution of auxin, a key hormone for plant development. Consequently, this leads to a reorientation of root growth towards the newly established gravity vector. This work provides new insights into granules' relocalization, the proteins associated with them, and the molecular processes triggered downstream.

Comments on revised submission:

In the previous review round, the reviewers noted that the authors had missed an opportunity to discuss the results presented in two recently published articles closely related to the topic of their manuscript. The authors have now referenced these articles in the current version of the manuscript, but the discussion remains rather brief. It would have been beneficial to summarize, identify, and highlight the similarities among these studies in a more comprehensive manner.

In Figure 1, it would have been more informative if the authors had provided specific information concerning the key time-points described in the graphs to visually illustrate the dynamics of PIN3 localization, intracellular calcium transients, and auxin reporter DII Venus. Including these images would have perfectly complemented panels E, F, and G.

The authors expressed concerns about overcrowding the figure. If the aesthetics of the figure were their primary concern, they could have included essential image frames for the data represented in the graphs in a supplementary figure. Alternatively, a detailed description of supplementary movie 3, highlighting the specific frames quantified in the graphs (Figure 1), could have sufficed.

Reviewer #2 (Public Review):

Summary:

This manuscript addresses what rapid molecular events underly the earliest responses after gravity-sensing via the sedimentation of starch-enriched amyloplasts in columella cells of the plant root cap. The LAZY or NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR) protein family is involved in this process and localizes to both the amyloplast and to the plasma membrane (PM) of columella cells.

This manuscript complements and extends a very recent study, (Nishimura et al., Science, 2023, August 10, 2023) that reported that the LZY3 and LZY4 proteins translocate from amyloplasts to the PM and that this translocation is likely necessary for the root gravitropic response. Kulich and colleagues describe the role of the LZY2 protein, also called NGR1, during this process, imaging its fast relocation and addressing additional novel points such as molecular mechanisms underlying NGR1 plasma membrane association as well as revealing the requirement of NGR1/LZY2, 3,4 for the polar localization of the AGCVIII D6 protein kinase at the PM of columella cells, in which NGR1/LZY2 acts redundantly with LZY3 and LZY4.

The authors initially monitored relocalization of functional NGR1-GFP in columella cells of the ngr1 ngr2 ngr3 triple mutant after 180 degree reorientation of the roots. Within 10 -15 min NGR1-GFP signal disappeared from the upper PM after reorientation and reappeared at the lower PM of the reoriented cells in close proximity to the sedimented amyloplasts. Reorientation of NGR1-GFP occurred substantially faster than PIN3-GFP reorientation, at about the same time or slightly later than a rise in a calcium sensor (GCaMP3) just preceding a change in D2-Venus auxin sensor alterations. Reorientation of NGR1-GFP proved to be fast and not dependent on a brefeldin A-sensitive ARF GEF-mediated vesicle trafficking, unlike the trafficking of PIN proteins, like PIN3, or the AGCVIII D6 protein kinase. Strikingly, the PM association of NGR1-GFP was highly sensitive to pharmacological interference with sterol composition or concentration and phosphatidylinositol (4)kinase inhibition as well as dithiothreitol (DTT) treatment interfering with thioester bond formation e.g. during S-acylation. Indeed, combined mutation of a palmitoylation site and polybasic regions of NRG1 abolished its PM but not its amyloplast localization and rendered the protein non-functional during the gravitropic response, suggesting NRG1 PM localization is essential for the gravitropic response. Targeting the protein to the PM via an artificially introduced N-terminal myristoylation and a ROP2-derived polybasic region and geranylgeranylation site partially restored its functionality in the gravitropic response.

Strengths:

This timely work should be of broad interest to plant, cell and developmental biologists across the field as gravity sensing and signaling may well be of general interest. The point that NGR1 is rapidly responsive to gravistimulation, polarizes at the PM in the vicinity to amyloplast and that this is required for repolarization of D6 protein kinase, prior to PIN relocation is really compelling. The manuscript is generally well written and accessible to a general readership, except for very minor language errors. The figures are clear and of high quality, the methods are sufficiently explained for reproduction of the experiments.

Comments on revised submission:

The authors have addressed my comments to a large part, however, while they write they have updated the statistical analysis as requested, they only did this for the main figures, but NOT for the supplementary images (except for Fig. S2) and their legends. These issues need fixing in order to correctly describe the data and let the reader know, which distributions actually differed. Some specific examples of concerns are:

In Figs. 3F and D we now know that a one-way ANOVA test was performed and that letters designate the statistically significant difference between distributions with p smaller 0.0001, but we still do not know what "n" in the displayed distributions is e.g. how many PM/cytoplasm ratios were measured i.e. e.g 112? (from 112 cells?). It is said that 8-15 roots were quantified, but the data points in the distributions are not 8-15 .... . They are many more, so, "n" must be the number of cells derived from 8-15 roots but what is "n" in the displayed distributions and is that the same value that was used for the Anova test?

This must be clarified as it has very well been done for Fig. 2 and Fig. S2B, E in the legends and by inserting a lettering for significance differences in the figures.

Similar information is still lacking for Fig. S3D, no number "n" of cells from which the PM/cytoplasm ratios are analyzed is given, no lettering for differences, no p -value. This leaves one to guess which distributions differ from each other.

This also needs to be fixed for Figs. S4 E, F (for G and H one can see the differences where the SDs do not overlap and it is explained what they are derived from).

Reviewer #3 (Public Review):

This reviewer appreciates the responses to previous notes. The authors attempted to address concerns mostly in writing, avoiding performing some of the experiments suggested in my previous review. Although some of the points were clarified, and the revised manuscript presents valuable insights into the implications of YBR238C and RMD9 on cellular function and yeast aging, my major concern still needs to be addressed. The gene expression signature significantly changes under different metabolic conditions. The media condition under which samples are collected for RNAseq analyses should match the media condition under which the lifespans of those KO strains are tested. This is the major confounding effect, and the conclusions are not informative based on the analysis done in this study.

To avoid experiments, the authors responded that yeast culture results in low optical density and does not reach the stationary phase under rapamycin treatment conditions; however, the simple solution is to grow the yeast cells until they reach the stationary phase and then rapamycin treatment can be done for certain hours - collect the cells for transcriptomics analysis then it can be compared to the CLS gene set.

Another example is chromosome copy number alteration, which can be easily analyzed using transcriptome data, and it is an important aspect to understand whether observed expression changes are also affected by this alteration in YBR238C KO cells. However, the authors ignore this important point as well.

After all, this is an interesting study "limited by subfield" and will be of general interest in the yeast aging field, again considering the lack of homology of the genes of interest in higher eukaryotes.

Reviewer #1 (Public Review):

Summary

This fascinating paper by M. Alfatah et al. describes work to uncover novel genes affecting lifespan in the budding yeast S. cerevisiae, eventually identifying and further characterizing a gene, YBR238C, now named AAG1 by the authors.<br /> The authors began by considering published gene sets pulled from the Saccharomyces genome database that described increases or decreases in either chronological lifespan or replicative lifespan in yeast. They also began with gene sets known to be downregulated upon treatment with the lifespan-extending TOR inhibitor rapamycin.

YBR283C was unique in being largely uncharacterized, downregulated upon rapamycin treatment and linked to both increased replicative lifespan and increased chronological lifespan upon deletion.

The authors show that YBR283C may act to negatively regulate mitochondrial function, in ways that are both dependent on and independent of the stress-responsive transcription factor Hap4, largely by looking at relative expression levels of relevant mitochondrial genes.

In a hard to fully interpret but well documented series of experiments the authors not that the two paralogues YBR283C and RMD9 (which have ~66% similarity) (a) have opposite effects when acting alone, and (b) appear to interact in that some phenotypes of ybr283c are dependent on RMD9.

A particularly interesting finding in light of the current literature and of the authors' strategy in identifying YBR283C is that changes in electron transport chain genes upon rapamycin treatment appear to be effected via YBR283C.<br /> Based on a series of experiments the authors move to conclude the existence of "a feedback loop between TORC1 and mitochondria (the TORC1-Mitochondria-TORC1 (TOMITO) signaling process) that regulates cellular aging processes."

Strengths

Overall, this study describes a great deal of new data from a large number of experiments, that shed light on the potential specific roles of YBR238C and its paralog RMD9 in aging in yeast, and also underscore the potential of an approach looking for "dark matter" such as uncharacterized genes when seining the increasing deluge of published datasets for new hypotheses to test. This work when revised will become a valuable addition to the field.

Weaknesses

A paralog of YBR283C, RMD9, also exists in the yeast genome. While the authors indicate that part of their interest in YBR283C lies in its uncharacterized nature, its paralogue, RMD9, is not uncharacterized but is named due to its phenotype of Required for Meiotic nuclear Division, which is not mentioned or discussed anywhere in the manuscript currently.

In the context of the current work, in addition to the cited Hillen, H.S et al. and Nouet C. et al, the authors might be very interested in the 2007 Genetics paper "Translation initiation in Saccharomyces cerevisiae mitochondria: functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase and novel protein Rmd9p" (PMID: 17194786), which does not appear to be cited or discussed in the current version of the manuscript.

Reviewer #2 (Public Review):

The effectors of cellular aging in yeast have not been fully elucidated. To address this, the authors curated gene expression studies to link genes influenced by rapamycin - a well-known mediator of longevity across model systems - to genes known to affect chronological and replicative lifespan (RLS) in yeast. Through their analyses, they find one gene, ybr238c, whose deletion increases both CLS and RLS upon deletion and that is downregulated by rapamycin. The authors follow up their cellular aging studies using CLS as a model throughout their study, demonstrating that deletion of ybr238c increases CLS across multiple yeast strains and through multiple assays. The authors also test the effects of YBR238C overexpression on lifespan and find the opposite effect, with overexpression yeast showing decreased survival relative to wild type cells, consistent with accelerated aging as the authors propose. The authors also note that ybr238c has a paralog, rmd9, whose deletion decreases CLS and seems to be epistatic to ybr238c, as a double ybr238c/rmd9 mutant has decreased CLS relative to a wild-type strain.

Collectively, the data presented by the authors convincingly demonstrate that ybr238c influences lifespan in a manner that is distinct from (and likely opposite to) rmd9. The authors then link the increased CLS in Δybr238c yeast to HAP4, a transcription factor that promotes mitochondrial biogenesis and oxidative phosphorylation. Through genetic studies, the authors suggest a model in which YBR238C negatively regulates HAP4 activity, and thus loss of HAP4 repression in Δybr238c yeast leads to elevated mitochondrial function. Notably, while the authors use various methods to test mitochondrial function, including the quantification of transcripts associated with oxidative phosphorylation, cellular ATP levels, and mtDNA, none of these fully test mitochondrial function. Thus, while the trends of these proxies are consistent with the model proposed by the authors, including data such as respirometry or assaying the activity of oxidative phosphorylation complexes would have bolstered these conclusions.

Finally, the authors tie the phenotypes of mitochondrial dysfunction caused by deletion of ybr238c to TORC1 signaling, as the gene is influenced by rapamycin. However, the data assaying mitochondrial function in these experiments, such as profiling the transcriptional changes in oxidative phosphorylation complexes or monitoring cellular ATP levels, do not directly measure mitochondrial function. Furthermore, many of the studies performed by the authors rely on genetic or pharmacological rescue of lifespan to establish the influence of YBR238C on TORC1 signaling and mitochondrial function. While valuable, these assays leave questions as to the molecular mechanisms by which YBR238C functions. As such, this manuscript establishes that ybr238c is rapamycin responsive and influences CLS, but the molecular mechanisms by which it affects mitochondrial activity and TORC1 signaling remain to be elucidated.

Reviewer #3 (Public Review):

Summary:<br /> Kobayashi et al identify MER21C as a common promoter of GPR1-AS/Liz in Euarchontoglires, which establishes a somatic DMR that controls ZFDB2 imprinting. In mice, MER21C appears to have diverged significantly from its primate counterparts and is no longer annotated as such.

Strengths:<br /> The authors used high-quality cross-species RNA-seq data to characterise GPR1-AS-like transcripts, which included generating new data in five different species. The association between MER21C/B elements and the promoter of GPR1-AS in most species is clear and convincing. The expression pattern of MER21C/B elements overall further supports their role in enabling correct temporal expression of GPR1-AS during embryonic development.

Weaknesses:<br /> A deeper comparison of syntenic regions to the GPR1-AS promoter could be performed to provide a clearer picture of how the MER21C/B element evolved. The use of alternative TE annotation software may also be helpful. These analyses would be particularly useful to drive home the conclusion that the mouse (Liz) promoter is derived from the same insertion.

Reviewer #1 (Public Review):

Summary:<br /> The study tests the conservation of imprinting of the ZBDF2 locus across mammals. ZDBF2 is known to be imprinted in mice, humans, and rats. The locus has a unique mechanism of imprinting: although imprinting is conferred by a germline DMR methylated in oocytes, the DMR is upstream to ZDBF2 (at GPR1) and monoallelic methylation of the gDMR does not persist beyond early developmental stages. Instead, a lncRNA (GPR1-AS, also known as Liz in mouse) initiating at the gDMR is expressed transiently in embryos and sets up a secondary DMR (by mechanisms not fully elucidated) that then confers monoallelic expression of ZDBF2 in somatic tissues.

In this study, the authors first interrogate existing placental RNA-seq datasets from multiple mammalian species, and detect GPR1-AS1 candidate transcripts in humans, baboons, macaques and mice, but not in about a dozen other mammals. Because of the varying depth, quality, and nature of these RNA-seq libraries, the ability to definitely detect the GPR1-AS1 lncRNA is not guaranteed; therefore, they generate their own deep, directional RNA-seq data from tissues/embryos from five species, finding evidence of GPR1-AS in rabbits and chimpanzees, but not bovine animals, pigs or opossums. From these surveys, the authors conclude that the lncRNA is present only in Euarchontoglires mammals. To test the association between GPR1-AS and ZDBF2 imprinting, they perform RT-PCR and sequencing in tissue from wallabies and cattle, finding biallelic expression of ZDBF2 in these species that also lack a detected GPR1-AS transcript. From inspection of the genomic location of the GPR1-AS first exon, the authors identify an overlap with a solo LTR of the MER21C retrotransposon family in those species in which the lncRNA is observed, except for some rodents, including mice. However, they do detect a degree of homology (46%) to the MER21C consensus at the first exon on Liz in mouse. Finally, the authors explore public RNA-seq datasets to show that GPR1-AS is expression transiently during human preimplantation development, an expression dynamic that would be consistent with the induction of monoallelic methylation of a somatic DMR at ZDBF2 and consequent monoallelic expression.

Strengths:<br /> -The analysis uncovers a novel mechanism by which a retrotransposon-derived LTR may be involved in genomic imprinting.<br /> -The genomic analysis is very well executed.<br /> -New directional and deeply-sequenced RNA-seq datasets from the placenta or the trophectoderm of five mammalian species and marsupial embryos, that will be of value to the community.

Weaknesses:<br /> Although the genomic analysis is very strong, the study remains entirely correlative. All of the data are descriptive, and much of the analysis is performed on RNA-seq and other datasets from the public domain; a small amount of primary data is generated by the authors.<br /> Evidence that the residual LTR in mouse is functionally relevant for Liz lncRNA expression is lacking.

Reviewer #2 (Public Review):

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

Strengths:<br /> (1) The authors convincingly demonstrate the existence of GPR1-AS orthologs in specific mammalian lineages using deeply sequenced, stranded, and paired-end RNA-seq libraries collected from diverse mammalian species.

Weaknesses:<br /> (1) The authors do not directly demonstrate imprinting of the ZDBF2 locus in rabbits and non-human primates, which would greatly strengthen their model linking ZDBF2 imprinting to transcription from MER21C.

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

Reviewer #1 (Public Review):

This work demonstrates a new technique to characterize the interaction between a crawling larva and the substrate on which it is crawling, at much higher temporal speed and spatial resolution than previously possible. While I have some questions about the interpretation of the data, both the demonstration of WARP microscopy to characterize small animal behavior and the discovery of new crawling-associated anatomical features and motor patterns make the paper worthy of attention.

I thank the authors for providing data underlying the figures. In these uncropped data sets, the deformation of the substrate due to the surface tension of an adhering water layer is visible. I would hope the authors would provide a subset of these images and some of the accompanying information (e.g. that the deformation of the gel due to the water layer cannot be accurately calculated due to too-rapid phase wrapping in the interferogram) as supplements to the text, to aid in interpretation and understanding of the data. It is also worth noting that in the data provided, under the larva, the integral of the stress on the gel is upward, despite the downward force exerted by the protopodia.

Future work using this exciting technique might address the role of surface tension and the balance of forces and might also produce direct evidence to show that the protopodia serve to "anchor" segments of the larva not in motion. Indeed, the most exciting aspect of this work is the number of new questions it both raises and provides a technological pathway towards resolving.

Reviewer #2 (Public Review):

The biology and dynamics is well-described. The ERISM and WARP methods are state-of-the-art. The most important new information is the highly accurate and detailed maps of displacement. The real achievements are the new locomotory dynamics uncovered with amazing displacement measurements. One key discovery is the broad but shallow anchoring of the posterior body when the anterior body undertakes a "head sweep". Another discovery is the tripod indentation at the tail at the beginning of peristalsis cycles. This paper describes the detailed dynamics of anchoring for the first time. Anchoring behavior now has to be included in the motor sequence for Drosophila larva locomotion in any comprehensive biomechanical or neural model.

Reviewer #1 (Public Review):

Understanding the ecology including the dietary ecology of enantiornithines is challenging by all means. This work explores the possible trophic diversity of the "opposite-bird" enantiornithines by referring to the body mass, jaw mechanical advantage, finite element analysis of the jaw bones, and morphometrics of the claws and skull of both fossil and extant avian species. By incorporation the dietary information of longipterygids and pengornithinds, the authors predicted a wide variety of foods for enantiornithine ancestors. This indicates the evolutionary successes of enantiornitine during Cretaceous is very likely to have been driven by the wide range of recipes. I believe this work represented the most comprehensive analysis of enantiornithines' diet and trophic diversity by far and the first systematic dietary analysis of bohaiornithids, though the analysis themselves are largely based on the indirect evidence including jaw bone morphologies and claw and skull morphometrics. Anyway, I believe the authors did most the paleontologists could do, and I do not know whether the conclusions could be further supported by incorporating some geochemical data, as most of the specimens the authors analyzed were recovered from a small geographic area. The results also indicate that the developmental trajectories of enantiornithines, at least for jaw bones, might also have been diverse to some extent in response to the diverse ecological niches they adapted. My only concern regarding the analysis is to what extent the conclusions are convincing by comparing specimens representing various ontogenetic stages. This concern has been addressed in the revised manuscript. I believe the authors have almost exhausted all available methods, and I congratulate the authors for the detailed study they conducted.

Reviewer #2 (Public Review):

Miller et al. take a variety of measurements and analytical techniques to assess the ecology of various species of the enantiornithine clade Bohaiornithidae. From this they suggest that the ancestral enantiornithine was a generalist and that the descendant clades occupied a breadth of niches similar to that of the radiation of derived birds after the K-Pg extinction.

Overall, I find the idea that enantiornithines had occupied a similar niche breadth to post-K-Pg derived birds to be a curious, thought-provoking proposal.

I am satisfied with the edits made by the authors and approve the revised version of the manuscript.

Reviewer #3 (Public Review):

Summary:<br /> The authors use several quantitative approaches to characterize the feeding ecologies of bohaiornithid enantiornithines, including allometric data, mechanical advantage and finite element analyses of the jaw, and morphometric analyses of the claws. The authors combine their results with data for other enantiornithines collected from the literature to shed new insight on the ecological evolution of Enantiornithes as a clade.

Although the authors have taken steps to improve their paper, I generally find improvements unsatisfying, especially regarding my comments.

My remaining concerns:

Teeth: My concern here is not whether having teeth limits available niche space compared to having a keratinous beak. Rather, my concern regards how exploitation of the same niche space might be differently reflected in parameter space between birds with teeth and birds with beaks. Can we reliably expect two species that both eat seeds to occupy the same parameter space if, for example, distribution of stress/strain is across a series of teeth vs. across a more uniform beak? In this manuscript, the authors are clearly making this assumption, but that assumption is not made explicit, let alone justified. The authors should discuss this.

Cranial kinesis: As with teeth, my concern here regards our ability to compare data between birds with and without a flexible beak to mitigate forces when foraging. I appreciate that the functional complexity of the kinetic neognath skull precludes our ability to account for it in analyses such as these, but when comparisons are made using these analyses *specifically among neognaths*, we can reliably assume that we are comparing like to like - that is, we can assume that both have kinetic skulls, and so kinesis is reflected similarly in the data for each bird. Similarly, even if a comparison between two neognaths focuses exclusively on the mandible - in which cranial kinesis is not directly reflected - we can assume that those mandibles serve as comparisons between functionally similar systems. However, we cannot necessarily make those same assumptions when comparing the kinetic skull of a neognath to the akinetic skull of an enantiornithine. Indeed, even when focusing just on the mandible, can we reliably assume that data collected from an akinetic enantiornithine reflect the same comparative context as data collected from kinetic neognaths? I appreciate that the authors added a call for better functional understanding of bird cranial kinesis - a call I enthusiastically endorse - but the authors should still discuss how that current lack of understanding impacts interpretations of the comparisons they draw.

Finally, I still find the discussion to be overly long and lacking clear focus and organization. I again urge the authors to minimally consider adding subheadings to better allow the reader to follow the flow of ideas, and I second Reviewer #1's suggestion to add a "Conclusions" section.

Reviewer #1 (Public Review):

In this work, the authors set out to ask whether the MYRF family of transcription factors, represented by myrf-1 and myrf-2 in C. elegans, have a role in the temporally controlled expression of the miRNA lin-4. The precisely timed onset of lin-4 expression in the late L1 stage is known to be a critical step in the developmental timing ("heterochronic") pathway, allowing worms to move from the L1 to the L2 stage of development. Despite the importance of this step of the pathway, the mechanisms that control the onset of lin-4 expression are not well understood.

Overall, the paper provides convincing evidence that MYRF factors have a role in the regulation of lin-4 expression. Using state-of-the-art techniques (knock-in reporters and conditional alleles), the authors show that MYRF factors are essential for lin-4 activation and act cell-autonomously. While there are some minor concerns about the use of unusual gain-of-function alleles, these are mitigated by consistent results using other approaches. The authors also provide evidence that MYRF factors activate lin-4 by directly activating its promoter. While their results are certainly consistent with this possibility, they rely on indirect measurements and are therefore not definitive. Further experiments will be necessary to determine whether this model is accurate.

Some details about the relative roles of the two C. elegans MYRF factors, myrf-1 and myrf-2, remain unclear. myrf-1 clearly seems to play the more important role lin-4 activation and the regulation of developmentally timed processes. However, there are numerous hints that myrf-2 may act in the opposite direction, either by inhibiting myrf-1 itself or its ability to activate its targets. Further work will be necessary to understand the genetic and mechanistic relationships between these two genes.

Overall, the findings in this paper are convincing, and the results will be of interest to a wide range of developmental biologists.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, the authors examine how temporal expression of the lin-4 microRNA is transcriptionally regulated.

In the revised manuscript, the authors have suitably addressed my original concerns.

Aims achieved: The aims of the work are now achieved.

Impact: This study shows that a single transcription factor (MYRF-1) is important for the regulation of multiple microRNAs that are expressed early in development to control developmental timing.

Reviewer #2 (Public Review):

The authors suggest that the African trypanosome endomembrane system has unusual organisation, in that the entire system is a single reticulated structure. It is not clear if this is thought to extend to the lysosome or MVB. There is also a suggestion that this unusual morphology serves as a trans-(post)Golgi network rather than the more canonical arrangement.

The updated manuscript is significantly improved. I remain at slight odds with the author's push for the lack of generality as important, and the new cell biology that we have been on the verge of for decades. However, that is a scholarly issue and is not grounds for any further revision of the present manuscript.

Reviewer #3 (Public Review):

Summary:<br /> A key element in the ability of trypanosomes to evade the mammalian host's immune system is its high rate of endocytosis. This rapid turnover of its surface enables the trypanosome to 'clean' its surface removing antibodies and other immune effectors that are subsequently degraded. The high rate of endocytosis is likely reflected in the organisation of the endosomal system in these parasites. Here, Link et al., sought to address this question using a range of light and three-dimensional electron microscopy approaches to define the endosomal organisation in this parasite.

Before this study, the vast majority of our information about the make-up of the trypanosome endosomal system was from thin section electron microscopy and immunofluorescence studies, which did not provide the necessary resolution and 3D information to address this issue. Therefore, it was not known how the different structures observed by EM were related. Link et al., have taken advantage of the advances in technology and used an impressive combination of approaches at the LM and EM level to study the endosomal system in these parasites. This innovative combination has now shown the interconnected-ness of this network and demonstrated that there are no 'classical' compartments within the endosomal system, with instead different regions of the network enriched in different protein markers (Rab5a, Rab7, Rab11). Overall, the authors have achieved their aims, with results supporting their conclusions.

This is a well written manuscript in which the authors use an impressive range of approaches to address the organisation of the endosomal system. The authors have clearly demonstrated that trypanosomes have a large interconnected endosomal network, without defined compartments and instead shows enrichment for specific Rabs within this network. I appreciate their inclusion of how they used a range of different light microscopy approaches even though for instance the dSTORM approach did not turn out to be as effective as hoped.

The methodological impact of this work has the potential to be large, as the authors have introduced a range of advanced EM techniques for the study of trypanosomes. Moreover, the study of fundamental biological processes such as endosomal trafficking in divergent eukaryotes is important to define the limits within which this process operates.

Reviewer #1 (Public Review):

Summary:<br /> This important study nicely integrates a breadth of experimental and computational data to address fundamental aspects of RNA methylation by an important for biology and health RNA methyltransferases (MTases). 



Strengths: The authors offer compelling and strong evidence, based on carefully performed with appropriate and well-established techniques to shed light on aspects of the methyl transfer mechanism of the methyltransferase-like protein 3 (METTL3), which is part of the methyltransferase-like proteins 3 & 14 (METTL3-14) complex. 


There are no weaknesses that we identified in the revised version.

Reviewer #2 (Public Review):

Summary:<br /> Caflisch and coworkers investigate the methyltransferase activity of the complex of methyltransferase-like proteins 3 and 14 (METTL3-14). To obtain an high resolution description of the complete catalytic cycle they have carefully designed a combination of experiments and simulations. Starting from the identification of bisubstrate analogues (BAs) as binder to stabilise a putative transition state of the reaction they have determined multiple crystal structures and validated relevant interactions by mutagenesis and enzymatic assays.

Using the resolved structure and classical MD simulations they obtained a kinetic picture of the binding and release of the substrates. Of note, they accumulate very good statistics on these processes using 16 simulation replicates over a time scale of 500 ns. To compare the time scale of the release of the products with that of the catalytic step they performed state-of-the-art QM/MM free energy calculations (testing multiple levels of theory) and obtain a free energy barrier that indicates how the release of the product is slower than the catalytic step.

Strengths:<br /> All the work proceeds through clear hypothesis testing based on a combination of literature and new results. Eventually, this allows them to present in Figure 10 a detailed step-by-step description of the catalytic cycle. The work is very well crafted and executed.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Coberski et al describes a combined experimental and computational study aimed to shed light on the catalytic mechanism in a methyltransferase that transfers a methyl group from S-adenosylmethionine (SAM) to a substrate adenosine to form N6-methyladenosine (m6A).

Strengths:<br /> The authors determine crystal structures in complex with so-called bi-substrate analogs that can bridge across the SAM and adenosine binding sites and mimic a transition state or intermediate of the methyl-transfer reaction. The crystal structures suggest dynamical motions of the substrate(s) that are examined further using classical MD simulations. The authors then use QM/MM calculations to study the methyl-transfer process. Together with biochemical assays of ligand/substrate binding and enzyme turnover, the authors use this information to suggest what the key steps are in the catalytic cycle. The manuscript is in most places easy to read.

Weaknesses:<br /> After revising the manuscript, there are few weaknesses beyond those listed in the paper.

Reviewer #1 (Public Review):

Summary:<br /> This paper provides strong evidence for the roles of JH in an ametabolous insect species. In particular, it demonstrates that:<br /> • JH shifts embryogenesis from a growth mode to a differentiation mode and is responsible for terminal differentiation during embryogenesis. This, and other JH roles, are first suggested as correlations, based on the timing of JH peaks, but then experimentally demonstrated using JH antagonists and rescue thereof with JH mimic. This is a robust approach and the experimental results are very convincing.<br /> • JH redirects ecdysone-induced molting to direct formation of a more mature cuticle<br /> • Kr-h1 is downstream of JH in Thermobia, as it is in other insects, and is a likely mediator of many JH effects<br /> • The results support the proposed model that an ancestral role of JH in promoting and maintaining differentiation was coopted during insect radiations to drive the evolution of metamorphosis. However, alternate evolutionary scenarios should also be considered.

Strengths:

Overall, this is a beautiful, in-depth student. The paper is well-written and clear. The background places the work in a broad context and shows its importance in understanding fundamental questions about insect biology. The researchers are leaders in the field, and a strength of this manuscript is their use of a variety of different approaches (enzymatic assays, gene expression, agonists & antagonists, analysis of morphology using different types of microscopy and detection, and more) to attack their research questions. The experimental data is clearly presented and carefully executed with appropriate controls and attention to detail. The 'multi-pronged' approach provides support for the conclusions from different angles, strengthening conclusions. In sum, the data presented are convincing and the conclusions about experimental outcomes are well-justified based on the results obtained.

Weaknesses:

This paper provides more detail than is likely needed for readers outside the field but also provides sufficient depth for those in the field. This is both a strength and a weakness. I would suggest the authors shorten some aspects of their text to make it more accessible to a broader audience. In particular, the discussion is very long and accompanied by two model figures. The discussion could be tightened up and much of the text used for a separate review article (perhaps along with Figure 11) that would bring more attention to the proposed evolution of JH roles.

Reviewer #2 (Public Review):

The authors have studied in detail the embryogenesis of the ametabolan insect Thermobia domestica. They have also measured the levels of the two most important hormones in insect development: juvenile hormone (JH) and ecdysteroids. The work then focuses on JH, whose occurrence concentrates in the final part (between 70 and 100%) of embryo development. Then, the authors used a precocene compound (7-ethoxyprecocene, or 7EP) to destroy the JH producing tissues in the embryo of the firebrat T. domestica, which allowed to unveil that this hormone is critically involved in the last steps of embryogenesis. The 7EP-treated embryos failed to resorb the extraembryonic fluid and did not hatch. More detailed observations showed that processes like the maturational growth of the eye, the lengthening of the foregut and posterior displacement of the midgut, and the detachment of the E2 cuticle, were impaired after the 7EP treatment. Importantly, a treatment with a JH mimic subsequent to the 7EP treatment restored the correct maturation of both the eye and the gut. It is worth noting that the timing of JH mimic application was essential for correcting the defects triggered by the treatment with 7EP.

This is a relevant result in itself since the role of JH in insect embryogenesis is a controversial topic. It seems to have an important role in hemimetabolan embryogenesis, but not so much in holometabolans. Intriguingly, it appears important for hatching, an observation made in hemimetabolan and in holometabolan embryos. Knowing that this role was already present in ametabolans is relevant from an evolutionary point of view, and knowing exactly why embryos do not hatch in the absence of JH, is relevant from the point of view of developmental biology.

Then, the authors describe a series of experiments applying the JH mimic in early embryogenesis, before the natural peak of JH occurs, and its effects on embryo development. Observations were made under different doses of JHm, and under different temporal windows of treatment. Higher doses triggered more severe effects, as expected, and different windows of application produced different effects. The most used combination was 1 ng JHm applied 1.5 days AEL, checking the effects 3 days later. Of note, 1.5 days AEL is about 15% embryonic development, whereas the natural peak of JH occurs around 85% embryonic development. In general, the ectopic application of JHm triggered a diversity of effects, generally leading to an arrest of development. Intriguingly, however, a number of embryos treated with 1 ng of JHm at 1.5 days AEL showed a precocious formation of myofibrils in the longitudinal muscles. Also, a number of embryos treated in the same way showed enhanced chitin deposition in the E1 procuticle and showed an advancement of at least a day in the deposition of the E2 cuticle.

While the experiments and observations are done with great care and are very exhaustive, I am not sure that the results reveal genuine JH functions. The effects triggered by a significant pulse of ectopic JHm when the embryo is 15% of the development will depend on the context: the transcriptome existing at that time, especially the cocktail of transcription factors. This explains why different application times produce different effects. This also explains why the timing of JHm application was essential for correcting the effects of 7EP treatment. In this reasoning, we must consider that the context at 85% development, when the JH peaks in natural conditions and plays its genuine functions, must be very different from the context at 15% development, when the JHm was applied in most of the experiments. In summary, I believe that the observations after the application of JHm reveal effects of the ectopic JHm, but not necessarily functions of the JH. If so, then the subsequent inferences made from the premise that these ectopic treatments with JHm revealed JH functions are uncertain and should be interpreted with caution.

Those inferences affect not only the "JH and the progressive nature of embryonic molts" section, but also, the "Modifications in JH function during the evolution of hemimetabolous and holometabolous life histories" section, and the entire "Discussion". In addition to inferences built on uncertain functions, the sections mentioned, especially the Discussion, I think suffer from too many poorly justified speculations. I love speculation in science, it is necessary and fruitful. But it must be practiced within limits of reasonableness, especially when expressed in a formal journal.

Finally, In the section "Modifications in JH function during the evolution of hemimetabolous and holometabolous life", it is not clear the bridge that connects the observations on the embryo of Thermobia and the evolution of modified life cycles, hemimetabolan and holometabolan.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors use inhibitors and mimetics of juvenile hormone (JH) to demonstrate that JH has a key role in late embryonic development in Thermobia, specifically in gut and eye development but also resorption of the extraembryonic fluid and hatching. They then exogenously apply JH early in development (when it is not normally present) to examine the biological effects of JH at these stages. This causes a plethora of defects including developmental arrest, deposition of chitin, limb development, and enhanced muscle differentiation. The authors interpret these early effects on development as JH being important for the shift from morphogenetic growth to differentiation - a role that they speculate may have facilitated the evolution of metamorphosis (hemi- and holo-metaboly). This paper will be of interest to insect evo-devo researchers, particularly those with interests in the evolution of metamorphosis.

Strengths:

The experiments are generally conducted very well with appropriate controls and the authors have included a very detailed analysis of the phenotypes.<br /> The manuscript significantly advances our understanding of Thermobia development and the role of JH in Thermobia development.<br /> The authors interpret this data to present some hypotheses regarding the role of JH in the evolution of metamorphosis, some aspects of which can be addressed by future studies.

Weaknesses:

The results are based on using inhibitors and mimetics of JH and there was no attempt to discern immediate effects of JH from downstream effects. The authors show, for instance, that the transcription of myoglianin is responsive to JH levels, it would have been interesting to see if any of the phenotypic effects are due to myoglianin upregulation/suppression (using RNAi for example). These kinds of experiments will be necessary to fully work out if and how the JH regulatory network has been co-opted into metamorphosis.

The results generally support the authors' conclusions. However, the discussion contains a lot of speculation and some far-reaching conclusions are made about the role of JH and how it became co-opted into controlling metamorphosis. There are some interesting hypotheses presented and the author's speculations are consistent with the data presented. However, it is difficult to make evolutionary inferences from a single data point as although Thermobia is a basally branching insect, the lineage giving rise to Thermobia diverged from the lineages giving rise to the holo- and hemimetabolous insects approx.. 400 mya and it is possible that the effects of JH seen in Thermobia reflect lineage-specific effects rather than the 'ancestral state'. The authors ignore the possibility that there has been substantial rewiring of the networks that are JH responsive across these 400 my. I would encourage the authors to temper some of the discussion of these hypotheses and include some of the limitations of their inferences regarding the role of JH in the evolution of metamorphosis in their discussion.

Reviewer #1 (Public Review):

The basic approach is that the authors first train an RBM on all MprF sequences, and then use this analysis to identify a subset of the family that catalyzes the addition of amino acids to PG. Then a second RBM is trained on this subset.

In the initial RBM training a particular hidden unit is identified that has a sparse and bimodal activation in response to the input sequences. The contribution of individual resides is shown in Figure 3c, which highlights one of the strengths of this RBM implementation - it is interpretable in a physically meaningful way. However, there are several decisions here, the justification of which is not entirely clear.

i) Some of the residues in Fig 3c are stated as "relevant" for aminoacylated PG production. But is this the only such hidden unit? Or are there others that are sparse, bimodal, and involve "relevant" AA?<br /> ii) In order to filter the sequences for the second stage, only those that produce an activation over +2.0 in this particular hidden unit were taken. How was this choice made?<br /> iii) How many sequences are in the set before and after this filtering? On the basis of the strength of the results that follow I expect that there are good reasons for these choices, but they should be more carefully discussed.<br /> iv) Do the authors think that this gets all of the aminoacylated PG enzymes? Or are some missed?

The authors show that they can classify members of the family by training a second RBM on the filtered sequences. They do this by identifying two hidden unit activations in particular (Figure 5b) which seem to be useful for determining lipid substrate specificity, and they test several variants that obtain different responses of these two hidden units by experimentally determining what lipids they produce (Table 2). However, some similar criticisms from the last point occur here as well, namely the selection of which weights should be used to classify the enzymes' function. Again the approach is to identify hidden unit activations that are sparse (with respect to the input sequence), have a high overall magnitude, and "involve residues which could be plausibly linked to the lipid binding specificity."

i) Two hidden units are identified as useful for classification, but how many candidates are there that pass the first two criteria? Indeed, how many hidden units are there?<br /> ii) The criterion "involve residues which could be plausibly linked to the lipid binding specificity" is again vague. Do all of the other candidate hidden units *not* involve significant contributions from substrate-binding residues? Maybe one of the other units does a better job of discriminating substrate specificity. (As indicated in Figure 8, there are examples of enzymes that confound the proposed classification.) Why combine the activations of two units for the classification, instead of 1 or 3 or...?

Reviewer #2 (Public Review):

In "Lipid discovery enabled by sequence statistics and machine learning" Christensen et al. address an important question: how can bacteria modify lipid charges to produce cationic lipids, prone to confer resistance to cationic antibiotics? One of the enzymes involved in this process is MprF, which can, through the transfer of amino acids, in particular, lysine, from charged tRNA modify the charge of anionic membrane phospholipid from negative to positive. Recent works have shown that MprF can also modify another substrate, glycolipid glucosyl-diacylglycerol, which is neutral. These findings immediately raise two questions: what are the determinants in the MrpF sequence controlling the lipid substrates it can modify? Are there other substrates for MrpF, so far unknown?

Christensen et al. address both of these questions in an elegant way, combining sequence analysis with machine-learning methods and experimental characterisation of the enzymatic products through mass spectrometry. Using restricted Boltzmann machines (RBM), an unsupervised architecture extracting statistical features from the sequence data, they identify putative amino-acid motifs along the MprF sequences possibly related to the substrate identity, select some bacterial species whose wild-type sequence contains those motifs, and validate the biological role of the motifs by identifying the produced lipids. Remarkably, with this approach, the authors find a novel cationic lipid with two glucosyl groups.

Besides these new results on MrpF and its operation, the present work is appealing, as it shows that the functional characterisation of a very small number of proteins (here, three!) combined with the guided classification of homologous sequence data with appropriate machine-learning methods can lead to the discovery of new functionalities.

Reviewer #3 (Public Review):

Summary:<br /> After the previous identification that the Streptococcus agalactiae MprF enzyme can synthesize also lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), besides the already known lysyl-phosphatidylglycerol (Lys-PG), the authors aim for the current manuscript was to investigate the molecular determinants of MprF lipid substrate specificity in a variety of bacterial species.

Strengths:<br /> - In general, the manuscript is well constructed and easy to follow, especially taking into account the multidisciplinary aspect of it (computational machine learning combined with lipid biology).<br /> -The added value of the Restricted Boltzmann machines (RBM) approach, in comparison to standard computational pairwise sequence statistics, becomes evident. This is exemplified by a successful, although not perfect, classification and categorization of MprF activity.<br /> - The MS analysis (monoisotopic mass, plus fragmentation pattern), convincingly shows the identification of a novel lipid species Lys-Glc2-DAG.

Weaknesses:<br /> -In many of the analyzed strains, the presence of the lipid species Lys-PG, Lys-Glc-DAG, and Lys-Glc2-DAG is correlated to the presence of the MprF enzyme(s), but one should keep in mind that a multitude of other membrane proteins are present that in theory could be involved in the synthesis as well. Therefore, there is no direct evidence that the MprF enzymes are linked to the synthesis of these lipid species. Although, it is unlikely that other enzymes are involved, this weakens the connection between the observed lipids and the type of MprF.<br /> -Related to this, in a few cases MprF activity is tested, but the manuscript does not contain any information on protein expression levels. Heterologous expression of membrane proteins is in general challenging and due to various reasons, proteins end up not being expressed at all. As an example, the absence of activity for the E. faecalis MprF1 and E. faecium MprF2 could very well be explained by the entire absence of the protein.

Overall, the authors largely achieved their goals, as the applied RBM approach led to specific sequence determinants in MprF enzymes that could categorize the specificity of these enzymes. The experimental data could largely confirm this categorization, although a stronger connection between synthesized lipids and enzyme activity would have further strengthened the observations.

The work now focuses only on MprF enzymes, but could in theory be expanded to other categories of lipid-synthesizing enzymes. In other words, the RBM approach could have an impact on the lipid synthesis field, if it would be a tool that is easily applicable. Moreover, the lipids synthesized by MprF (Lys-PG, but also other cationic lipids) play an important role in bacterial resistance against certain antibiotics.

Reviewer #1 (Public Review):

This paper describes a new method for sequence-based remote homology detection. Such methods are essential for the annotation of uncharacterized proteins and for studies of protein evolution.

The main strength and novelty of the proposed approach lies in the idea of combining state-of-the-art sequence-based (HHpred and HMMER) and structure-based (Foldseek) homology detection methods with protein language models (the ESM2 model was used). The authors show that high-dimensional, information-rich representations extracted from the ESM2 model can be efficiently combined with the aforementioned tools.

The benchmarking of the new approach is convincing and shows that it is suitable for homology detection at very low sequence similarity. The method is also fast because it does not require the computation of multiple sequence alignments for profile calculation or structure prediction.

Overall, this is an interesting and useful paper that proposes an alternative direction for the problem of distant homology detection.

Reviewer #1 (Public Review):

The current manuscript focusses on the adenine phosphoribosyltransferase (Aprt) and how the lack of its function affects nervous system function. It puts it into the context of Lesch-Nyhan disease, a rare hereditary disease linked to hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Since HGPRT appears absent in Drosophila, the study focusses initially on Aprt and shows that aprt mutants have a decreased life-span and altered uric acid levels (the latter can be attenuated by allopurinol treatment). Moreover, aprt mutants show defects in locomotor reactivity behaviors. A comparable phenotype can be observed when specifically knocking down aprt in dopaminergic cells (in an adult-specific fashion). Interestingly, also glia-specific knock-down caused a similar behavioral defect, which could not be restored when re-expressing UAS-aprt, while neuronal re-expression did restore the mutant phenotype. Moreover, mutants, pan-neuronal and glia-specific RNAi for aprt caused sleep-defects. Based on immunostainings Dopamine levels are increased; UPLC shows that adenosine levels are reduced and PCR showed in increase of Ent2 levels are increased (but not AdoR). Moreover, aprt mutants display seizure-like behaviros, which can be partly restored by purine feeding (adenosine and N6-methyladenosine). Finally, expression of the human HGPRT also causes locomotor defects.

The authors provide a wide range of genetic experimental data to assess behavior and some molecular assessment on how the defects may emerge. It is clearly written, and the arguments follow the experimental evidence that is provided.

The findings provide a new example of how manipulating specific genes in the fruit fly allow the study of fundamental molecular processes that are linked to a human disease.

Reviewer #2 (Public Review):

The manuscript by Petitgas et al demonstrates that loss of function for the only enzyme responsible for the purine salvage pathway in fruit-flies reproduces the metabolic and neurologic phenotypes of human patients with Lesch-Nyhan disease (LND). LND is caused by mutations in the enzyme HGPRT, but this enzyme does not exist in fruit-flies, which instead only have Aprt for purine recycling. They demonstrate that mutants lacking the Aprt enzyme accumulate uric acid, which like in humans can be rescued by feeding flies allopurinol, and have decreased longevity, locomotion and sleep impairments and seizures, with striking resemblance to HGPRT loss of function in humans. They demonstrate that both loss of function throughout development or specifically in the adult ubiquitously or in all neurons, or dopaminergic neurons, mushroom body neurons or glia, can reproduce the phenotypes (although knock-down in glia does not affect sleep). They show that the phenotypes can be rescued by over-expressing a wild-type form of the Aprt gene in neurons. They identify a decrease in adenosine levels as the cause underlying these phenotypes, as adenosine is a neurotransmitter functioning via the purinergic adenosine receptor in neurons. In fact, feeding flies throughout development and in the adult with either adenosine or m6A could prevent seizures. They also demonstrate that loss of adenosine caused a secondary up-regulation of ENT nucleoside transporters and of dopamine levels, that could explain the phenotypes of decreased sleep and hyperactivity and night. Finally, they provide the remarkable finding that over-expression of the human mutant HGPRT gene but not its wild-type form in neurons impaired locomotion and induced seizures. This means that the human mutant enzyme does not simply lack enzymatic activity, but it is toxic to neurons in some gain-of-function form. Altogether, these are very important and fundamental findings that convincingly demonstrate the establishment of a Drosophila model for the scientific community to investigate LND, to carry out drug testing screens and find cures.

The authors have dealt with my concerns satisfactorily and have explained the instances in which resolving experimentally the criticisms raised would require a work effort well beyond the scope of a revision for this manuscript.

Reviewer #3 (Public Review):

The revised study provides better evidence to suggest that loss of Aprt activity in Drosophila provides a model for the loss of HGPRT activity in humans, which is causative for LND. Analysis of Drosophila Aprt mutations and RNAi-mediated knockdown reveals similar phenotypes to LND, particularly neurological defects, reduced nighttime sleep, and potentially seizures. LND is currently resistant to treatments and screening of a limited number of compounds in Drosophila has not identified a compound that can reduce all of the associated phenotypes. It is appropriate, therefore, that claims to have developed a clinically exploitable model for human LND have been toned down. Future drug screening may well prove profitable, but currently the evidence that Drosophila Aprt will be a suitable model for LND remains speculative.

The second approach adopted is to express a 'humanised mutated' form of HGPRT in Drosophila, which holds more promise for the development of a pharmacological screen. In particular, the locomotor defect is recapitulated but the seizure-like activity, whilst reported as being recapitulated, is debatable. A recovery time of 2.3 seconds is very much less than timings for typical seizure mutants. Nevertheless, the SING behaviour could be sufficient to screen against. However, this is not explored. With respect the short seizure duration, the authors cite similar findings for porin loss of function, but the cited study similarly did not employ anti-seizure drug exposure to validate that this phenotype is seizure related.

In summary, this is a largely descriptive study reporting the behavioural effects of an Aprt loss-of-function mutation. RNAi KD and rescue expression studies suggest that a mix of neuronal (particularly dopaminergic and possibly adenosinergic signalling pathways) and glia are involved in the behavioural phenotypes affecting locomotion, sleep and seizure. There remains insufficient evidence to have full confidence that the Arpt fly model will prove valuable for understanding / treating LND.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Clark et. al. used electrophysiology approaches to measure MEC neuron activity while mice performed spatial memory tasks in one-dimensional virtual tracks, where the mice must stop in a specific reward zone for a reward. The authors identified that grid cell activity could either be anchored to the track reference frame ('task-anchored') or can maintain a periodic firing pattern independent of the track reference frame ('task-independent'). They found that in the task that requires path integration, good task performance is specifically associated with task-anchored grid cell activity.

Strength:<br /> This study took advantage of the variation in neural activity and navigation task behaviors to answer an important question: how grid cell activity is associated with performance of spatial tasks. The mice performed individual trials where they must stop in a specific reward zone for a reward. Individual behavioral sessions could include three types of trials: (1) a visual cue at the reward location (beaconed trials), (2) no cue at the reward location (non-beaconed trials), and (3) no cue and no reward regardless of stopping (probe trials). The authors found that, interestingly, grid cell activity pattern could be anchored to task reference frame or maintain a periodic pattern independent of the reference frame. The anchoring of activity patterns could switch within a behavioral session. On the other hand, spatial firing of non-grid cells was either coherent with the grid population or was stably anchored to the task reference frame. Combining grid cell activity feature with task behaviors, they uncovered an association between the task-anchoring of grid cell activity with good performance in spatial navigation tasks that requires path integration (non-beaconed and probe trials). This work suggests the contribution of grid firing to path integration-dependent navigation.

Weakness:<br /> It would be interesting to find out that on the trial-by-trial basis, whether the activity anchoring switched first, or the task behaviors altered first, or whether they happened within the same trial. This will potentially determine whether the encoding is causal for the behavior, or the other way around. However, based the authors explanation, their experimental design lacks sufficient statistical power to address the timing of mode switches within a trial, because task mode switching is relatively infrequent (so the n for switching is low) and only a subset of trials are uncued (making the relevant n even lower), while at a trial level the behavioral outcome is variable (increasing the required n for adequate power).

In addition, the authors reported that the activity anchoring of some non-grid cells coherently switched with grid cells, while others do not. They propose that the MEC implement multiple coding schemes. However, it is unclear whether and how the coding scheme is associated with behavior. It would be interesting to further investigate this question.

Reviewer #2 (Public Review):

Clark and Nolan's study aims to test whether the stability of grid cell firing fields is associated with better spatial behavior performance on a virtual task. Mice were trained to stop at a rewarded location along a virtual linear track. The rewarded location could be marked by distinct visual stimuli or be unmarked. When the rewarded location was unmarked, the animal had to estimate its distance run from the beginning of the trial to know where to stop. When the mouse reached the end of the virtual track, it was teleported back to the start of the virtual track.

The authors found that grid cells could fire in at least two modes. In the "task-anchored" mode, grid firing fields had stable positions relative to the virtual track. In the "task-independent" mode, grid fields were decoupled from the virtual cues and appeared to be located as a function of distance run on the track. Importantly, on trials in which the rewarded location was unmarked, the behavioral performance of mice was better when grid cells fired in the "task-anchored" mode. When a unique visual cue marked the reward location, navigation performance was not correlated with the grid cells' firing mode.

This study is very timely as there is a pressing need to identify/delimit the contribution of grid cells to spatial behaviors. More studies are needed in which grid cell activity is linked to navigational abilities. The link proposed by Clark and Nolan between "task-anchored" coding by grid cells and navigational performance is a significant step toward better understanding how grid cell activity might support behavioral behavior. The results also highlight that some forms of navigation (approaching a location marked by a visual cue) might be less dependent on the anchoring of grid cells.

It should be noted that the study by Clark and Nolan is correlative. Therefore, the effect of selective manipulations of grid cell activity on the virtual task will be needed to evaluate whether the activity of grid cells is causally linked to the behavioral performance on this task. A previous study by the same research group showed that inactivating the synaptic output of stellate cells of the medial entorhinal cortex affected mice's performance of the same virtual task (Tennant et al., 2018). Although this manipulation likely affects non-grid cells, it is still one of the most selective manipulations of grid cells that are currently available.

It is interesting to consider how grid cells remain anchored to virtual cues. Recent work shows that grid cell activity spans the surface of a torus (Gardner et al., 2022). A run on the track can be mapped to a trajectory on the torus. Assuming that grid cell activity is updated primarily from self-motion cues on the track and that the grid cell period is unlikely to be an integer of the virtual track length, having stable firing fields on the virtual track likely requires a resetting mechanism taking place on each trial. During this resetting event, the active location on the torus is likely to jump to a new toroidal location, independently of self-motion cues. Future studies in which large numbers of grid cells are recorded could pinpoint at which moment such resetting event occurs on each trial.

Reviewer #1 (Public Review):

Summary:<br /> This paper presents a compelling and comprehensive study of decision-making under uncertainty. It addresses a fundamental distinction between belief-based (cognitive neuroscience) formulations of choice behaviour with reward-based (behavioural psychology) accounts. Specifically, it asks whether active inference provides a better account of planning and decision-making, relative to reinforcement learning. To do this, the authors use a simple but elegant paradigm that includes choices about whether to seek both information and rewards. They then assess the evidence for active inference and reinforcement learning models of choice behaviour, respectively. After demonstrating that active inference provides a better explanation of behavioural responses, the neuronal correlates of epistemic and instrumental value (under an optimised active inference model) are characterised using EEG. Significant neuronal correlates of both kinds of value were found in sensor and source space. The source space correlates are then discussed sensibly, in relation to the existing literature on the functional anatomy of perceptual and instrumental decision-making under uncertainty.

Strengths:<br /> The strengths of this work rest upon the theoretical underpinnings and careful deconstruction of the various determinants of choice behaviour using active inference. A particular strength here is that the experimental paradigm is designed carefully to elicit both information-seeking and reward-seeking behaviour; where the information-seeking is itself separated into resolving uncertainty about the context (i.e., latent states) and the contingencies (i.e., latent parameters), under which choices are made. In other words, the paradigm - and its subsequent modelling - addresses both inference and learning as necessary belief and knowledge-updating processes that underwrite decisions.

The authors were then able to model belief updating using active inference and then look for the neuronal correlates of the implicit planning or policy selection. This speaks to a further strength of this study; it provides some construct validity for the modelling of belief updating and decision-making; in terms of the functional anatomy as revealed by EEG. Empirically, the source space analysis of the neuronal correlates licences some discussion of functional specialisation and integration at various stages in the choices and decision-making.

In short, the strengths of this work rest upon a (first) principles account of decision-making under uncertainty in terms of belief updating that allows them to model or fit choice behaviour in terms of Bayesian belief updating - and then use relatively state-of-the-art source reconstruction to examine the neuronal correlates of the implicit cognitive processing.

Weaknesses:<br /> The main weaknesses of this report lies in the communication of the ideas and procedures. Although the language is generally excellent, there are some grammatical lapses that make the text difficult to read. More importantly, the authors are not consistent in their use of some terms; for example, uncertainty and information gain are sometimes conflated in a way that might confuse readers. Furthermore, the descriptions of the modelling and data analysis are incomplete. These shortcomings could be addressed in the following way.

First, it would be useful to unpack the various interpretations of information and goal-seeking offered in the (active inference) framework examined in this study. For example, it will be good to include the following paragraph:

"In contrast to behaviourist approaches to planning and decision-making, active inference formulates the requisite cognitive processing in terms of belief updating in which choices are made based upon their expected free energy. Expected free energy can be regarded as a universal objective function, specifying the relative likelihood of alternative choices. In brief, expected free energy can be regarded as the surprise expected following some action, where the expected surprise comes in two flavours. First, the expected surprise is uncertainty, which means that policies with a low expected free energy resolve uncertainty and promote information seeking. However, one can also minimise expected surprise by avoiding surprising, aversive outcomes. This leads to goal-seeking behaviour, where the goals can be regarded as prior preferences or rewarding outcomes.

Technically, expected free energy can be expressed in terms of risk plus ambiguity - or rearranged to be expressed in terms of expected information gain plus expected value, where value corresponds to (log) prior preferences. We will refer to both decompositions in what follows; noting that both decompositions accommodate information and goal-seeking imperatives. That is, resolving ambiguity and maximising information gain have epistemic value, while minimising risk or maximising expected value have pragmatic or instrumental value. These two kinds of values are sometimes referred to in terms of intrinsic and extrinsic value, respectively [1-4]."

The description of the modelling of choice behaviour needs to be unpacked and motivated more carefully. Perhaps along the following lines:

"To assess the evidence for active inference over reinforcement learning, we fit active inference and reinforcement learning models to the choice behaviour of each subject. Effectively, this involved optimising the free parameters of active inference and reinforcement learning models to maximise the likelihood of empirical choices. The resulting (marginal) likelihood was then used as the evidence for each model. The free parameters for the active inference model scaled the contribution of the three terms that constitute the expected free energy (in Equation 6). These coefficients can be regarded as precisions that characterise each subjects' prior beliefs about contingencies and rewards. For example, increasing the precision or the epistemic value associated with model parameters means the subject would update her beliefs about reward contingencies more quickly than a subject who has precise prior beliefs about reward distributions. Similarly, subjects with a high precision over prior preferences or extrinsic value can be read as having more precise beliefs that she will be rewarded. The free parameters for the reinforcement learning model included..."

In terms of the time-dependent correlations with expected free energy - and its constituent terms - I think the report would benefit from overviewing these analyses with something like the following:

"In the final analysis of the neuronal correlates of belief updating - as quantified by the epistemic and intrinsic values of expected free energy - we present a series of analyses in source space. These analyses tested for correlations between constituent terms in expected free energy and neuronal responses in source space. These correlations were over trials (and subjects). Because we were dealing with two-second timeseries, we were able to identify the periods of time during decision-making when the correlates were expressed.

In these analyses, we focused on the induced power of neuronal activity at each point in time, at each brain source. To illustrate the functional specialisation of these neuronal correlates, we present whole-brain maps of correlation coefficients and pick out the most significant correlation for reporting fluctuations in selected correlations over two-second periods. These analyses are presented in a descriptive fashion to highlight the nature and variety of the neuronal correlates, which we unpack in relation to the existing EEG literature in the discussion. Note that we did not attempt to correct for multiple comparisons; largely, because the correlations observed were sustained over considerable time periods, which would be almost impossible under the null hypothesis of no correlations."

There was a slight misdirection in the discussion of priors in the active inference framework. The notion that active inference requires a pre-specification of priors is a common misconception. Furthermore, it misses the point that the utility of Bayesian modelling is to identify the priors that each subject brings to the table. This could be easily addressed with something like the following in the discussion:

"It is a common misconception that Bayesian approaches to choice behaviour (including active inference) are limited by a particular choice of priors. As illustrated in our fitting of choice behaviour above, priors are a strength of Bayesian approaches in the following sense: under the complete class theorem [5, 6], any pair of choice behaviours and reward functions can be described in terms of ideal Bayesian decision-making with particular priors. In other words, there always exists a description of choice behaviour in terms of some priors. This means that one can, in principle, characterise any given behaviour in terms of the priors that explain that behaviour. In our example, these were effectively priors over the precision of various preferences or beliefs about contingencies that underwrite expected free energy."

(1) Oudeyer, P.-Y. and F. Kaplan, What is intrinsic motivation? a typology of computational approaches. Frontiers in Neurorobotics, 2007. 1: p. 6.<br /> (2) Schmidhuber, J., Formal Theory of Creativity, Fun, and Intrinsic Motivation (1990-2010). Ieee Transactions on Autonomous Mental Development, 2010. 2(3): p. 230-247.<br /> (3) Barto, A., M. Mirolli, and G. Baldassarre, Novelty or surprise? Front Psychol, 2013. 4: p. 907.<br /> (4) Schwartenbeck, P., et al., Computational mechanisms of curiosity and goal-directed exploration. Elife, 2019. 8: p. e41703.<br /> (5) Wald, A., An Essentially Complete Class of Admissible Decision Functions. Annals of Mathematical Statistics, 1947. 18(4): p. 549-555.<br /> (6) Brown, L.D., A Complete Class Theorem for Statistical Problems with Finite-Sample Spaces. Annals of Statistics, 1981. 9(6): p. 1289-1300.

Reviewer #2 (Public Review):

Summary:<br /> Zhang and colleagues use a combination of behavioral, neural, and computational analyses to test an active inference model of exploration in a novel reinforcement learning task.

Strengths:<br /> The paper addresses an important question (validation of active inference models of exploration). The combination of behavior, neuroimaging, and modeling is potentially powerful for answering this question.

Weaknesses:<br /> The paper does not discuss relevant work on contextual bandits by Schulz, Collins, and others. It also does not mention the neuroimaging study of Tomov et al. (2020) using a risky/safe bandit task.

The statistical reporting is inadequate. In most cases, only p-values are reported, not the relevant statistics, degrees of freedom, etc. It was also not clear if any corrections for multiple comparisons were applied. Many of the EEG results are described as "strong" or "robust" with significance levels of p<0.05; I am skeptical in the absence of more details, particularly given the fact that the corresponding plots do not seem particularly strong to me.

The authors compare their active inference model to a "model-free RL" model. This model is not described anywhere, as far as I can tell. Thus, I have no idea how it was fit, how many parameters it has, etc. The active inference model fitting is also not described anywhere. Moreover, you cannot compare models based on log-likelihood, unless you are talking about held-out data. You need to penalize for model complexity. Finally, even if active inference outperforms a model-free RL model (doubtful given the error bars in Fig. 4c), I don't see how this is strong evidence for active inference per se. I would want to see a much more extensive model comparison, including model-based RL algorithms which are not based on active inference, as well as model recovery analyses confirming that the models can actually be distinguished on the basis of the experimental data.

Another aspect of the behavioral modeling that's missing is a direct descriptive comparison between model and human behavior, beyond just plotting log-likelihoods (which are a very impoverished measure of what's going on).

The EEG results are intriguing, but it wasn't clear that these provide strong evidence specifically for the active inference model. No alternative models of the EEG data are evaluated.

Overall, the central claim in the Discussion ("we demonstrated that the active inference model framework effectively describes real-world decision-making") remains unvalidated in my opinion.

Reviewer #3 (Public Review):

Summary:<br /> This paper aims to investigate how the human brain represents different forms of value and uncertainty that participate in active inference within a free-energy framework, in a two-stage decision task involving contextual information sampling, and choices between safe and risky rewards, which promotes a shift from exploration to exploitation. They examine neural correlates by recording EEG and comparing activity in the first vs second half of trials and between trials in which subjects did and did not sample contextual information, and perform a regression with free-energy-related regressors against data "mapped to source space." Their results show effects in various regions, which they take to indicate that the brain does perform this task through the theorised active inference scheme.

Strengths:<br /> This is an interesting two-stage paradigm that incorporates several interesting processes of learning, exploration/exploitation, and information sampling. Although scalp/brain regions showing sensitivity to the active-inference-related quantities do not necessarily suggest what role they play, it can be illuminating and useful to search for such effects as candidates for further investigation. The aims are ambitious, and methodologically it is impressive to include extensive free-energy theory, behavioural modelling, and EEG source-level analysis in one paper.

Weaknesses:<br /> Though I could surmise the above general aims, I could not follow the important details of what quantities were being distinguished and sought in the EEG and why. Some of this is down to theoretical complexity - the dizzying array of constructs and terms with complex interrelationships, which may simply be part and parcel of free-energy-based theories of active inference - but much of it is down to missing or ambiguous details.

In general, an insufficient effort has been made to make the paper accessible to readers not steeped in the free energy principle and active inference. There are critical inconsistencies in key terminology; for example, the introduction states that aim 1 is to distinguish the EEG correlates of three different types of uncertainty: ambiguity, risk, and unexpected uncertainty. But the abstract instead highlights distinctions in EEG correlates between "uncertainty... and... risk" and between "expected free energy .. and ... uncertainty." There are also inconsistencies in mathematical labelling (e.g. in one place 'p(s|o)' and 'q(s)' swap their meanings from one sentence to the very next).

Some basic but important task information is missing, and makes a huge difference to how decision quantities can be decoded from EEG. For example:<br /> - How do the subjects press the left/right buttons - with different hands or different fingers on the same hand?<br /> - Was the presentation of the Stay/cue and safe/risky options on the left/right sides counterbalanced? If not, decisions can be formed well in advance especially once a policy is in place.<br /> - What were the actual reward distributions ("magnitude X with probability p, magnitude y with probability 1-p") in the risky option? 

The EEG analysis is not sufficiently detailed and motivated. For example,<br /> - why the high lower-filter cutoff of 1 Hz, and shouldn't it be acknowledged that this removes from the EEG any sustained, iteratively updated representation that evolves with learning across trials?<br /> - Since the EEG analysis was done using an array of free-energy-related variables in a regression, was multicollinearity checked between these variables?<br /> - In the initial comparison of the first/second half, why just 5 clusters of electrodes, and why these particular clusters? How many different variables are systematically different in the first vs second half, and how do you rule out less interesting time-on-task effects such as engagement or alertness? In what time windows are these amplitudes being measured? In the comparison of asked and not-asked trials, what trial stage and time window is being measured? Again, how many different variables, of the many estimated per trial in the active inference model, are different in the asked and not-asked trials, and how can you know which of these differences is the one reflected in the EEG effects? The authors choose to interpret that on not-asked trials the subjects are more uncertain because the cue doesn't give them the context, but you could equally argue that they don't ask because they are more certain of the possible hidden states.<br /> - The EEG regressors are not fully explained. For example, an "active learning" regressor is listed as one of the 4 at the beginning of section 3.3, but it is the first mention of this term in the paper and the term does not arise once in the methods.<br /> - In general, it is not clear how one can know that the EEG results reflect that the brain is purposefully encoding these very parameters while implementing this very mechanism, and not other, possibly simpler, factors that correlate with them since there is no engagement with such potential confounds or alternative models. For example, a model-free reinforcement learning model is fit to behaviour for comparison. Why not the EEG?

Reviewer #1 (Public Review):

The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain. Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, Zambo and coworkers use a powerful technique, called native holdup, to measure the affinity of the SH3 domain of BIN1 for cellular partners. Using this assay, they combine data using cellular proteins and proline-containing fragments in these proteins to identify 97 distinct direct binding partners of BIN1. They also compare the binding interactome of the BIN1 SH3 domain to the interactome of several other SH3 domains, showing varying levels of promiscuity among SH3 domains. The authors then use pathway analysis of BIN1 binding partners to show that BIN1 may be involved in mitosis. Finally, the authors examine the impact of clinically relevant mutations of the BIN1 SH3 domain on the cellular interactome. The authors were able to compare the interactome of several different SH3 domains and provide novel insight into the cellular function of BIN1. Generally, the data supports the conclusions, although the reliance on one technique and the low number of replicates in each experiment is a weakness of the study.

Strengths:<br /> The major strength of this paper is the use of holdup and native holdup assays to measure the affinity of SH3 domains to cellular partners. The use of both assays using cell-derived proteins and peptides derived from identified binding partners allows the authors to better identify direct binding partners. This assay has some complexity but does hold the possibility of being used to measure the affinity of the cellular interactome of other proteins and protein domains. Beyond the utility of the technique, this study also provides significant insight into the cellular function of BIN1. The authors have strong evidence that BIN1 might have an undiscovered function in cellular mitosis, which potentially highlights BIN1 as a drug target. Finally, the study provides outstanding data on the cellular binding properties and partners of seven distinct SH3 domains, showing surprising differences in the promiscuity of these proteins.

Weaknesses:<br /> There are three major weaknesses of the study. First, the authors rely completely on a single technique to measure the affinity of the cellular interactome. The native holdup is a relatively new technique that is powerful yet relatively unproven. However, it appears to have the capacity to measure the relative affinity of proteins. Second, the authors appear to use a relatively small number of replicates for the holdup assays. There is no information in the legends about the number of replicates but the materials and methods suggest the native holdup data is from a single experimental replicate with multiple technical replicates. Finally, the authors' data using cellular proteins and fragments show that the affinity of the whole proteins is 5-20 fold lower than individual proline-containing fragments. The authors state that this difference suggests that there is cooperativity between different proline-rich sites of the binding partners of BIN1, yet BIN1 only has one SH3 domain. It is unclear what the molecular mechanism of the cooperative interaction would be exactly since there would be only one SH3 domain to bind the partner. An alternative interpretation would be that the BIN 1 SH3 domain requires sequences outside of the short proline-rich regions for high-affinity interactions with cellular partners, a hypothesis that is supported by other studies.

Reviewer #2 (Public Review):

Summary:

Chew et al describe interaction of the flavivirus protein NS1 with HDL using primarily cryoEM and mass spec. The NS1 was secreted from dengue virus infected Vero cells, and the HDL were derived from the 3% FBS in the culture media. NS1 is a virulence factor/toxin and is a biomarker for dengue infection in patients. The mechanisms of its various activities in the host are incompletely understood. NS1 has been seen in dimer, tetramer and hexamer forms. It is well established to interact with membrane surfaces, presumably through a hydrophobic surface of the dimer form, and the recombinant protein has been shown to bind HDL. In this study, cryoEM and crosslinking-mass spec are used to examine NS1 secreted from virus-infected cells, with the conclusion that the sNS1 is predominantly/exclusively HDL-associated through specific contacts with the ApoA1 protein.

Strengths: The experimental results are consistent with previously published data.

Weaknesses:

CryoEM:<br /> Some of the neg-stain 2D class averages for sNS1 in Fig S1 clearly show 1 or 2 NS1 dimers on the surface of a spherical object, presumably HDL, and indicate the possibility of high-quality cryoEM results. However, the cryoEM results are disappointing. The cryo 2D class averages and refined EM map in Fig S4 are of poor quality, indicating sub-optimal grid preparation or some other sample problem. Some of the FSC curves (2 in Fig S7 and 1 in Fig S6) have extremely peculiar shapes, suggesting something amiss in the map refinement. The sharp drop in the "corrected" FSC curves in Figs S5c and S6c (upper) indicate severe problems. The stated resolutions (3.42 & 3.82 Å) for the sNS1ts-Fab56.2 are wildly incompatible with the images of the refined maps in Figs 3 & S7. At those resolutions, clear secondary structural elements should be visible throughout the map. From the 2D averages and 3D maps shown in the figures, this does not seem to be the case. Local resolution maps should be shown for each structure.

The samples were clearly challenging for cryoEM, leading to poor quality maps that were difficult to interpret. None of the figures are convincing that NS1, Ab56.2 or Fab56.2 are correctly fit into EM maps. There is no indication of ApoA1 helices. Details of the fit of models to density for key regions of the higher-resolution EM maps should be shown and the models should be deposited in the PDB. An example of modeling difficulty is clear in the sNS1ts dimer with bound Fab56.2 (figs 3c & S7e). For this complex, the orientation of the Fab56.2 relative to the sNS1ts dimer in this submission (Fig 3c) is substantially different than in the bioRxiv preprint (Fig 3c). Regions of empty density in Fig 3c also illustrate the challenge of building a model into this map.

Mass spec:<br /> Crosslinking-mass spec was used to detect contacts between NS1 and ApoA1, providing strong validation of the sNS1-HDL association. As the crosslinks were detected in a bulk sample, they show that NS1 is near ApoA1 in many/most HDL particles, but they do not indicate a specific protein-protein complex. Thus, the data do not support the model of an NS1-ApoA1 complex in Fig 4d. Further, a specific NS1-ApoA1 interaction should have evidence in the EM maps (helical density for ApoA1), but none is shown or mentioned. If such exists, it could perhaps be visualized after focused refinement of the map for sNS1ts-HDL with Fab56.2 (Fig S7d). The finding that sNS1-ApoA1 crosslinks involved residues on the hydrophobic surface of the NS1 dimer confirms previous data that this NS1 surface engages with membranes and lipids.

Sample quality:<br /> The paper lacks any validation that the purified sNS1 retains established functions, for example the ability to enhance virus infectivity or to promote endothelial dysfunction. Peculiarities include the gel filtration profiles (Fig 2a), which indicate identical elution volumes (apparent MWs) for sNS1wt-HDL bound to Ab562 (~150 kDa) and to the ~3X smaller Fab56.2 (~50 kDa). There should also be some indication of sNS1wt-HDL pairs crosslinked by the full-length Ab, as can be seen in the raw cryoEM micrograph (Fig S5b).

Obtaining high quality structures is often more demanding of sample integrity than are activity assays. Given the low quality of the cryoEM maps, it's possible that the acidification step in immunoaffinity purification damaged the HDL complex. No validation of HDL integrity, for example with acid-treated HDL, is reported. Acid treatment is perhaps discounted by a statement (line 464) that another group also used immunoaffinity purification in a recent study (ref 20) reporting sNS1 bound to HDL. However the statement is incorrect; the cited study used affinity purification via a strep-tag on recombinant sNS1.

Discussion:<br /> The Discussion reflects a view that the NS1 secreted from virus-infected cells is a 1:1 sNS1dimer:HDL complex with the specific NS1-ApoA1 contacts detected by crosslinking mass spec. This is inconsistent with both the neg-stain 2D class average with 2 sNS1 dimers on an HDL (Fig S1c) and with the recent study of Flamand & co-workers showing 1-3 NS1 dimers per HDL (ref 20). It also ignores the propensity of NS1 to associate with membranes and lipids. It is far more likely that NS1 association with HDL is driven by these hydrophobic interactions than by specific protein-protein contacts. A lengthy Discussion section (lines 461-522) includes several chemically dubious or inconsistent statements, all based on the assumption that specific ApoA1 contacts are essential to NS1 association with HDL and that sNS1 oligomers higher than the dimer necessarily involve ApoA1 interaction, conclusions that are not established by the data in this paper.

Additional comments on the revised manuscript:

Comments on the structures:

The authors kindly provided their fitted atomic models for the 2 reported structures. The EM maps are available in the EMDB. Based on these materials, the derived structures are not well supported due to problems with the models, the maps, and the fit of models to maps.

Quick inspection revealed that the models for both structures are implausible due to a large steric clash of Fab56.2 and the end of the NS1. The Fab and NS1 protein backbones interpenetrate by nearly 20 Å. This substantial overlap exists for all 3 Fab56.2-NS1 interactions in the 2 structures, and is also visible in the perpendicular views of the NS1 dimer with 2 bound Fab56.2 in Fig. 2c. It appears that the Fab56.2 model was jammed into the NS1 model in order to bring all domains inside the density envelope at the threshold chosen to display the map. The poor fit of model to map is also clear in several protruding density regions without any model.

The fits of both atomic models to the maps are questionable because<br /> - The maps suffer from severe preferred orientation problems, as seen in the streaky tubes of density. The streaks in both maps do not match the NS1 beta strands of the fitted models.<br /> - The shape of the modeled ApoA1 helical ring surrounding the HDL does not match the shape of the EM density. In some regions, the ApoA1 helices are inside the rather strong density for the spherical HDL, but in other regions the helices are outside the density.<br /> - Both maps have regions of strong density that are adjacent to NS1 but lack any protein model, while other parts of the structure, including the beta-roll domain, lack density.<br /> - The claimed 4.3-Å resolution of the NS1-Fab56.2 complex is wildly overstated. The local resolution of ~2.5 Å for the "best" part of the structure (Supp Fig. 7E) appears to pertain to the beta strands at the center of the NS1 dimer. However, these density streaks do not match the beta strands of the fit model.<br /> - The manuscript lacks statistics on the fit of model to map. A standard cryo-EM "Table 1" should include more than is presented in Supp Table 1. The fitted model for at least the higher resolution structure should be deposited in the PDB.

Comments on the structure interpretation:

By now it should be abundantly clear that the oligomer state of NS1 is dynamic and highly sensitive to environmental conditions and to each sample's "history". For the reasons pointed out by reviewer 1, it is not clear that the immunoaffinity purification method captured all forms of sNS1 equally. Thus, the authors insistence that NS1 secreted from virus-infected cells is predominantly bound to HDL particles in a ratio of 1 NS1 dimer per HDL is not well supported. They employ similar arguments to challenge the discovery of sNS1 as a lipoprotein particle (PNAS 2011), contending that the 2011 finding was an artefact of recombinant NS1 production and is irrelevant to sNS1 from a virus infection. The several published structures of NS1 oligomers reveal a large degree of asymmetry in dimer-dimer interaction, consistent with the ability of NS1 to dynamically associate with a variety of hydrophobic entities.

Reviewer #1 (Public Review):

The authors of this study seek to visualize NS1 purified from dengue virus infected cells. They infect vero cells with DV2-WT and DV2 NS1-T164S (a mutant virus previously characterized by the authors). The authors utilize an anti-NS1 antibody to immunoprecipitate NS1 from cell supernatants and then elute the antibody/NS1 complex with acid. The authors evaluate the eluted NS1 by SDS-PAGE, Native Page, mass spec, negative-stain EM, and eventually Cryo-EM. SDS-PAGE, mas spec, and native page reveal a >250 Kd species containing both NS1 and the proteinaceous component of HDL (ApoA1). The authors produce evidence to suggest that this population is predominantly NS1 in complex with ApoA1. This contrasts with recombinantly produced NS1 (obtained from a collaborator) which did not appear to be in complex with or contain ApoA1 (Figure 1C). The authors then visualize their NS1 stock in complex with their monoclonal antibody by CryoEM. For NS1-WT, the major species visualized by the authors was a ternary complex of an HDL particle in complex with an NS1 dimer bound to their mAB. For their mutant NS1-T164S, they find similar structures, but in contrast to NS1-WT, they visualize free NS1 dimers in complex with 2 Fabs (similar to what's been reported previously) as one of the major species. This highlights that different NS1 species have markedly divergent structural dynamics. It's important to note that the electron density maps for their structures do appear to be a bit overfitted since there are many regions with electron density that do not have a predicted fit and their HDL structure does not appear to have any predicted secondary structure for ApoA1. The authors then map the interaction between NS1 and ApoA1 using cross-linking mass spectrometry revealing numerous NS1-ApoA1 contact sites in the beta-roll and wing domain. The authors find that NS1 isolated from DENV infected mice is also present as a >250 kD species containing ApoA1. They further determine that immunoprecipitation of ApoA1 out of the sera from a single dengue patient correlates with levels of NS1 (presumably COIPed by ApoA1) in a dose-dependent manner.

In the end, the authors make some useful observations for the NS1 field (mostly confirmatory) providing additional insight into the propensity of NS1 to interact with HDL and ApoA1. The study does not provide any functional assays to demonstrate activity of their proteins or conduct mutagenesis (or any other assays) to support their interaction predications. The authors assertion that higher-order NS1 exists primarily as a NS1 dimer in complex with HDL is not well supported as their purification methodology of NS1 likely introduces bias as to what NS1 complexes are isolated. While their results clearly reveal NS1 in complex with ApoA1, the lack of other NS1 homo-oligomers may be explained by how they purify NS1 from virally infected supernatant. Because NS1 produced during viral infection is not tagged, the authors use an anti-NS1 monoclonal antibody to purify NS1. This introduces a source of bias since only NS1 oligomers with their mAb epitope exposed will be purified. Further, the use of acid to elute NS1 may denature or alter NS1 structure and the authors do not include controls to test functionality of their NS1 stocks (capacity to trigger endothelial dysfunction or immune cell activation). The acid elution may force NS1 homo-oligomers into dimers which then reassociate with ApoA1 in a manner that is not reflective of native conditions. Conducting CryoEM of NS1 stocks only in the presence of full-length mAbs or Fabs also severely biases what species of NS1 is visualized since any NS1 oligomers without the B-ladder domain exposed will not be visualized. If the residues obscured by their mAb are involved in formation of higher-order oligomers then this antibody would functionally inhibit these species from forming. The absence of critical controls, use of one mAb, and acid elution for protein purification severely limits the interpretation of these data and do not paint a clear picture of if NS1 produced during infection is structurally distinct from recombinant NS1. Certainly there is novelty in purifying NS1 from virally infected cells, but without using a few different NS1 antibodies to purify NS1 stocks (or better yet a polyclonal population of antibodies) it's unclear if the results of the authors are simply a consequence of the mAb they selected.

Data produced from numerous labs studying structure and function of flavivirus NS1 proteins provide diverse lines of evidence that the oligomeric state of NS1 is dynamic and can shift depending on context and environment. This means that the methodology used for NS1 production and purification will strongly impact the results of a study. The data in this manuscript certainly capture one of these dynamic states and overall support the general model of a dynamic NS1 oligomer that can associate with both host proteins as well as itself but the assertions of this manuscript are overall too strong given their data, as there is little evidence in this manuscript, and none available in the large body of existing literature, to support that NS1 exists only as a dimer associated with ApoA1. More likely the results of this paper are a result of their NS1 purification methodology.

Comments on revised version:

The authors have not adequately addressed my concerns from the original review. My major concerns are that the binding modality of NS1 to ApoA1/HDL was not validated using a mutagenesis approach and that the overarching conclusion drawn by the authors, that the major species of NS1 in vivo is a dimer in complex with ApoA1, is not supported by the data in this study given the methodology of using a single monoclonal antibody to immunoprecipitate NS1. Certainly, the structures in this manuscript are valuable in confirming that NS1 interacts with HDL and captures a snapshot of NS1/HDL interaction dynamics, but the use of only a single antibody is a major source of bias that makes it challenging to draw conclusions about the oligomeric state of NS1. Further on this point, a critically important control that is missing from this study is to determine if the anti-NS1 mAb 56.2 prevents NS1 from interacting with cells, triggering the release of proinflammatory cytokines from immune cells, or mediating endothelial dysfunction of endothelial cells. If this antibody inhibits these NS1-triggered events (linked to pathogenesis), it would suggest that the NS1 within this ternary complex is not active. Presumably some protective anti-NS1 antibodies may function by modulating the oligomeric state of NS1.

Reviewer #1 (Public Review):

Summary:<br /> This technical report by Kugler et al., expands the application of a fluorescence-based reporter to study the conformational state of various kinases. This reporter, named KinCon (Kinase Conformation), interrogates the conformational state of a kinase (i.e., active vs. inactive) based on engineering complementary fusion proteins that fluoresce upon interaction. This assay has several advantages as it allows studying full-length kinases, that is, the kinase domain and regulatory domains, inside the cell and under various experimental conditions such as the presence of inhibitors or activator proteins, and in wildtype and mutants involved in disease states.

Strengths:

One major strength of this study is that it is quite comprehensive. The authors use KinCon for four different kinases, BRAF, LKB1, RIP, and CDK4/6. These kinases have very different regulatory elements and associated proteins, which the authors explore to study their conformational state. Moreover, they use small molecule inhibitors or mutations to further dissect how the conformational state of the kinase in disease states. The collective set of results strongly suggests that KinCon is a versatile tool that can be used to study many kinases of biomedical and fundamental importance. Given that kinases are extensively studied by researchers in academia or industry, KinCon could have a broad impact as well.

Weaknesses:<br /> This manuscript, however, also has several weaknesses. These include:

- The manuscript is exceedingly long. For instance, the introduction provides background information for each kinase that is further expanded in the results section. I think the background information for each kinase in the Introduction and Results sections could be significantly reduced to highlight the major points. Otherwise, not only does the manuscript become too long, but the main points get diluted.

- The figure legends are very long, providing information that is already in the main text or Methods. In the legend, the authors should provide only the essential information to understand the figure.

- A major concern throughout the manuscript is the use of the word "dynamics," which is used in the text in various contexts. The authors should clarify what they understand about the dynamics of conformation. Are they measuring how the time-dependent process by which the kinase is interconverting between active and inactive states? It seems to me that the assays in this report evaluate a population of kinases that are in an open or close conformation (i.e., a particular state in each experimental condition) but there is no direct information on how the kinase goes from one state to the other. In that sense, the use of the word dynamics is unclear. Also, the use of the word dynamics in different sentences is ambiguous.

- There are various other issues with terminology and presentation that also affect the overall level of impact of the manuscript.

Reviewer #2 (Public Review):

Summary:<br /> Protein kinases have been very successfully targeted with small molecules for several decades, with many compounds (including clinical drugs) bringing about conformational changes that are also relevant to broader interactions with the cellular signaling networks that they control. The authors set out to develop a targeted biosensor approach to evaluate distinct kinase conformations in cells for multiple kinases in the context of incoming signals, other proteins, and small molecule binding, with a broad goal of using the KinCon assay to confirm (and perhaps predict) how drug binding or signal perception changes conformations and outputs in the presence of cellular complexes. This work will likely impact on the field with cellular reporters of kinase conformations a useful addition to the toolbox.

Strengths:<br /> The KinCon reporter platform has previously been validated for well-known kinases; in this study, the team evaluates how to employ a full-length kinase (often containing a known pathlogical mutation). The sensitive detection method is based on a Renilla luciferase (RLuc)protein fragment complementation assay, where individual RLuc fragments are present at the N and the C terminus of the kinase. This report, which is both technical and practical in nature, co-expresses the kinase with known interactors (at low levels) in a high throughput format and then performs pharmacological evaluation with known small molecule kinase modulators. This is explained nicely in Figure 1, as are the signaling pathways that are being evaluated. Data demonstrate that V600E BRAF iexposed to vemurafenib is converted to the inactive conformation, as expected. In contrast, the more closed STRAD𝛼 and LKB1 KinCon conformations appear to represent the more active state of the complexed kinase, and a W308C mutation (evaluated alongside others) reverses this effect. The authors then evaluated necroptotic signaling in the context of RIPK1/3 under conditions where RIPK1 and RIPK3 are active, confirming that the reporters highlight the active states of both kinases. Exposure to compounds that are known to engage with the RIPK1 arm of the pathway induce bioluminescence changes consistent with the opening (inactivation) of the kinase. Finally, the authors move to an important drug target for which clinical drugs have arrived relatively recently; the CDK4/6 complexes. These are of additional importance because kinase-independent functions also exist for CDK6, and the effects of drugs in cells usually rely on a downstream marker, rather than demonstration of direct protein complex engagement. The data presented are interpreted as the formation of complexes with the CDK inhibitor p16INK4a; reducing the affinity of the interaction through mutations drives an inactive conformation, whilst the application of CDK4/6 inhibitors does not, implying binding to the active conformation.

Weaknesses:<br /> (1) The work is very solid, uses examples from the literature, and also extends into new experimental space. An obvious weakness is mentioned by the authors for the CKDK data, in that measurements with Cyclin D (the activating subunit) are not characterised, although Cyclin D might be assumed to be present.

(2) The work with the trimeric LKB1 complex involves pseudokinase, STRADalpha, whose conformation is also examined as a function of LKB1 status; since STRAD is an activator of LKB1. A future goal should be the evaluation of the complex in the presence of STRAD inhibitory/activating small molecules.

Reviewer #1 (Public Review):

Summary:<br /> This study provides convincing data showing that expression of the PIK3R1(delta Exon11) dominant negative mutation in Activated PI3K Delta Syndrome 1/2 (APDS1/2) patient-derived cells reduces AKT activation and p110δ protein levels. Using a 3T3-L1 model cell system, the authors show that overexpressed p85α delta Exon 11) displays reduced association with the p110α catalytic subunit but strongly interacts with Irs1/2. Overexpression of PIK3R1 dominant negative mutants inhibits AKT phosphorylation and reduces cellular differentiation of preadipocytes. The strength of this article is the clear results derived from Western blots analysis of cell signaling markers (e.g. pAKT1), and co-immunoprecipitation of PI3K holoenzyme complexes and associated regulatory factors (e.g. Irs1/2). The experimental design, interpretation, and quantification broadly support the authors' conclusions.

Strengths:<br /> The authors analyze a variety of PIK3R1 mutants (i.e. delta Exon11, E489K, R649W, and Y657X), which reveals a range of phenotypes that support the proposed model for dominant negative activity. The use of clonal cell lines with doxycycline-induced expression of the PIK3R1 mutants (Exon 11, R649W, and Y657X) provides convincing experimental data concerning the relationship between p85α mutant expression and AKT phosphorylation in vivo. The authors convincingly show that p85α delta Exon11, R649W, or Y657X) is unable to associate with p110α but instead more strongly associates with Irs1/2 compared to wild type p85α. This helps explain why the authors were unable to purify the recombinant p110α/p85α delta Exon 11) heterodimeric complex from insect cells.

Weaknesses:<br /> Future experimentation will be needed to reconcile the cell type specific differences (e.g. APDS2 patient-derived cells vs. the 3T3-L1 cell model system) in PIK3R1 mutant behavior reported by the authors. An unbiased proteomic study that broadly evaluates the cell signaling landscape could provide a more holistic understanding of the APDS2 and SHORT mutants compared to a candidate-based approach. Additional biochemical analysis of p110α/p85α delta Exon 11) complex is needed to explain why this mutant regulatory subunit does not strongly associate with the p110 catalytic subunit. It remains unclear why p85α delta Exon 11) expression reduces p110δ protein levels in APDS2 patient-derived dermal fibroblasts. This study would benefit from a more comprehensive biochemical analysis of the described p110α/p85α, p110β/p85α, and p110δ/p85α mutant protein complexes. The current limitation of this study to the use of a single endpoint assay to measure PI3K lipid kinase activity in the presence of a single regulatory input (i.e. RTK-derived pY peptide). A broader biochemical analysis of the mutant PI3K complexes across the canonical signaling landscape will be important for establishing how competition between wild-type and mutant regulatory subunits is regulated in different cell signaling pathways.

Reviewer #2 (Public Review):

Summary:<br /> Patsy R. Tomlinson et al; investigated the impact of different p85 alpha variants associated with SHORT syndrome or APDS2 on insulin-mediated signaling in dermal fibroblasts and preadipocytes. They find no evidence of hyperactive PI3K signalling monitored by pAKT in APDS2 patient-derived dermal fibroblast cells. In these cells p110 alpha protein levels were comparable to levels in control cells, however, the p110 delta protein levels were strongly reduced. Remarkably, the truncated APDS2-causal p85 alpha variant was less abundant in these cells than p85 alpha wildtype. Afterwards, they studied the impact of ectopically expressed p85 alpha variants on insulin-mediated PI3K signaling in 3T3-L1 preadipocytes. Interestingly they found that the truncated APDS2-causal p85 alpha variant impaired insulin-induced signaling. Using immunoprecipitation of p110 alpha they did not find truncated APDS2-causal p85 alpha variant in p110 alpha precipitates. Furthermore, by immunoprecipitating IRS1 and IRS2, they observed that the truncated APDS2-causal p85 alpha variant was very abundant in IRS1 and IRS2 precipitates, even in the absence of insulin stimulation. These important findings add in an interesting way possible mechanistic explanation for the growing number of APDS2 patients described with features of SHORT syndrome.

Strengths:<br /> Based on state-of-the-art functional investigation the authors propose indicating a loss-of-function activity of the APDS2-disease causing p85 alpha variant in preadipocytes providing a possible mechanistic explanation for the growing number of APDS2 patients described with features of SHORT syndrome.

Weaknesses:<br /> Related to Figure 1: PIK3R1 expression not only by Western blotting but also by quantifying the RNA transcripts, e.g. mutant and wildtype transcripts, was not performed. RNA expression analysis would further strengthen the suggested impaired stabilization/binding.

Related to Figure 2: As mentioned by the authors in the manuscript the expression of p110 delta but also p110 beta in 3T3-L1 preadipocytes ectopically expressing p85 alpha variants has not been analyzed.

Furthermore, a direct comparison of the truncated APDS2-causal p85 alpha variant with SHORT syndrome -causal p85 alpha variants in regard to pAKT level, and p85 alpha expression level has not been performed.

These investigations would further strengthen the data.

Related to Figure 3:<br /> The E489K and Y657X p85 alpha variants should be also tested in combination with p110 delta in the PI3K activity in vitro assay. This would help to further decipher the overall impact, especially of the E489K variant.

Reviewer #1 (Public Review):

Summary:<br /> This study is valuable in that it may lead to the discovery of future OA markers, etc., in that changes in glycan metabolism in chondrocytes are involved in the initiation of cartilage degeneration and early OA via hypertrophic differentiation of chondrocytes. However, more robust results would be obtained by analyzing the mechanisms and pathways by which changes in glycosylation lead to cartilage degeneration.

Strengths:<br /> This study is important because it indicates that glycan metabolism may be associated with pre-OA and may lead to the elucidation of the cause and diagnosis of pre-OA.

Weaknesses:<br /> More robust results would be obtained by analyzing the mechanism by which cartilage degeneration induced by changes in glycometabolism occurs.

Reviewer #2 (Public Review):

Summary:<br /> This paper consists of mostly descriptive data, judged from alpha-mannosidase-treated samples, in which they found an increase in core fucose, a product of Fut 8.

Strengths:<br /> This paper is interesting in the clinical field, but unfortunately the data is mostly descriptive and does not have a significant impact on the scientific community in general.

Weaknesses:<br /> If core fucose is increased, at least the target glycan molecules of core fucose should be evaluated. They also found an increase in NO, suggesting that inflammatory processes also play an important role in OA in addition to glycan changes.<br /> It has already been reported that core fucose is decreased by administration of alpha-mannosidase inhibitors. Therefore, it is expected that alphaa-mannosidase administration increases core fucose.

Review #1 (Public Review)

Single-molecule visualization of chromatin remodelers on long chromatin templates-a long sought-after goal-is still in its infancy. This work describes the behaviors of two remodelers RSC and ISW2, from SWI/SNF and ISWI families respectively, with well-conducted experiments and rigorous quantitative analysis, thus representing a significant advance in the field of chromatin biology and biophysics.

Review #2 (Public Review)

The authors use a dual optical trap instrument combined with 2-color fluorescence imaging to analyze the diffusion of RSC and ISW2 on DNA, both in the presence and absence of nucleosomes, as well as long-range nucleosome sliding by these remodelers. This allowed them to demonstrate that both enzymes can participate in 1D diffusion along DNA for rather long ranges, with ISW2 predominantly tracking the DNA strand, while RSC diffusion involves hopping. In an elegant two-color assay, the authors were able to analyze interactions of diffusing remodeler molecules, both of the same or different types, observing their collisions, co-diffusion and bypassing. The authors demonstrate that nucleosomes act as barriers for remodeler diffusion, either repelling or sequestering them upon collision. In the presence of ATP, they observed surprisingly processive unidirectional nucleosome sliding with a strong bias in the direction opposite to where the remodeler approached the nucleosome from for ISW2. These results have fundamentally important implications for the mechanism of nucleosome positioning at promoters in vivo, will be of great interest for the scientific community, and will undoubtedly spark exciting future research

Reviewer #1 (Public Review):

Summary: Here, the authors were attempting to use molecular simulation or probe the nature of how lipids, especially PIP lipids, bind to a medically-important ion channel. In particular, they look at how this binding impacts the function of the channel.

Strengths: The study is very well written and composed. The techniques are used appropriately, with plenty of sampling and analysis. The findings are compelling, and provide clear insights into the biology of the system.

Weaknesses: A few of the analyses are hard to understand/follow, and rely on "in house" scripts. This is particularly the case for the lipid binding events, which can be difficult to compute accurately. However the provision of these scripts on github means that these can be assessed by the reader if desired. Additionally, a lack of experimental validation, or coupling to existing experimental data, limits the study.

It is my view that the authors have achieved their aims, and their findings are compelling and believable. Their findings should have impacts on how researchers understand the functioning of the Nav1.4 channel, as well as on the study of other ion channels and how they interact with membrane lipids.

Reviewer #2 (Public Review):

Summary:<br /> Lin Y., Tao E., et al. used multiscale MD simulations to show that PI(4,5)P2 binds stably to an inactivated state of Nav channels at a conserved site within the DIV S4-S5 linker, which couples the voltage sensing domain (VSD) to the pore. The authors hypothesized that PI(4,5)P2 prolongs inactivation by binding to the same site where the C-terminal tail is proposed to bind during recovery from inactivation. They convincingly showed that PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, thus slowing the conformational changes required for the channel to recover to the resting state. They also conducted MD simulations to show that phosphoinositides bind to VSD gating charges in the resting state of Nav channels. These interactions may anchor VDS at the resting state and impede its activation. Their results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the recovery rate from inactivation, an important step for developing novel therapies to treat Nav-related diseases. However, the study is incomplete lacks the expected confirmatory studies which are relevant to such proposals.

Strengths:<br /> The authors identified a novel binding between phosphoinositides and the VSD of Nav and showed that the strength of this interaction is state-dependent. Based on their work, the affinity of PIPs to the inactivated state is higher than the resting state. This work will help pave the way for designing novel therapeutics that may help relieve pain or treat diseases like arrhythmia, which may result from a leftward shift of the channel's activation.

Weaknesses:<br /> However, the study lacks the expected confirmatory studies relevant to such proposals. For example, one would expect that the authors would mutate the positive residues that they claim to make interactions with phosphoinositides to show that there are much fewer interactions once they make these mutations. Another point is that the authors found that the main interaction site of PIPs with Nav1.4 is the VSD-DIV and DIII-DIV linker. This interaction is expected to delay fast inactivation if it happens at the resting state. The authors should make a resting state model of the Nav1.4 channel to explain the recent experimental data showing that PIP2 delays the activation of Nav1.4, with almost no effect on the voltage dependence of fast inactivation.

The reviewers answered most of my concerns about the first version of the manuscript.

Reviewer #3 (Public Review):

Summary:<br /> This work uses multiscale molecular dynamics simulations to demonstrate molecular mechanism(s) for phosphatidylinositol regulation of voltage gated sodium channel (Nav1.4) gating. Recent experimental work by Gada et al. JGP 2023 showed altered Nav1.4 gating when Nav1.4 current was recorded with simultaneous application of PI(4,5)P2 dephosphorylate. Here the authors revealed probable molecular mechanism that can explain PI(4,5)P2 modulation of Nav1.4 gating. They found PIP lipids interacting with the gating charges - potentially making it harder to move the voltage sensor domain and altering the channels voltage sensitivity. They also found a stable PIP binding site that reaches the D_IV S4-S5 linker, reducing the mobility of the linker and potentially competing with the C-terminal domain.

Strengths:<br /> Using multiscale simulations with course-grained simulations to capture lipid-protein interactions and the overall protein lipid fingerprint and then all-atom simulations to verify atomistic details for specific lipid-protein interactions is extremely appropriate for the question at hand. Overall, the types of simulation and their length are suitable for the questions the authors pose and a thorough set of analysis was done which illustrates the observed PIP-protein interactions.

Weaknesses:<br /> Although the set of current simulations and analysis supports the conclusions drawn nicely, the course-grained simulations have further utility than that utilized by the authors. With the 4to1 heavy atoms bead mapping in Martini 2 some detailed chemical specificity is averaged out but parameters for different PIP family members do exist - including specific PIP(4,5)P2 vs PIP(3,4)P2, and could have been explored at the course-grained level. However, performing more detailed all-atom simulation, as done in this manuscript, is always advisable to extend and/or confirm course-grained results.

Reviewer #1 (Public Review):

Summary:<br /> Cyclic Nucleotide Binding (CNB) domains are pervasive structural components involved in signaling pathways across eukaryotes and prokaryotes. Despite their similar structures, CNB domains exhibit distinct ligand-sensing capabilities. The manuscript offers a thorough and convincing investigation that clarifies numerous puzzling aspects of nucleotide binding in Trypanosoma.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript clearly shows that Trypanosoma PKA is controlled by nucleoside analogues rather than cyclic nucleotides, which are the primary allosteric effectors of human PKA and PKG. The authors demonstrate that the inosine, guanosine, and adenosine nucleosides bind with high affinity and activate PKA in the tropical pathogens T. brucei, T. cruzi and Leishmania. The underlying determinants of nucleoside binding and selectivity are dissected by solving the crystal structure of T. cruzi PKAR(200-503) and T. brucei PKAR(199-499) bound to inosine at 1.4 Å and 2.1 Å resolution and through comparative mutational analyses. Of particular interest is the identification of a minimal subset of 2-3 residues that controls nucleoside vs. cyclic nucleotide specificity.

Reviewer #1 (Public Review):

Summary of Author's Objectives:<br /> The authors aimed to explore JMJD6's role in MYC-driven neuroblastoma, particularly in the interplay between pre-mRNA splicing and cancer metabolism, and to investigate the potential for targeting this pathway.

Strengths:<br /> (1) The study employs a diverse range of experimental techniques, including molecular biology assays, next-generation sequencing, interactome profiling, and metabolic analysis. Moreover, the authors specifically focused on gained chromosome 17q in neuroblastoma, in combination with analyzing cancer dependency genes screened with Crispr/Cas9 library, analyzing the association of gene expression with prognosis of neuroblastoma patients with large clinical cohort. This comprehensive approach strengthens the credibility of the findings. The identification of the link between JMJD6-mediated pre-mRNA splicing and metabolic reprogramming in MYC-driven cancer cells is innovative.<br /> (2) The authors effectively integrate data from multiple sources, such as gene expression analysis, RNA splicing analysis, JMJD6 interactome assay, and metabolic profiling. This holistic approach provides a more complete understanding of JMJD6's role.<br /> (3) The identification of JMJD6 as a potential therapeutic target and its correlation with the response to indisulam have significant clinical implications, addressing an unmet need in cancer treatment.

Weaknesses:<br /> It would be beneficial to explore whether treatment with JMJD6 inhibitors, both in vitro and in vivo, can effectively target the enhanced pre-mRNA splicing of metabolic genes in MYC-driven cancer cells. However, the authors have noted that there are currently no potent and selective JMJD6 inhibitors available.

Appraisal of Achievement and Conclusion Support:<br /> The authors have effectively met their objectives by offering valuable insights into JMJD6's role in MYC-driven neuroblastoma. The results robustly underpin their conclusions about JMJD6's contribution to metabolic reprogramming through alternative splicing and its connection to the therapeutic response to indisulam.

Likely Impact on the Field and Utility of Methods/Data:<br /> The study's findings have the potential to significantly impact the field of cancer research by identifying JMJD6 as a promising therapeutic target for MYC-driven cancers. The methods and data presented in the manuscript offer valuable resources to the research community for further investigations into cancer metabolism and splicing regulation.

Additional Context for Interpretation:<br /> Understanding the complex interplay between cancer metabolism and splicing regulation is crucial for developing effective cancer treatments. This study sheds light on a previously poorly understood aspect of MYC-driven cancers and opens new avenues for targeted therapies. However, the transition from preclinical findings to clinical applications may face challenges, which should be considered in future research and clinical trials.

Reviewer #2 (Public Review):

Summary:

Jablonowski and colleagues explored altered pre-mRNA splicing and metabolism in MYC-driven neuroblastoma cell lines. They focused on the role of JMJD6 assessing cellular transformation, for example through interactions with RNA-binding proteins. Moreover, the study examined JMJD6's impact on the splicing of glutaminase (GLS), crucial in neuroblastoma cell metabolism. It also connected JMJD6 to the anti-proliferative effects of indisulam, a compound targeting RBM39 (splicing factor interacting with JMJD6).

Overall, the findings presented by Jablonowski et al. begin to illuminate a cancer-promoting metabolic, and potentially, a protein synthesis suppression program that may be linked to alternative pre-mRNA splicing through the action of JMJD6 - downstream of MYC. This discovery can provide further evidence for considering JMJD6 as a potential therapeutic target for the treatment of MYC-driven cancers.

Strengths:

Alternative Splicing Induced by JMJD6 Knockdown: the study presents evidence for the role of JMJD6 in alternative splicing in neuroblastoma cells. Specifically, the RNA immunoprecipitation experiments demonstrated a significant shift from the GAC to the KGA GLS isoform upon JMJD6 knockdown. Moreover, a significant correlation between JMJD6 levels and GAC/KGA isoform expression was identified in two distinct neuroblastoma cohorts. This suggests a causative link between JMJD6 activity and isoform prevalence.

Physical Interaction of JMJD6 in Neuroblastoma Cells: The paper provides preliminary insight into the physical interactome of JMJD6 in neuroblastoma cells. This offers a potential mechanistic avenue for the observed effects on metabolism and protein synthesis and could be exploited for a deeper investigation into the exact nature, and implications of neuroblastoma-specific JMJD6 protein-protein interactions.

Weaknesses:

There are several areas that would benefit from improvements with regards to the neuroblastoma modelling strategy, lack of in vivo data, and depth of mechanistic investigation. While the need for additional experimental evidence in these areas remains (as highlighted in the initial review), the authors have now acknowledged several relevant limitations and provided a paragraph discussing future experimental work.

Reviewer #1 (Public Review):

Summary<br /> In this study, Xu et al. provide insights into the substrate divergence of caspase 3 and 7 (CASP3 and CASP7) for gasdermin E (GSDME) cleavage and activation during evolution in vertebrates. Using a diverse set of biochemical assays, domain swapping, site-directed mutagenesis, and bioinformatics tools, the authors demonstrate that the human GSDME C-terminal region and the S234 residue of human CASP7 are the key determinants that impede the cleavage of human GSDME by human CASP7. Their findings suggest that mutations affecting the function of caspases have enabled the functional divergence of distinct caspase family members to specialize in controlling complicated cellular functions in mammals.

Strengths<br /> The authors made an important contribution to the field by demonstrating how human CASP7 has functionally diverged to lose the ability to cleave GSDME and showing that reverse-mutations in CASP7 can restore GSDME cleavage. The use of multiple methods to support their conclusions strengthens the authors' findings. The unbiased mutagenesis screen performed to identify S234 in huCASP7 as the determinant of its GSDME cleavability is also a strength.

Weaknesses<br /> While the authors employed a comprehensive experimental setup to investigate the CASP7-mediated GSDME cleavage across evolution, future studies will be required to fully understand the physiological implications of this evolutionary divergence.

Reviewer #2 (Public Review):

The authors wanted to address the differential processing of GSDME by caspase 3 and 7, finding that while in humans GSDME is only processed by CASP3, Takifugu GSDME, and other mammalian can be processed by CASP3 and 7. This is due to a change in a residue in the human CAPS7 active site that abrogates GSDME cleavage. This phenomenon is present in humans and other primates, but not in other mammals such as cats or rodents. This study sheds light on the evolutionary changes inside CASP7, using sequences from different species. Although the study is somehow interesting and elegantly provides strong evidence of this observation, it lacks the physiological relevance of this finding, i.e. on human side, mouse side, and fish what are the consequences of CASP3/7 vs CASP3 cleavage of GSDME.

Fish also present a duplication of GSDME gene and Takifugu present GSDMEa and GSDMEb. It is not clear in the whole study if when referring to TrGSDME is the a or b. This should be stated in the text and discussed in the differential function of both GSDME in fish physiology (i.e. PMIDs: 34252476, 32111733 or 36685536).

Joint Public Review:

Previous findings by authors show that heliomycin induces autophagy to inhibit cancer progression, while its water-soluble analogs induce apoptosis. Here, they show that one of the analogs, 4-dmH, binds to tNOX, a NADH oxidase which supports SirT1 activity, in addition to SirT1, while heliomycin only binds to SirtT1 but not tNOX, using CETSA and in silico molecular docking studies, in human oral cancer cells. The additional binding activity of 4-dmH to tNOX might explain the different biological outcome from heliomycin. 4-dmH induces ubiquitination and degradation of tNOX protein, in dependent of p53 status. The tumor suppressive effect of 4-dmH (by intra-tumoral injections) is better than heliomycin. TCGA data base analysis suggests that high tNOX mRNA expression is correlated with poor prognosis of oral cancer patients.

This group has been a leading lab of chemical and biological characterization of heliomycin and its analogs. Their findings are interesting and advance their previous findings. The revised manuscript well responded to the reviewers' concerns.

Reviewer #1 (Public Review):

Strengths:

- The paper is clearly written, and all the conclusions stem from a set of 3 principles: circular topology, rotational symmetry, and noise minimization. The derivations are sound and such rigor by itself is commendable.

- The authors provide a compelling argument on why evolution might have picked an eight-column circuit for path-integration, which is a great example of how theory can inform our thinking about the organization of neural systems for a specific purpose.

- The authors provide a self-consistency argument on how cosine-like activity supports cosine-like connectivity with a simple Hebbian rule. However, their framework doesn't answer the question of how this system integrates angular velocity with the correct gain in the absence of allothetic cues to produce a heading estimate (more on that on point 3 below).

Weaknesses:

- The authors make simplifying assumptions to arrive at the cosine activity/cosine connectivity circuit. Among those are the linear activation function, and cosine driving activity u. The authors provide justification for the linearization in methods 3.1, however, this ignores the well-established fact that bump amplitude is modulated by angular velocity in the fly head direction system (Turner-Evans et al 2017). In such a case, nonlinearities in the activation function cannot be ignored and would introduce harmonics in the activity. Furthermore, even though activity has been reported to be cosine-like, in fact in the fruit fly it takes the form of a somewhat concentrated activity bump (~80-100 degrees, Seelig & Jayaraman 2015; Turner-Evans et al 2017), and one has to take into account the smoothing effect of calcium dynamics too which might make the bump appear more cosine-like. So in general, it would be nice to see how the conclusions extend if the driving activity is more square-like, which would also introduce further harmonics. Overall, it would be interesting to see whether, despite the harmonics introduced by these two factors interacting in the learning rule, Oja's rule can still pick up the "base" frequency and produce sinusoidal weights (as mentioned in methods 3.8). At this point, the examples shown in Figure 5 (tabula rasa and slightly perturbed weights) are quite simple. Such a demonstration would greatly enhance the generality of the results.

- The match of the theoretical prediction of cosine-like connectivity profiles with the connectivity data is somewhat lacking. In the locust the fit is almost perfect, however, the low net path count combined with the lack of knowledge about synaptic strengths makes this a motivating example in my opinion. In the fruit fly, the fit is not as good, and the function-fitting comparison (Methods Figure 6) is not as convincing. First, some function choices clearly are not a good fit (f1+2, f2). Second, the profile seems to be better fit by a Gaussian or other localized function, however the extra parameter of the Gaussian results in the worst AIC and AICc. To better get at the question of whether the shape of the connectivity profile matches a cosine or a Gaussian, the authors could try for example to fix the width of the Gaussian (e.g. to the variance of the best-fit cosine, which seems to match the data very well even though it wasn't itself fit), and then fit the two other parameters to the data. In that case, no AIC or AICc is needed. And then do the same for a circular distribution, e.g. von Mises. In addition, the theoretical prediction of cosine-like connectivity is not clearly stated in the abstract, introduction, or discussion. As a prediction, I believe it should be center forward, as it might be revisited again in the future in lieu of e.g. new experimental data.

- I find the authors' claim that Oja's rule suffices to learn the insect head direction circuit (l. 273-5) somewhat misleading/vague. The authors seem to not be learning angular integration here at all. First, it is unclear to me what is the form of u(t). Is it the desired activity in the network at time t given angular velocity? This is different than modelling a population of PEN neurons jointly tuned to head direction and angular velocity, and learning weights so as to integrate angular velocity with the correct gain (Vafidis et al 2022). The learning rule here establishes a self-consistency between sinusoidal weights and activity, however, it does not learn the weights from PEN to EPG neurons so as to perform angular integration. Similar simple Hebbian rules have been used before to learn angular integration (Stringer et al 2002), however, they failed to learn the correct gain. Therefore, the authors should limit the statement that their simpler learning rule is enough to learn the circuit (l. 273-5), making sure to outline differences with the current literature (Vafidis et al 2022).

Reviewer #2 (Public Review):

Summary:<br /> Proteins that bind to double-stranded RNA regulate various cellular processes, including gene expression and viral recognition. Such proteins often contain multiple double-stranded RNA-binding domains (dsRBDs) that play an important role in target search and recognition. In this work, Chug and colleagues have characterized the backbone dynamics of one of the dsRBDs of a protein called TRBP2, which carries two tandem dsRBDs. Using solution NMR spectroscopy, the authors characterize the backbone motions of dsRBD2 in the absence and presence of dsRNA and compare these with their previously published results on dsRBD1. The authors show that dsRBD2 is comparatively more rigid than dsRBD1 and claim that these differences in backbone motions are important for target recognition.

Strengths:<br /> The strengths of this study are multiple solution NMR measurements to characterize the backbone motions of dsRBD2. These include 15N-R1, R2, and HetNOE experiments in the absence and presence of RNA and the analysis of these data using an extended-model-free approach; HARD-15N-experiments and their analysis to characterize the kex. The authors also report differences in binding affinities of dsRBD1 and dsRBD2 using ITC and have performed MD simulations to probe the differential flexibility of these two domains.

Weaknesses:<br /> While it may be true that dsRBD2 is more rigid than dsRBD1, the manuscript lacks conclusive and decisive proof that such changes in backbone dynamics are responsible for target search and recognition and the diffusion of TRBP2 along the RNA molecule. To conclusively prove the central claim of this manuscript, the authors could have considered a larger construct that carries both RBDs. With such a construct, authors can probe the characteristics of these two tandem domains (e.g., semi-independent tumbling) and their interactions with the RNA. Additionally, mutational experiments may be carried out where specific residues are altered to change the conformational dynamics of these two domains. The corresponding changes in interactions with RNA will provide additional evidence for the model presented in Figure 8 of the manuscript. Finally, there are inconsistencies in the reported data between different figures and tables.

Reviewer #1 (Public Review):

Summary:<br /> In the manuscript entitled "Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding," Chugh and co-workers utilize a suite of NMR relaxation methods to probe the dynamic landscape of the TAR RNA binding protein (TRBP) double-stranded RNA-binding domain 2 (dsRBD2) and compare these to their previously published results on TRBP dsRBD1. The authors show that, unlike dsRBD1, dsRBD2 is a rigid protein with minimal ps-ns or us-ms time scale dynamics in the absence of RNA. They then show that dsRBD2 binds to canonical A-form dsRNA with a higher affinity compared to dsRBD1 and does so without much alteration in protein dynamics. Using their previously published data, the authors propose a model whereby dsRBD2 recognizes dsRNA first and brings dsRBD1 into proximity to search for RNA bulge and internal loop structures.

Strengths:<br /> The authors expertly use a variety of NMR techniques to probe protein motions over six orders of magnitude in time. Other NMR titration experiments and ITC data support the RNA-binding model.

Weaknesses:<br /> The data collection and analysis are sound. The only weakness in the manuscript is the lack of context with the much broader field of RNA-binding proteins. For example, many studies have shown that RNA recognition motif (RRM) domains have similar dynamic characteristics when binding diverse RNA substrates. Furthermore, there was no discussion about the entropy of binding derived from ITC. It might be interesting to compare with dynamics from NMR.

Reviewer #1 (Public Review):

Summary:<br /> Wang and co-workers characterise the fossil of Beretella spinosa from the early Cambrian, Yanjiahe Formation, South China. Combining morphological analyses with phylogenetic reconstructions, the authors conclude that B. spinosa is closely related to Saccorhytus, an enigmatic fossil recently ascribed to Ecdysozoa, or moulting animals, as an extinct "basal" lineage. Finding additional representatives of the clade Saccorhytida strengthens the idea that there existed a diversity of body plans previously underappreciated in Ecdysozoa, which may have implications for our understanding of the earliest steps in the evolution of this major animal group.

Strengths:<br /> I'm not a paleobiologist; therefore, I cannot give an expert opinion on the descriptions of the fossils. However, the similarities with Saccorhytus seem evident, and the phylogenetic reconstructions are adequate. Evolutionary interpretations are generally justified, and the consolidation of Saccorhytida as the extinct sister lineage to extant Ecdysozoans will have significant implications for our understanding of this major animal clade.

Weaknesses:<br /> While I generally agree with the author's interpretations, the idea of Saccorhytida as a divergent, simplified off-shot is slightly contradictory with a probably non-vermiform ecdysozoan ancestor. The author's analyses do not discard the possibility of a vermiform ecdysozoan ancestor (importantly, Supplementary Table 4 does not reconstruct that character), and outgroup comparison with Spiralia (and even Deuterostomia for Protostomia as a whole) indicates that a more or less anteroposteriorly elongated (i.e., vermiform) body is likely common and ancestral to all major bilaterian groups, including Ecdysozoa. Indeed, Figure 4b depicts the potential ancestor as a "worm". The authors argue that the simplification of Saccorhytida from a vermiform ancestor is unlikely "because it would involve considerable anatomical transformations such as the loss of vermiform organization, introvert, and pharynx in addition to that of the digestive system". However, their data support the introvert as a specialisation of Scalidophora (Figure 4a and Supplementary Table 4), and a pharyngeal structure cannot be ruled out in Saccorhytida. Likewise, loss of an anus is not uncommon in Bilateria. Moreover, this can easily become a semantics discussion (to what extent can an animal be defined as "vermiform"? Where is the limit?). Therefore, I suggest to leave the evolutionary scenario more open. Supporting Saccorhytida as a true group at the early steps of Ecdysozoa evolution is important and demonstrates that animal body plans are more plastic than previously appreciated. However, with the current data, it is unlikely that Saccorhytida represents the ancestral state for Ecdysozoa (as the authors admit), and a vermiform nature is not ruled out (and even likely) in this animal group. Suggesting that the ancestral Ecdysozoan might have been small and meiobenthic is perhaps more interesting and supported by the current data (phylogeny and outgroup comparison with Spiralia).

Reviewer #2 (Public Review):

Summary:<br /> This work provides important anatomical features of a new species from the Lower Cambrian, which helps advance our understanding of the evolutionary origins of animal body plans. The authors interpreted that the new species possessed a bilateral body covered with cuticular polygonal reticulation and a ventral mouth. Based on cladistic analyses using maximum likelihood, Bayesian, and parsimony, the new species was placed, along with Saccorhytus, in a sister group ("Saccorhytida") of the Ecdysozoa. The phylogenetic position of Saccorhytida suggests a new scenario of the evolutionary origin of the crown ecdysozoan body plan.

Strengths:<br /> Although the new species reported in this paper show strange morphologies, the interpretation of anatomical features was based on detailed observations of multiple fossil specimens, thereby convincing at the moment. Morphological data about fossil taxa in the Ediacaran and Early Cambrian are quite important for our understanding of the evolution of body plans (and origins of phyla) in paleontology and evolutionary developmental biology, and this paper represents a valuable contribution to such research fields.

Weaknesses:<br /> The preservations of the specimens, in particular on the putative ventral side, are not good, and the interpretation of the anatomical features needs to be tested with additional specimens in the future. The monophyly of Cycloneuralia (Nematoida + Scalidophora) was not necessarily well-supported by cladistic analyses, and the evolutionary scenario (Figure 4) also needs to be tested in future works.

Reviewer #3 (Public Review):

Summary:<br /> The authors of this manuscript identified the fossils of the newly designated species Beretella spinosa and analyzed its phylogenetic position in relation to the extinct described species and extant species. Their analysis placed the newly described species Beretella spinosa and Saccorhytus as an independent clade from the rest of the ecdysozoans. Remarkably, these species are non-vermiform, and the resulting evolutionary scenario assumes non-vermiform as early ecdysozoans.

Strengths:<br /> The study presents outstanding, novel data and provides new insights into the evolution of animal forms especially regarding their morphological diversity after the Cambrian explosion.

Weaknesses:<br /> I, as a paleontology non-expert, experienced several difficulties in reading the manuscript. This should be taken into consideration when assuming a wide range of readers including non-experts.

Reviewer #1 (Public Review):

Summary:<br /> The evolution of non-shivering thermogenesis is of fundamental importance to understand. Here, in small mammals, the contractile apparatus of the muscle is shown to increase energy expenditure upon a drop in ambient temperature. Additionally, in the state of torpor, small hibernators did not show an increase in energy expenditure under the same challenge.

Strengths:<br /> The authors have conducted a very well-planned study that has sampled the muscles of large and small hibernators from two continents. Multiple approaches were then used to identify the state of the contractile apparatus, and its energy expenditure under torpor or otherwise.

Weaknesses:<br /> There was only one site of biopsy from the animals used (leg). It would be interesting to know if non-shivering thermogenesis is something that is regionally different in the animal, given the core body and distal limbs have different temperatures.

Reviewer #2 (Public Review):

Summary:<br /> The authors utilized (permeabilized) fibers from muscle samples obtained from brown and black bears, squirrels, and Garden dormice, to provide interesting and valuable data regarding changes in myosin conformational states and energetics during hibernation and different types of activity in summer and winter. Assuming that myosin structure is similar between species then its role as a regulator of metabolism would be similar and not different, yet the data reveal some interesting and perplexing differences between the selected hibernating species.

Strengths:<br /> The experiments on the permeabilized fibers are complementary, sophisticated, and well-performed, providing new information regarding the characteristics of skeletal muscle fibers between selected hibernating mammalian species under different conditions (summer, interarousal, and winter).

The studies involve complementary assessments of muscle fiber biochemistry, sarcomeric structure using X-ray diffraction, and proteomic analyses of posttranslational modifications.

Weaknesses:<br /> It would be helpful to put these findings on permeabilized fibers into context with the other anatomical/metabolic differences between the species to determine the relative contribution of myosin energetics (with these other contributors) to overall metabolism in these different species, including factors such as fat volume/distribution.

Reviewer #3 (Public Review):

Summary and strengths:<br /> The manuscript, "Remodelling of skeletal muscle myosin metabolic states in hibernating mammals", by Lewis et al, investigates whether myosin ATP activity may differ between states of hibernation and activity in both large and small mammals. The study interrogates (primarily) permeabilized muscle strips or myofibrils using several state-of-the-art assays, including the mant-ATP assay to investigate ATP utilization of myosin, X-ray diffraction of muscles, proteomics studies, metabolic tests, and computational simulations. The overall data suggests that ATP utilization of myosin during hibernation is different than in active conditions.

A clear strength of this study is the use of multiple animals that utilize two different states of hibernation or torpor. Two large animal hibernators (Eurasian Brown Bear, American Black Bear) represent large animal hibernators that typically undergo prolonged hibernation. Two small animal hibernators (Garden Dormouse, 13 Lined Ground Squirrel) undergo torpor with more substantial reductions in heart rate and body temperature, but whose torpor bouts are interrupted by short arousals that bring the animals back to near-summer-like metabolic conditions.

Especially interesting, the investigators analyze the impact that body temperature may have on myosin ATP utilization by performing assays at two different temperatures (8 and 20 degrees C, in 13 Lined Ground Squirrels).

The multiple assays utilized provide a more comprehensive set of methods with which to test their hypothesis that muscle myosins change their metabolic efficiency during hibernation.

Suggestions and potential weaknesses:<br /> While the samples and assays provide a robust and comprehensive coverage of metabolic needs and testing, the data is less categorical. Some of these may be dependent on sample size or statistical analysis while others may be dependent on interpretation.

(1) Statistical Analysis<br /> (1.a) The results of this study often cannot be assessed properly due to a lack of clarity in the statistical tests.<br /> For example, the results related to the large animal hibernators (Figure 1) do not describe the statistical test (in the text of the results, methods, or figure legends). (Similarly for figure 6 and Supplemental Figure 1). Further, it is not clear whether or when the analysis was performed with paired samples. As the methods described, it appears that the Eurasian Brown Bear data should be paired per animal.

(1.b) The statistical methods state that non-parametric testing was utilized "where data was unevenly distributed". Please clarify when this was used.

(1.c) While there are two different myosin isoforms, the isoform may be considered a factor. It is unclear why a one-way ANOVA is generally used for most of the mant-ATP chase data.

(1.d) While the technical replicates on studies such as the mant-ATP chase assay are well done, the total biological replicates are small. A consideration of the sample power should be included.

(1.e) An analysis of the biological vs statistical significance should be considered, especially for the mant-ATP chase data from the American Black Bear, where there appear to be shifts between the summer and winter data.

(2) Consistency of DRX/SRX data.<br /> (2.a) The investigators performed both mant-ATP chase and x-ray diffraction studies to investigate whether myosin heads are in an "on" or "off" state. The results of these two studies do not appear to be fully consistent with each other, which should not be a surprise. The recent work of Mohran et al (PMID 38103642) suggests that the mant-ATP-predicted SRX:DRX proportions are inconsistent with the position of the myosin heads. The discussion appears to lack a detailed assessment of this prior work and lack a substantive assessment contrasting the differing results of the two assays in the current study. i.e. why the current study's mant-ATP chase and x-ray diffraction results differ.

(2.b) The discussion of the current study's x-ray diffraction data relating to the I_1,1/I_1,0 ratio and how substantially different this is to the M6 results merits discussion. i.e. how can myosin both be more primed to contract during IBA versus torpor (according to intensity ratio), but also have less mass near the thick filament (M6).

(3) Possible interactions with Heat Shock Proteins<br /> Heat Shock Proteins (HSPs), such as HSP70, have been shown to be differential during torpor vs active states. A brief search of HSP and myosin reveals HPSs related to thick filament assembly and Heat Shock Cognate 70 interacting with myosin binding protein C. Especially given the author's discussion of protein stability and the potential interaction with myosin binding protein C and the SRX state, the limitation of not assessing HSPs should be discussed. (While HSP's relation to thick filament assembly might conceivably modify the interpretation of the M3 x-ray diffraction results, this reviewer acknowledges that possibility as a leap.)

Despite the above substantial concerns/weaknesses, this reviewer believes that this manuscript represents a valuable data set.

Other comments related to interpretation:<br /> (4) The authors briefly mention the study by Toepher et al [Ref 25] and that it utilizes cardiac muscles. There would benefit from increased discussion regarding the possible differences in energetics between cardiac and skeletal muscle in these states.

(5) The author's analysis of temperature is somewhat limited.<br /> (5.a) First, the authors use 20 degrees C (room temperature), not 37 degrees C, a more physiologic body temperature for large mammals. While it is true that limbs are likely at a lower temperature, 20 degrees C seems substantially outside of a normal range. Thus, temperature differences may have been minimized by the author's protocol.

(5.b) Second, the authors discuss the possibility of myosin contributing to non-shivering thermogenesis. The magnitude of this impact should be discussed. The suggestion of myosin ATP utilization also implies that there is some basal muscle tone (contraction), as the myosin ATPase utilizes ATP to release from actin, before binding and hydrolyzing again. Evidence of this tone should be discussed.

Reviewer #1 (Public Review):

Ps observed 24 objects and were asked which afforded particular actions (14 action types). Affordances for each object were represented by a 14-item vector, values reflecting the percentage of Ps who agreed on a particular action being afforded by the object. An affordance similarity matrix was generated which reflected similarity in affordances between pairs of objects. Two clusters emerged, reflecting correlations between affordance ratings in objects smaller than body size and larger than body size. These clusters did not correlate themselves. There was a trough in similarity ratings between objects ~105 cm and ~130 cm, arguably reflecting the body size boundary. The authors subsequently provide some evidence that this clear demarcation is not simply an incidental reflection of body size, but likely causally related. This evidence comes in the flavour of requiring Ps to imagine themselves as small as a cat or as large as an elephant and showing a predicted shift in the affordance boundary. The manuscript further demonstrates that ChatGPT (theoretically interesting because it's trained on language alone without sensorimotor information; trained now on words rather than images) showed a similar boundary.

The authors also conducted a small MRI study task where Ps decide whether a probe action was affordable (graspable?) and created a congruency factor according to the answer (yes/no). There was an effect of congruency in posterior fusiform and superior parietal lobule for objects within body size range, but not outside. No effects in LOC or M1.

The major strength of this manuscript in my opinion is the methodological novelty. I felt the correlation matrices were a clever method for demonstrating these demarcations, the imagination manipulation was also exciting, and the ChatGPT analysis provided excellent food for thought. These findings are important for our understanding of the interactions between action and perception, and hence for researchers from a range of domains of cognitive neuroscience.

The major element that limits conclusions is that an MRI study with 12 P in this context can really only provide pilot data. Certainly the effects are not strong enough for 12 P to generate much confidence. The others of my concerns have been addressed in the revision.

Reviewer #2 (Public Review):

Summary

In this work, the authors seek to test a version of an old idea, which is that our perception of the world and our understanding of the objects in it are deeply influenced by the nature of our bodies and the kinds of behaviours and actions that those objects afford. The studies presented here muster three kinds of evidence for a discontinuity in the encoding of objects, with a mental "border" between objects roughly of human body scale or smaller, which tend to relate to similar kinds of actions that are yet distinct from the kinds of actions implied by human-or-larger scale objects. This is demonstrated through observers' judgments of the kinds of actions different objects afford; through similar questioning of AI large-language models (LLMs); and through a neuroimaging study examining how brain regions implicated in object understanding make distinctions between kinds of objects at human and larger-than-human scales.

Strengths 

The authors address questions of longstanding interest in the cognitive neurosciences -- namely how we encode and interact with the many diverse kinds of objects we see and use in daily life. A key strength of the work lies in the application of multiple approaches. Examining the correlations among kinds of objects, with respect to their suitability for different action kinds, is novel, as are the complementary tests of judgments made by LLMs. The authors include a clever manipulation in which participants are asked to judge action-object pairs, having first adopted the imagined size of either a cat or an elephant, showing that the discontinuity in similarity judgments effectively moved to a new boundary closer to the imagined scale than the veridical human scale. The dynamic nature of the discontinuity hints that action affordances may be computed dynamically, "on the fly", during actual action behaviours with objects in the real world.

Weaknesses 

A limitation of the tests of LLMs may be that it is not always known what kinds of training material was used to build these models, leading to a possible "black box" problem. Further, presuming that those models are largely trained on previous human-written material, it may not necessarily be theoretically telling that the "judgments" of these models about action-object pairs shows human-like discontinuities. Indeed, verbal descriptions of actions are very likely to mainly refer to typical human behaviour, and so the finding that these models demonstrate an affordance discontinuity may simply reflect those statistics, rather than providing independent evidence for affordance boundaries.

The relatively small sample size of the brain imaging experiment, and some design features (such as the task participants performed, and the relatively narrow range of objects tested) provide some limits on the extent to which it can be taken as support for the authors' claims.

Reviewer #3 (Public Review):

Summary:<br /> Feng et al. test the hypothesis that human body size constrains the perception of object affordances, whereby only objects that are smaller than the body size will be perceived as useful and manipulable parts of the environment, whereas larger objects will be perceived as "less interesting components."

To test this idea, the study employs a multi-method approach consisting of three parts:

In the first part, human observers classify a set of 24 objects that vary systematically in size (e.g., ball, piano, airplane) based on 14 different affordances (e.g., sit, throw, grasp). Based on the average agreement of ratings across participants, the authors compute the similarity of affordance profiles between all object pairs. They report evidence for two homogenous object clusters that are separated based on their size with the boundary between clusters roughly coinciding with the average human body size. In follow-up experiments, the authors show that this boundary is larger/smaller in separate groups of participants who are instructed to imagine themselves as an elephant/cat.

In the second part, the authors ask different large language models (LLMs) to provide ratings for the same set of objects and affordances and conduct equivalent analyses on the obtained data. Some, but not all, of the models produce patterns of ratings that appear to show similar boundary effects, though less pronounced and at a different boundary size than in humans.

In the third part, the authors conduct an fMRI experiment. Human observers are presented with four different objects of different sizes and asked if these objects afford a small set of specific actions. Affordances are either congruent or incongruent with objects. Contrasting brain activity on incongruent trials against brain activity on congruent trials yields significant effects in regions within the ventral and dorsal visual stream, but only for small objects and not for large objects.

The authors interpret their findings as support for their hypothesis that human body size constrains object perception. They further conclude that this effect is cognitively penetrable, and only partly relies on sensorimotor interaction with the environment (and partly on linguistic abilities).

Strengths:<br /> The authors examine an interesting and relevant question and articulate a plausible (though somewhat underspecified) hypothesis that certainly seems worth testing. Providing more detailed insights into how object affordances shape perception would be highly desirable. Their method of analyzing similarity ratings between sets of objects seems useful and the multi-method approach is original and interesting.

Weaknesses:<br /> The study presents several shortcomings that clearly weaken the link between the obtained evidence and the drawn conclusions. Below I outline my concerns in no particular order:

(1) It is not entirely clear to me what the authors are proposing and to what extent the conducted work actually speaks to this. For example, in the introduction, the authors write that they seek to test if body size serves not merely as a reference for object manipulation but also "plays a pivotal role in shaping the representation of objects." This motivation seems rather vague motivation and it is not clear to me how it could be falsified.

Overall, the lack of theoretical precision makes it difficult to judge the appropriateness of the approaches and the persuasiveness of the obtained results. I would strongly suggest clarifying the theoretical rationale and explaining in more detail how the chosen experiments allow them to test falsifiable predictions.

(2) The authors used only a very small set of objects and affordances in their study and they do not describe in sufficient detail how these stimuli were selected. This renders the results rather exploratory and clearly limits their potential to discover general principles of human perception. Much larger sets of objects and affordances and explicit data-driven approaches for their selection would provide a more convincing approach and allow the authors to rule out that their results are just a consequence of the selected set of objects and actions.

(3) Relatedly, the authors could be more thorough in ruling out potential alternative explanations. Object size likely correlates with other variables that could shape human similarity judgments and the estimated boundary is quite broad (depending on the method, either between 80 and 150 cm or between 105 to 130 cm). More precise estimates of the boundary and more rigorous tests of alternative explanations would add a lot to strengthen the authors' interpretation.

(4) While I appreciate the manipulation of imagined body size, as a clever way to solidify the link between body size and affordance perception, I find it unfortunate that it is implemented in a between-subjects design, as this clearly leaves open the possibility of pre-existing differences between groups. I certainly disagree with the authors' statement that their findings suggest "a causal link between body size and affordance perception."

(5) The use of LLMs in the current study is not clearly motivated and I find it hard to understand what exactly the authors are trying to test through their inclusion. As it currently stands, I find it hard to discern how the presence of perceptual boundaries in LLMs could constitute evidence for affordance-based perception.

(6) Along the same lines, the fMRI study also provides little evidence to support the authors' claims. The use of congruency effects as a way of probing affordance perception is not well motivated. Importantly (and related to comment 2 above), the very small set of objects and affordances in this experiment heavily complicates any conclusions about object size being the crucial variable determining the occurrence of congruency effects.

Overall, I consider the main conclusions of the paper to be far beyond the reported data. Articulating a clearer theoretical framework with more specific hypotheses as well as conducting more principled analyses on more comprehensive data sets could help the authors obtain stronger tests of their ideas.

Reviewer #1 (Public Review):

Summary:<br /> The authors sought to understand the stage-dependent regulation of exophergenesis, a process thought to contribute to promoting neuronal proteostasis in C. elegans. Focusing on the ALMR neuron, they show that the frequency of exopher production correlates with the timing of reproduction. Using many genetic tools, they dissect the requirements of this pathway to eventually find that occupancy of the uterus acts as a signal to induce exophergenesis. Interestingly, the physical proximity of neurons to the egg zone correlates with exophergenesis frequency. The authors conclude that communication between the uterus and proximal neurons occurs through the sensing of mechanic forces of expansion normally provided by egg occupancy to coordinate exophergenesis with reproduction.

Strengths:<br /> The genetic data presented is thorough and solid, and the observation is novel.

Weaknesses:<br /> The main weakness of the study is that the detection of exophers is based on the overexpression of a fluorescent protein in touch neurons, and it is not clear whether this process is actually stimulated in wild-type animals, or if neurons have accumulated damaged proteins in relatively young day 2 animals.

Reviewer #2 (Public Review):

Summary:<br /> This paper reports that mechanical stress from egg accumulation is a biological stimulus that drives the formation of extruded vesicles from the neurons of C. elegans ALMR touch neurons. Using powerful genetic experiments only readily available in the C. elegans system, the authors manipulate oocyte production, fertilization, embryo accumulation, and egg-laying behavior, providing convincing evidence that exopher production is driven by stretch-dependent feedback of fertilized, intact eggs in the adult uterus. Shifting the timing of egg production and egg laying alters the onset of observed exophers. Pharmacological manipulation of egg laying has the predicted effects, with animals retaining fewer eggs having fewer exophers and animals with increased egg accumulation having more. The authors show that egg production and accumulation have dramatic consequences for the viscera, and moving the ALMR process away from eggs prevents the formation of exophers. This effect is not unique to ALMR but is also observed in other touch neurons, with a clear bias toward neurons whose cell bodies are adjacent to the filled uterus. Embryos lacking an intact eggshell with reduced rigidity have impaired exopher production. Acute injection into the uterus to mimic the stretch that accompanies egg production causes a similar induction of exopher release. Together these results are consistent with a model where stretch caused by fertilized embryo accumulation, and not chemical signals from the eggs themselves or egg release, underlies ALMR exopher production seen in adult animals.

Strengths:<br /> Overall, the experiments are very convincing, using a battery of RNAi and mutant approaches to distinguish direct from indirect effects. Indeed, these experiments provide a model generally for how one would methodically test different models for exopher production. The paper is well-written and easy to understand. I had been skeptical of the origin and purpose of exophers, concerned they were an artefact of imaging conditions, caused by deranged calcium activity under stressful conditions, or as evidence for impaired animal health overall. As this study addresses how and when they form in the animal using otherwise physiologically meaningful manipulations, the stage is now set to address at a cellular level how exophers like these are made and what their functions are.

Weaknesses:<br /> Not many. The experiments are about as good as could be done. Some of the n's on the more difficult-to-work strains or experiments are comparatively low, but this is not a significant concern because of the number of different, complementary approaches used. The microinjection experiment in Figure 7 is very interesting, there are missing details that would confirm whether this is a sound experiment.

Reviewer #3 (Public Review):

Summary:<br /> In this paper, the authors use the C. elegans system to explore how already-stressed neurons respond to additional mechanical stress. Exophers are large extracellular vesicles secreted by cells, which can contain protein aggregates and organelles. These can be a way of getting rid of cellular debris, but as they are endocytosed by other cells can also pass protein, lipid, and RNA to recipient cells. The authors find that when the uterus fills with eggs or otherwise expands, a nearby neuron (ALMR) is far more likely to secrete exophers. This paper highlights the importance of the mechanical environment in the behavior of neurons and may be relevant to the response of neurons exposed to traumatic injury.

Strengths:<br /> The paper has a logical flow and a compelling narrative supported by crisp and clear figures.

The evidence that egg accumulation leads to exopher production is strong. The authors use a variety of genetic and pharmacological methods to show that increasing pressure leads to more exopher production, and reducing pressure leads to lower exopher production. For example, egg-laying defective animals, which retain eggs in the uterus, produce many more exophers, and hyperactive egg-laying is accompanied by low exopher production. The authors even inject fluid into the uterus and observe the production of exophers.

Weaknesses:<br /> The main weakness of the paper is that it does not explore the molecular mechanism by which the mechanical signals are received or responded to by the neuron, but this could easily be the subject of a follow-up study.

I was intrigued by this paper, and have many questions. I list a few below, which could be addressed in this paper or which could be the subject of follow-up studies.

- Why do such a low percentage of ALMR neurons produce exophers (5-20%)? Does it have to do with the variability of the proteostress?<br /> - Why does the production of exophers lag the peak in progeny production by 24-48 hours? Especially when the injection method produces exophers right away?<br /> - As mentioned in the discussion, it would be interesting to know if PEZO-1/PIEZO is required for uterine stretching to activate exophergenesis. pezo-1 animals accumulate crushed oocytes in the uterus.

Reviewer #1 (Public Review):

Summary:<br /> Kimura et al performed a saturation mutagenesis study of CDKN2A to assess the functionality of all possible missense variants and compare them to previously identified pathogenic variants. They also compared their assay result with those from in silico predictors.

Strengths:<br /> CDKN2A is an important gene that modulates cell cycle and apoptosis, therefore it is critical to accurately assess the functionality of missense variants. Overall, the paper reads well and touches upon major discoveries in a logical manner.

Weaknesses:<br /> The paper lacks proper details for experiments and basic data, leaving the results less convincing. Analyses are superficial and do not provide variant-level resolution.

Reviewer #2 (Public Review):

This study describes a deep mutational scan across CDKN2A using suppression of cell proliferation in pancreatic adenocarcinoma cells as a readout for CDKN2A function. The results are also compared to in silico variant predictors currently utilized by the current diagnostic frameworks to gauge these predictors' performance. The authors also functionally classify CDKN2A somatic mutations in cancers across different tissues.

This study is a potentially important contribution to the field of cancer variant interpretation for CDKN2A, but is almost impossible to review because of the severe lack of details regarding the methods and incompleteness of the data provided with the paper. We do believe that the cell proliferation suppression assay is robust and works, but when it comes to the screening of the library of CDKN2A variants the lack of primary data and experimental detail prevents assessment of the scientific merit and experimental rigor.

Reviewer #1 (Public Review):

Summary:<br /> This study investigated the co-option of IGF2BP2, an RNA-binding protein by ZIKV proteins. Designed experiments evaluated if IFG2BP2 co-localized to sites of viral RNA replication, interacted with ZIKV proteins, and how ZIKV infection changed the IGF2BP2 interactome.

Strengths:<br /> The authors have used multiple interdisciplinary techniques to address several questions regarding the interaction of ZIKV proteins and IGF2BP2.<br /> The findings could be exciting, specifically regarding how ZIKV infection alters the interactome of IGF2BP2.

Weaknesses:<br /> Significant concerns regarding the current state of the figures, descriptions in the figure legends, and the quality of the immunofluorescence and electron microscopy exist.

Reviewer #2 (Public Review):

Clément Mazeaud et al. identified the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a proviral cellular protein that regulates Zika virus RNA replication by modulating the biogenesis of virus-induced replication organelles.

The absence of IGF2BP2 specifically dampens ZIKV replication without having a major impact on DENV replication. The authors show that ZIKV infection changes IGF2BP2 cellular distribution, which relocates to the perinuclear viral replication compartment. These assays were conducted by infecting cells with an MOI of 10 for 48 hours. Considering the ZIKV life cycle, it is noteworthy that at this time there may be a cytopathic effect. One point of concern arises regarding how the authors can ascertain that the observed change in localization is a consequence of the infection rather than of the cytopathic effect. To address this concern, shorter infection periods (e.g., 24 hours post-infection) or additional controls, such as assessing cellular proteins that do not change their localization or infecting with another flavivirus lacking the IGF2BP2 effect, could be incorporated into their experiments.

By performing co-immunoprecipitation assays on mock and infected cells that express HA-tagged IGF2BP2, the authors propose that the observed change in IGF2BP2 localization results from its recruitment to the replication compartment by the viral NS5 polymerase and associated with the viral RNA. Given that both IGF2BP2 and NS5 are RNA-binding proteins, it is plausible that their interaction is mediated indirectly through the RNA molecule. Notably, the authors do not address the treatment of lysates with RNAse before the IP assay, leaving open the possibility of this indirect interaction between IGF2BP2 and NS5.

In in vitro binding assays, the authors demonstrate that the RNA-recognition motifs of the IGF2BP2 protein specifically bind to the 3' nontranslated region (NTR) of the ZIKV genome, excluding binding to the 5' NTR. However, they cannot rule out the possibility of this host protein associating with other regions of the viral genome. Using a reporter ZIKV subgenomic replicon system in IGF2BP2 knock-down cells, they additionally demonstrate that IGF2BP2 enhances viral genome replication. Despite its proviral function, the authors note that the "overexpression of IGF2BP2 had no impact on total vRNA levels." However, the authors do not delve into a discussion of this latter statement.

In this study, the authors extend their findings by illustrating that ZIKV infection triggers a remodeling of IGF2BP2 ribonucleoprotein complex. They initially evaluate the impact of ZIKV infection on IGF2BP2's interaction with its endogenous mRNA ligands. Their results reveal that viral infection alters the binding of specific mRNA ligands, yet the physiological consequences of this loss of binding in the cell remain unexplored. Additionally, the authors demonstrate that ZIKV infection modifies the IGF2BP2 interactome. Through proteomic assays, they identified 62 altered partners of IGF2BP2 following ZIKV infection, with proteins associated with mRNA splicing and ribosome biogenesis being the most represented. In particular, the authors focused their research on the heightened interaction between IGF2BP2 and Atlastin 2, an ER-shaping protein reported to be involved in flavivirus vesicle packet formation. The validation of this interaction by Western blot assays prompted an analysis of the effect of ZIKV on organelle biogenesis using a newly described replication-independent vesicle packet induction system. Consequently, the authors demonstrate that IGF2BP2 plays a regulatory role in the biogenesis of ZIKV replication organelles.

Based on these findings and previously published data, the authors propose a model outlining the role of IGF2BP2 in ZIKV infectious cycle, detailing the changes in IGF2BP2 interactions with both cellular and viral proteins and RNAs that occur during viral infection.

The conclusions drawn in this paper are generally well substantiated by the data. However, it is worth noting that the majority of infections were conducted at a high MOI for 48 hours, spanning more than one infectious cycle. To enhance the robustness of their findings and mitigate potential cell stress, it would be valuable to observe these effects at shorter time intervals, such as 24 hours post-infection.<br /> Furthermore, the assertion regarding the association of IGF2BP2 with NS5 could be strengthened through additional immunoprecipitation (IP) assays. These assays, performed in the presence of RNAse treatment, would help exclude the possibility of an indirect interaction between IGF2BP2 and NS5 (both RNA-binding proteins) through viral RNA, thus providing more confidence in the observed association.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Mazeaud and colleagues pursued a small-scale screen of a targeted RNAi library to identify novel players involved in Zika (ZIKV) and dengue (DENV) virus replication. Loss-of-function of IGF2BP2 resulted in reduced titers for ZIKV of the Asian and African lineages in hepatic Huh7.5 cells, but not for either of the four DENV serotypes nor West Nile virus (WNV). The phenotype was further confirmed in two additional cell lines and using a ZIKV reporter virus. In addition, using immunoprecipitation assays the interaction between IGF2BP2 and ZIKV NS5 protein and RNA genome was detected. The work addressed the role of IGF2BP2 in the infected cell combining confocal microscopy imaging, and proteomic analysis. The approach indicated an altered distribution of IGF2BP2 in infected cells and changes in the protein interactome including disrupted association with partner mRNAs and modulation of the abundance of a specific set of protein partners in IGF2BP2 immunoprecipitated ribonucleoprotein (RNP) complexes. Finally, based on the changes in IGF2BP2 interactome and specifically the increment in the abundance of Atlastin 2, the biogenesis of ZIKV replication organelles (vRO) is investigated using a genetic system that allows virus replication-independent assembly of vRO. Electron microscopy showed that knockdown of IGF2BP2 expression reduced the number of cells with vRO.

Strengths:<br /> The role of IGF2BP2 as a proviral factor for ZIKV replication is novel.

The study follows a logical flow of experiments that altogether support the assembly of a specialized RNP complex containing IGF2BP2 and ZIKV NS5 and RNA genome.

Weaknesses:<br /> The statistical analysis should clearly indicate the number of biological replicates of experiments to support statistical significance.

The claim that IGF2BP2 knockdown impairs de novo viral organelle biogenesis and viral RNA synthesis is built upon data that show a reduction in RNA synthesis <0.5-fold as assessed using a reporter replicon, thus suggesting a limited impact of the knockdown on RNA replication.

Validation of IGF2BP2 partners that are modulated upon ZIKV infection (i.e. virus yield in knocked down cells) can be relevant especially for partners such as Atlastin 2, as the hypothesis of a role for IGF2BP2 RNP in vRO biogenesis is based on the observed increase in the abundance of Atlastin 2 in the RNP complex preciìtated from infected cells.

Reviewer #1 (Public Review):

The issue:<br /> The ciliates are a zoo of genetic codes, where there have been many reassignments of stop codons, sometimes with conditional meanings which include retention of termination function, and thus > 1 meaning. Thus ciliate coding provides a hotspot for the study of genetic code reassignments.

The particular issue here is the suggestion that translation of a stop (UGA) in Blastocritihidia has been attributed to a joint change in the protein release factor that reads UGA's and also breaking a base pair at the top of the anticodon stem of tRNATrp (Nature 613, 751, 2023).

The work:<br /> However, Swart, et al have looked into this suggestion, and find that the recently suggested mechanism is overly complicated.

The broken pairing at the top of the anticodon stem of tRNATrp indeed accompanies the reading of UGA as Trp as previously suggested. It changes the codon translated even though the anticodon remains CCA, complementary to UGG. A compelling point is that this misreading matches previous mutational studies of E coli tRNA's, in which breaking the same base pair in a mutant tRNATrp suppressor tRNA stimulated the same kind of miscoding.

But the amino acid change in release factor eRF1, the protein that catalyzes termination of protein biosynthesis at UGA is broadly distributed. There are about 9 organisms where this mutation can be compared with the meaning of UGA, and the changes are not highly correlated with a change in the meaning of the codon. Therefore, because UGA can be translated as Trp with or without the eRF1 mutation, Swart et al suggest that the tRNA anticodon stem change is the principal cause of the coding change.

The review:<br /> Swart et al have a good argument. I would only add that eRF1 participation is not ruled out, because finding that UGA encodes Trp does not distinguish between encoding Trp 90% of the time and encoding it 99% of the time. The release factor could still play a measurable quantitative role, but the major inference here seems convincing.

Reviewer #2 (Public Review):

The manuscript raises interesting observations about the potential evolution of release factors and tRNA to readdress the meaning of stop codons. The manuscript is divided into two parts: The first consists of revealing that the presence of a trp tRNA with an AS of 5bp in Condylostoma magnum is probably linked to contamination in the databases by sequences from bacteria. This is an interesting point which seems to be well supported by the data provided. It highlights the difficulty of identifying active tRNA genes from poorly annotated or incompletely assembled genomes. The second part criticises the fact that a mutation at position S67 of eRF1 is required to allow the UGA codon to be reassigned as a sense codon. As supporting evidence, they provide a phylogenetic study of the eRF1 factor showing that there are numerous ciliates in which this position is mutated, whereas the organism shows no trace of the reassignment of the UGA codon into a sense codon. While this criticism seems valid at first glance, it suffers from the lack of information on the level of translation of UGA codons in the organisms considered. It has been clearly shown that S67G or S67A mutations allow a strong increase in the reading of UGA codons by tRNAs, so this point is not in doubt. However, this has been demonstrated in model organisms, and we now need to determine whether other changes in the translational apparatus could accompany this mutation by modifying its impact on the UGA codon. This is a point partly raised at the end of the manuscript. Indeed, it is quite possible that in these organisms the UGA codon is both used to complete translation and is subject to a high level of readthrough. Actually, in the presence of a mutation at position 67 (or elsewhere), the reading of the UGA can be tolerated under specific stress conditions (nutrient deficiency, oxidative stress, etc.), so the presence of this mutation could allow translational control of the expression of certain genes. On the other hand, it seems obvious to me that there are other ways of reading through a stop codon without mutating eRF1 at position S67. So the absence of a mutation at this position is not really indicative of a level of reading of the UGA codon. Before writing such a strong assertion as that found on page 3, experiments should be carried out. The authors should therefore moderate their assertion.

To make a definitive conclusion, we would need to be able to measure the level of termination and readthrough in these organisms. So, from my point of view, all the arguments seem rather weak. Moreover, the authors themselves indicate that the conjunction between a Trp tRNA that is efficient at reading the UGA codon and an eRF1 factor that is not efficient at recognising this stop codon could be the key to reassignment.

Reviewer #1 (Public Review):

Summary: In this manuscript, the authors performed single nucleus RNA-seq for perirenal adipose tissue (PRAT) at different ages. They concluded a distinct subpopulation of adipocytes arises through beige-to-white conversion and can convert to a thermogenic phenotype upon cold exposure.

Strengths: PRAT adipose tissue has been reported as an adipose tissue that undergoes browning. This study confirms that beige-to-white and white-to-beige conversions also exist in PRAT, as previously reported in the subcutaneous adipose tissue.

Weaknesses:<br /> (1) There is overall a disconnection between single nucleus RNA-seq data and the lineage chasing data. No specific markers of this population have been validated by staining.<br /> (2) It would be nice to provide more evidence to support the conclusion shown in lines 243 to 245 "These results indicated that new BAs induced by cold exposure were mainly derived from UCP1- adipocytes rather than de novo ASPC differentiation in puPRAT". Pdgfra-negative progenitor cells may also contribute to these new beige adipocytes.<br /> (3) The UCP1Cre-ERT2; Ai14 system should be validated by showing Tomato and UCP1 co-staining right after the Tamoxifen treatment.

Reviewer #2 (Public Review):

Summary:<br /> In the present manuscript, Zhang et al utilize single-nuclei RNA-Seq to investigate the heterogeneity of perirenal adipose tissue. The perirenal depot is interesting because it contains both brown and white adipocytes, a subset of which undergo functional "whitening" during early development. While adipocyte thermogenic transdifferentiation has been previously reported, there remains many unanswered questions regarding this phenomenon and the mechanisms by which it is regulated.

Strengths:<br /> The combination of UCP1-lineage tracing with the single nuclei analysis allowed the authors to identify four populations of adipocytes with differing thermogenic potential, including an "whitened" adipocyte (mPRAT-ad2) that retains the capacity to rapidly revert to a brown phenotype upon cold exposure. They also identify two populations of white adipocytes that do not undergo browning with acute cold exposure.

Anatomically distinct adipose depots display interesting functional differences, and this work contributes to our understanding of one of the few brown depots present in humans.

Weaknesses:<br /> The most interesting aspect of this work is the identification of a highly plastic mature adipocyte population with the capacity to switch between a white and brown phenotype. The authors attempt to identify the transcriptional signature of this ad2 subpopulation, however the limited sequencing depth of single nuclei somewhat lessens the impact of these findings. Furthermore, the lack of any form of mechanistic investigation into the regulation of mPRAT whitening limits the utility of this manuscript. However, the combination of well-executed lineage tracing with comprehensive cross-depot single-nuclei presented in this manuscript could still serve as a useful reference for the field.

Reviewer #1 (Public Review):

Summary:<br /> In their revised manuscript, the authors analyze the evolution of the gasdermin family and observe that the GSDMA proteins from birds, reptiles and amphibians does not form a clade with the mammalian GSDMAs. Moreover, the non-mammalian GSDMA proteins share a conserved caspase-1 cleavage motif at the predicted activation site. The authors provide several series of experiments showing that the non-mammalian GSDMA proteins can indeed be activated by caspase-1 and that this activation leads to cell death (in human cells). They also investigate the role of the caspase-1 recognition tetrapeptide for cleavage by caspase-1 and for the pathogen-derived protease SpeB.

Strengths:<br /> The evolutionary analysis performed in this manuscript appears to use a broader data basis than what has been used in other published work. An interesting result of this analysis is the suggestion that GSDMA is evolutionary older than the main mammalian pyroptotic GSDMD, and that birds, reptiles and amphibians lack GSDMD but use GSDMA for the same purpose. The consequence that bird GSDMA should be activated by an inflammatory caspase (=caspase1) is convincingly supported by the experiments provided in the manuscript.

The changes made by the authors in response to the previous reviewer comments are (in my opinion) sufficient.

Reviewer #2 (Public Review):

Summary:

The authors investigated the molecular evolution of members of the gasdermin (GSDM) family. By adding the evolutionary time axis of animals, they created a new molecular phylogenetic tree different from previous ones. The analyzed result verified that non-mammalian GSDMAs and mammalian GSDMAs have diverged into completely different and separate clades. Furthermore, by biochemical analyses, the authors demonstrated non-mammalian GSDMA proteins are cleaved by the host-encoded caspase-1. They also showed mammalian GSDMAs have lost the cleavage site recognized by caspase-1. Instead, the authors proposed that the newly appeared GSDMD is now cleaved by caspase-1.

Through this study, we have been able to understand the changes in the molecular evolution of GSDMs, and by presenting the cleavage of GSDMAs through biochemical experiments, we have become able to grasp the comprehensive picture of this family molecules. However, there are some parts where explanations are insufficient, so supplementary explanations and experiments seem to be necessary.

Strengths:

It has a strong impact in advancing ideas into the study of pyroptotic cell death and even inflammatory responses involving caspase-1.

Weaknesses:

Based on the position of mammalian GSDMA shown in the molecular phylogenetic tree (Figure 1), it may be difficult to completely agree with the authors' explanation of the evolution of GSDMA.

(1) Focusing on mammalian GSDMA, this group and mammalian GSDMD diverged into two clades, and before that, GSDMA/D groups and mammalian GSDMC separated into two, more before that, GSDMB, and further before that, non-mammalian GSDMA, when we checked Figure 1. In the molecular phylogenetic tree, it is impossible that GSDMA appears during evolution again. Mammalian GSDMAs are clearly paralogous molecules to non-mammalian GSDMAs in the figure. If they are bona fide orthologous, the mammalian GSDMA group should show a sub-clade in the non-mammalian GSDMA clade. It is better to describe the plausibility of the divergence in the molecular evolution of mammalian GSDMA in the Discussion section.

(2) Regarding (1), it is recommended that the authors reconsider the validity of estimates of divergence dates by focusing on mammalian species divergence. Because the validity of this estimation requires recheck of the molecular phylogenetic tree, including alignment.

(3) If GSDMB and/or GSDMC between non-mammalian GSDMA and mammalian GSDMD as shown in the molecular phylogenetic tree would be cleaved by caspase-1, the story of this study becomes clearer. The authors should try that possibility.

Reviewer #1 (Public Review):

Summary:

Spinal cord injury (SCI) causes immediate and prolonged bladder dysfunction, for which there are poor treatments. Following up on evidence that AMPA glutamatergic receptors play a key role in bladder function, the authors induced spinal cord injury and its attendant bladder dysfunction and examined the effects of graded doses of allosteric AMPA receptor activators (ampakines). They show that ampakines ameliorate several prominent derangements in bladder function resulting from SCI, improving voiding intervals and pressure thresholds for voiding and sphincter function.

Strengths:

Well performed studies on a relevant model system. The authors induced SCI reproducibly and showed that they had achieved their model. The drugs revealed clear and striking effects. Notably, in some mice which had such bad SCI that they could not void, the drug appeared to restore voiding function.

Weaknesses:

The studies are well conducted, but it would be helpful to include information on the kinetics of the drugs used, their half-life and how long they are present in rats after administration. What blood levels of the drugs are achieved after infusion? How do these compare with blood levels achieved when these drugs are used in humans?

Reviewer #2 (Public Review):

Summary:

In this study, Rana and colleagues present interesting findings demonstrating potential beneficial effects of AMPA receptor modulator with ampakines in the context of neurogenic bladder following acute spinal cord injury. Neurogenic bladder dysfunction is characterized by urinary retention and/or incontinence, with limited treatments available. Based on recent observations showing that ampakines improved respiratory function in rats with SCI, the authors explored the use of ampakine CX1739 on bladder and external urethral sphincter (EUS) function and coordination early after mid-thoracic contusion injury. Using continuous flow cystometry and EUS myography the authors showed that ampakine treatment led to decreased peak pressures, threshold pressure, intercontraction interval and voided volume in SCI rats versus vehicle-treated controls. Although CX1739 did not alter EUS EMG burst duration, treatment did lead to EUS EMG bursting at lower bladder pressure compared to baseline. In a subset of rats that did not show regular cystometric voiding, CX1739 treatment diminished non-voiding contractions and improved coordinated EUS EMG bursting. Based on these findings the authors conclude that ampakines may have utility in recovery of bladder function following SCI.

Strengths and Weaknesses:

The experimental design is thoughtful and rigorous, providing evaluation of both the bladder and external urethral sphincter function in the absence and presence of ampakine treatment. The data in support of a role for CX1789 treatment in the context of neurogenic bladder are presented clearly, and the conclusions are adequately supported by the findings. The authors have addressed essentially all of the weaknesses related to translational significance, CX1789 half-life, and the use of female animals only in this study.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Rana and colleagues examined the effect of a "low impact" ampakine, an AMPA receptor allosteric modulator, on the voiding function of rats subjected to midline T9 spinal cord contusion injury. Previous studies have shown that the micturition reflex fully depends on AMPA glutaminergic signaling, and, that the glutaminergic circuits are reorganized after spinal cord injury. In chronic paraplegic rats, other circuits (no glutaminergic) become engage in the spinal reflex mechanism controlling micturition. The authors employed continuous flow cystometry and external urethral sphincter electromyography to assess bladder function and bladder-urethral sphincter coordination in naïve rats (control) and rats subjected to spinal cord injury (SCI). In the acute phase after SCI, rats exhibit larger voids with lower frequency than naïve rats. This study shows that CX1739 improves, in a dose-dependent manner, bladder function in rats with SCI. The interval between voids and the voided volume were reduced in rat with SCI when compared to controls. In summary, this is an interesting study that describes a potential treatment for patients with SCI.

Strengths:

The findings described in this manuscript are significant because neurogenic bladder predisposes patients with SCI to urinary tract infections, hydronephrosis and kidney failure. The manuscript is clearly written. The study is technically outstanding, and the conclusions are well justified by the data.

Weaknesses:

The study was conducted 5 days after spinal cord contusion when the bladder is underactive. In rats with chronic SCI, the bladder is overactive. Therefore, the therapeutic approach described here is expected to be effective only in the underactive bladder phase of SCI. The mechanism and site of action of CX1739 is not defined.

Reviewer #1 (Public Review):

Summary:<br /> 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 the 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:<br /> (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:<br /> (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 #2 (Public Review):

Summary:<br /> This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, the authors showed that TNXB knockdown leads to chondrocyte apoptosis, matrix degeneration, and subchondral bone loss in vivo/vitro. Furthermore, AKT agonists promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:<br /> In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:<br /> Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Dr. Chen et al. investigates the genes that are differentially methylated and associated with cartilage degeneration in hemophilia patients. The study demonstrates the functional mechanisms of the TNXB gene in chondrocytes and F8-/- mice. The authors first showed significant DNA methylation differences between hemophilic arthritis (HA) and osteoarthritis through genome-wide DNA methylation analysis. Subsequently, they showed a decreased expression of the differentially methylated TNXB gene in cartilage from HA patients and mice. By knocking down TNXB in vivo and in vitro, the results indicated that TNXB regulates extracellular matrix homeostasis and apoptosis by modulating p-AKT. The findings are novel and interesting, and the study presents valuable information in blood-induced arthritis research.

Strengths:<br /> The authors adopted a comprehensive approach by combining genome-wide DNA methylation analysis, in vivo and in vitro experiments using human and mouse samples to illustrate the molecular mechanisms involved in HA progression, which is crucial for developing targeted therapeutic strategies. The study identifies Tenascin XB (TNXB) as a central mediator in cartilage matrix degradation. It provides mechanistic insights into how TNXB influences cartilage matrix degradation by regulating the activation of AKT. It opens avenues for future research and potential therapeutic interventions using AKT agonists for cartilage protection in hemophilic arthropathy. The conclusions drawn from the study are clear and directly tied to the findings.

Weaknesses:<br /> (1) The study utilizes a small sample size (N=5 for both osteoarthritis and hemophilic arthropathy). A larger sample size would enhance the generalizability and statistical power of the findings.<br /> (2) The use of an animal model (F8-/- mouse) to investigate the role of TNXB may not fully capture the complexity of human hemophilic arthropathy. Differences in the biology between species may affect the translatability of the findings to human patients.<br /> (3) The study primarily focuses on TNXB as a central mediator, but it might overlook other potentially relevant factors contributing to cartilage degradation in hemophilic arthropathy. A more holistic exploration of genetic and molecular factors could provide a broader understanding of the condition.

Reviewer #2 (Public Review):

Summary:<br /> The manuscript by Morel et al. aims at identifying some potential mechano-regulators of transendothelial cell macro-aperture (TEM). Guided by the recognized role of caveolar invaginations in buffering the membrane tension of cells, the authors focused on caveolin-1 and associated regulator PTRF. They report a comprehensive in vitro work based on siRNA knockdown and optical imaging approach complemented with an in vivo work on mice, a biophysical assay allowing to measure the mechanical properties of membranes and a theoretical analysis inspired from soft matter physics.

The authors should be complimented for this multi-facetted and rigorous work. The accumulation of pieces of evidence collected from each type of approach makes very convincing the conclusion drawn by the authors on the new role of cavolin-1 as an individual protein instead of the main molecular component of caveolae. On a personal note, I was very impressed by the quality of STORM images (Fig. 2) which are very illuminating and useful, in particular for validating some hypotheses of the theoretical analysis.

While this work pins down the key role of caveolin-1, its mechanism remains to be further investigated. The hypotheses proposed by the authors in the discussions about the link between caveolin and lipids/cholesterol are very plausible though challenging. Even though we may feel slightly frustrated by the absence of data in this direction, the quality and merit of this paper remain.

The analogy with dewetting processes drawn to derive the theoretical model is very attractive. However, and although part of the model has already been published several times by the same group of authors, the validity of the Helfrich formalism is a key assumption that has to be explained clearly. Here, for the first time, thanks to these STORM analysis, the authors show that HUVECs intoxicated by ExoC3 exhibit a loose and defective cortex with a significantly increase mesh size, which supports this hypothesis.

Reviewer #1 (Public Review):

Summary<br /> A new method, tCFS, is introduced to offer richer and more efficient measurement of interocular suppression. It generates a new index, the suppression depth, based on the contrast difference between the up-ramped contrast for the target to breakthrough suppression and the down-ramped contrast for the target to disappear into suppression. A uniform suppression depth regardless of image types (e.g., faces, gratings and scrambles) was discovered in the paper, favoring an early-stage mechanism involving in CFS. Discussions about claims of unconscious processing and the related mechanisms.

Strength<br /> The tCFS method adds to the existing bCFS paradigms by providing the (re-)suppression threshold and thereafter the depression depth. Benefiting from adaptive procedures with continuous trials, the tCFS is able to give fast and efficient measurements. It also provides a new opportunity to test theories and models about how information is processed outside visual awareness.

Weakness:<br /> This paper reports the surprising finding of uniform suppression depth over a variety of stimuli. This is novel and interesting. But given the limited samples being tested, the claim of uniformity suppression depth needs to be further examined, with respect to different complexities and semantic meanings.<br /> From an intuitive aspect, the results challenged previous views about "preferential processing" for certain categories, though it invites further research to explore what exactly could suppression depth tell us about unconscious visual processing. The authors discussed about the possibility of gaining awareness according to different CRF functions in V1 and V4 neurons. But it confuses me about how the logic goes, especially from Line 713 to Line 718.

Reviewer #2 (Public Review):

The following review for a revised manuscript is updated where appropriate and otherwise unchanged for completeness.

Summary<br /> The paper concerns the phenomenon of continuous flash suppression (CFS), relevant to questions about the extent and nature of subconscious visual processing. Whereas standard CFS studies only measure the breakthrough threshold-the contrast at which an initially suppressed target stimulus with steadily increasing contrast becomes visible-the authors also measure the re-suppression threshold, the contrast at which a visible target with decreasing contrast becomes suppressed. Thus, the authors could calculate suppression depth, the ratio between the breakthrough and re-suppression thresholds. To measure both thresholds, the authors introduce the tracking-CFS method, a continuous-trial design that results in faster, better controlled, and lower-variance threshold estimates compared to the discrete trials standard in the literature. The study finds that suppression depths are similar for different image categories, providing an interesting contrast to previous results that breakthrough thresholds differ for different image categories. The new finding calls for a reassessment of interpretations based solely on the breakthrough threshold that subconscious visual processing is category-specific.

Strengths<br /> (1) The tCFS method quickly estimates breakthrough and re-suppression thresholds using continuous trials, which also better control for slowly varying factors such as adaptation and attention. Indeed, tCFS produces estimates with lower across-subject variance than the standard discrete-trial method (Fig. 2). The tCFS method is straightforward to adopt in future research on CFS and binocular rivalry.

(2) The CFS literature has lacked re-suppression threshold measurements. By measuring both breakthrough and re-suppression thresholds, this work calculated suppression depth (i.e., the difference between the two thresholds), which warrants different interpretations from the breakthrough threshold alone.

(3) The work found that different image categories show similar suppression depths, suggesting some aspects of CFS are not category-specific. This result enriches previous findings that breakthrough thresholds vary with image categories. Re-suppression thresholds vary symmetrically, such that their differences are constant.

Weakness<br /> The following concern remains from my initial review. Reviewer #3 raised a similar point in the last revision round, and I believe the authors do not fully address either comment. Thus, here I paraphrase my initial concern with reference to the authors' reply and discuss why it needs further elaboration.

I do not follow the authors' reasoning as to why the suppression depth is a better (or fuller, superior, more informative) indication of subconscious visual processing than the breakthrough threshold alone. To my previous round of comments, the authors replied that 'breakthrough provides only half of the needed information.' I do not understand this. One cannot infer the suppression depth from the breakthrough threshold alone, but *one cannot obtain the breakthrough threshold from the suppression depth alone*, either. The two measures are complementary. (To be sure, given *both* the suppression depth and the re-suppression threshold, one can trivially recover the breakthrough threshold. The discussion concerns the suppression depth *alone* and the breakthrough threshold *alone*.) I am fully open to being convinced that there is a good reason why the suppression depth may be more informative than the breakthrough threshold about a specific topic, e.g., inter-ocular suppression or subconscious visual processing. I only request that the authors make such an argument explicit. Preferably, this argument will precede claims that require it. For example, in the significance statement, the authors write, 'all images show equal suppression when both thresholds are measured. We *thus* find no evidence of differential unconscious processing and *conclude* reliance on breakthrough thresholds is misleading' (emphasis added). Just what supports the 'thus' and the 'conclude'? Similarly, at the end of the introduction, the authors write, '[...] suppression depth was constant for faces, objects, gratings and visual noise. *In other words*, we find no evidence to support differential unconscious processing among these particular, diverse categories of suppressed images' (emphasis added). I believe the statements before and after the period have not been shown to be equivalent. In the abstract, the authors revised, 'variations in bCFS thresholds alone are insufficient for inferring whether the barrier to achieving awareness exerted by interocular suppression is weaker for some categories of visual stimuli compared to others.' While I appreciate the added specificity, this claim still needs more support because the authors have not established that suppression depth is a better index than the breakthrough threshold of 'the barrier to achieving awareness exerted by interocular suppression.'

The authors' reply included a discussion of neural CRFs, which may explain why the bCFS thresholds differ across image categories. However, CRFs do not explain why the bCFS threshold does not implicate some component of subconscious processing. For example, the bCFS threshold may reflect the aspect of subconscious visual processing that corresponds to V1/V4 neural responses.

Reviewer #3 (Public Review):

Summary:<br /> In the 'bCFS' paradigm, a monocular target gradually increases in contrast until it breaks interocular suppression by a rich monocular suppressor in the other eye. The present authors extend the bCFS paradigm by allowing the target to reduce back down in contrast until it becomes suppressed again. The main variable of interest is the contrast difference between breaking suppression and (re) entering suppression. The authors find this difference to be constant across a range of target types, even ones that differ substantially in the contrast at which they break interocular suppression (the variable conventionally measured in bCFS). They also measure how the difference changes as a function of other manipulations. Interpretation is in terms of the processing of unconscious visual content, as well as in terms of the mechanism of interocular suppression.

Strengths:<br /> Interpretation of bCFS findings is mired in controversy, and this is an ingenuous effort to move beyond the paradigm's exclusive focus on breaking suppression. The notion of using the contrast difference between breaking and entering suppression as an index of suppression depth is interesting.

Reviewer #2 (Public Review):

As a report of the first structure of VMAT2, indeed the first structure of any vesicular monoamine transporter, this manuscript represents an important milestone in the field of neurotransmitter transport. VMAT2 belongs to a large family (the major facilitator superfamily, MFS) containing transporters from all living species. There is a wealth of information relating to the way that MFS transporters bind substrates, undergo conformational changes to transport them across the membrane and couple these events to the transmembrane movement of ions. VMAT2 couples the movement of protons out of synaptic vesicles to the vesicular uptake of biogenic amines (serotonin, dopamine and norepinephrine) from the cytoplasm. The new structure presented in this manuscript can be expected to contribute to an understanding of this proton/amine antiport process.

The structure contains a molecule of the inhibitor TBZ bound in a central cavity, with no access to either luminal or cytoplasmic compartments. The authors carefully analyze which residues interact with bound TBZ and measure TBZ binding to VMAT2 mutated at some of those residues. These measurements allow well-reasoned conclusions about the differences in inhibitor selectivity between VMAT1 and VMAT2 and differences in affinity between TBZ derivatives.

The structure also reveals polar networks within the protein and hydrophobic residues in positions that may allow them to open and close pathways between the central binding site and the cytoplasm or the vesicle lumen. The authors propose involvement of these networks and hydrophobic residues in coupling of transport to proton translocation and conformational changes. However, these proposals are quite speculative in the absence of supporting structures and experimentation that would test specific mechanistic details.

Reviewer #3 (Public Review):

Summary:

The vesicular monoamine transporter is a key component in neuronal signaling and is implicated in diseases such as Parkinson's. Understanding of monoamine processing and our ability to target that process therapeutically has been to date provided by structural modeling and extensive biochemical studies. However, structural data is required to establish these findings more firmly.

Strengths:

Dalton et al resolved a structure of VMAT2 in the presence of an important inhibitor, tetrabenazine, with the protein in detergent micelles, using cryo-EM and with the aid of protein domains fused to its N- and C-terminal ends, including one fluorescent protein that facilitated protein screening and purification. The resolution of the maps allows clear assignment of the amino acids in the core of the protein. The structure is in good agreement with a wealth of experimental and structural prediction data, and provides important insights into the binding site for tetrabenazine and selectivity relative to analogous compounds. The authors provide additional biochemical analyses that further support their findings. The comparison with AlphaFold models is enlightening.

Weaknesses:

The authors follow up their structures with molecular dynamics simulations of the tetrabenazine-bound state, and test several protonation states of acidic residues in the binding pocket, but not all possible combinations; thus, it is not clear the extent to which tetrabenazine rearrangements observed in these simulations are meaningful. Additional simulations of the substrate dopamine docked into this structure were also carried out, although it is unclear whether this "dead-end" occluded state is a relevant state for dopamine binding. The authors report release of dopamine during these simulations, but it is notable that this only occurs when all four acidic binding site residues were protonated and when an enhanced sampling approach was applied.

Reviewer #1 (Public Review):

Summary:

This study presents fundamental new insights into vesicular monoamine transport and the binding pose of the clinical drug tetrabenazine (TBZ) to the mammalian VMAT2 transporter. Specifically, this study reports the first structure for the mammalian VMAT (SLC18) family of vesicular monoamine transporters. It provides insights into the mechanism by which this inhibitor traps VMAT2 into a 'dead-end' conformation. The structure also provides some evidence for a novel gating mechanism within VMAT2, which may have wider implications for understanding the mechanism of transport in the wider SLC18 family.

Strengths:

The structure is high quality, and the method used to determine the structure via fusing mVenus and the anti-GFP nanobody to the amino and carboxyl termini is novel. The binding and transport data are convincing and provide new insights into the role of conserved side chains within the SLC18 members. The binding position of TBZ is of high value, given its role in treating Huntington's chorea and for being a 'dead-end' inhibitor for VMAT2.

Reviewer #1 (Public Review):

Summary:

This manuscript introduces an exciting way to measure SARS-CoV-2 aerosolized shedding using a disposable exhaled breath condensate collection device (EBCD). The paper draws the conclusion that the contagious shedding of the virus via aerosol route persists at a high level 8 days after symptoms.

Strengths:

The methodology is potentially of high importance and the paper is clearly written. The study design is clever. If aerosolized viral load kinetics truly differed from those of nasal swabs, then this would be a very important finding.

Weaknesses:

The study conclusions are not entirely supported by the data for several reasons:

(1) Most data points in the study are relatively late during infection when viral loads from other compartments (nasal and oral swabs) are typically much lower than peak viral loads which often occur in the pre-symptomatic or early symptomatic phase of infection. Moreover, the generation time for SARS-CoV-2 has been estimated to be 3-4 days on average meaning that most infections occur before or very early during symptoms. Therefore, the available epidemiologic data does not support 12 days of infection (day 8 symptoms) as important for most transmissions. Therefore, many of the measurement timepoints in this study may not be relevant for transmission.

(2) Fig 1A would be more powerful as a correlation plot between viral load from nasal samples (x-axis) and aerosol (y-axis). One would expect at least a rough correlation (as has been seen between viral loads in oral and nasal samples) and deviations from this correlation would provide crucial information about how and when aerosol shedding is discordant from nasal samples (ie early vs late time points, low versus high viral loads< etc...). It is too strong to state correspondence is 100% when viral load is only measured in one compartment and nasal swabs are reduced to the oversimplified "positive or negative".

(3) Results are reported in RNA copies which is fine but particle-forming units (pfu, or quantitative culture) are likely a more accurate surrogate of infectivity. It is quite possible that all of these samples would have been negative for pfu given that the ratio of RNA: pfu is often >1000 (though also dynamic over time during infection). This could be another indicator that most samples in the study were collected too late during infection to represent contagious time points.

(4) Individual kinetic curves should be shown for participants with more than three time points to demonstrate whether there are clear kinetic trends within individuals that would help further validate this approach. The inclusion of single samples from individuals is less informative.

(5) The S-shaped model in 2A is somewhat misleading as it is fit to means but there is tremendous variability within the data. Therefore the 8-day threshold should be listed clearly as a mean but not a rule for all individuals. The statement that viral RNA copies do not decrease until 8 days from symptom onset is unlikely to be true for all infected people and can't be made based on the available data in this study given that many people contributed only one datapoint.

(6) The incubation period for SARS-CoV-2 is highly variable. Therefore duration of symptoms is a rather poor correlate of the duration of infection. This further diminishes the interpretive value of positive samples from individuals who were only sampled once.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Lane and colleagues measured the abundance of SARS-CoV-2 on breath in 60 outpatients after the development of COVID-19 symptoms using a novel breath collection apparatus. They found that, overall, viral abundance remains high for approximately eight days following the development of symptoms, after which viral abundance on breath drops to a low level that may persist for approximately 20 days or more. They did not identify significant differences in viral shedding on breath by vaccination status or viral variant. They also noted substantial variation in the degree and duration of shedding across individuals.

Strengths:

The primary strengths of this study are (1) the focus on breath, rather than the more traditional nasal/oropharyngeal swabs, and (2) the fact that the data were collected at multiple time points for each infection. This allows the authors to characterize not only mean viral abundance across individuals but also how that abundance changes over time, allowing for a better understanding of the potential duration of infectiousness of SARS-CoV-2.

Weaknesses:

The sample size is moderate (60) and focuses only on outpatients. While these are minor weaknesses (as the authors note, the majority of SARS-CoV-2 transmission likely occurs among those with symptoms below the threshold of hospitalization), it would nevertheless be useful to have a fuller understanding of variation in viral shedding across clinical groups. Furthermore, the study lacks information on viral shedding prior to the development of symptoms, which may be a critical period for transmission. Since the samples were collected at home by study participants using a novel apparatus, it is difficult to assess the degree to which actual variation in viral abundance, user variability, and/or measurement variation is inherent to the apparatus.

Reviewer #1 (Public Review):

This manuscript uses 3 large neuroimaging datasets - which together span childhood to late adulthood - to model the relationship between birthweight (BW) and cortical anatomy over time. The authors separately consider BW associations with the "height" of cortical anatomy trajectories (intercept effects) vs. BW associations with trajectory shape. They authors also distinguish between BW associations with cortical surface area (SA) and cortical thickness (CT), which together determine cortical volume (CV). Prior studies have firmly established robust positive associations between BW and cortical SA, but this study adds evidence for the protracted lifespan persistence of these associations, and the degree to which BW associations with cortical change over time are much weaker.

The study has several strengths including: clearly motivation of this work in the Introduction and contextualization of the results in Discussion; use of three large neuroimaging datasets; inclusion of sensible sensitivity analyses; disambiguation of SA and CT findings; and use of formal spatial analysis to quantify the reproducibility of effects across cohorts.

The primary way in which this work seeks to extend beyond established findings is to determine if BW is associated with differences in cortical change over time. The results presented clearly establish that such BW-change associations are much more localized and less consistent across cohorts that BW-intercept associations. The authors use multiple complementary approaches to verify the robustness of this inference to dataset subsampling and variation in statistical methods.

Overall, this work provides a valuable new data point in our understanding of the profound and protracted influences that prenatal developmental features can have on postnatal outcomes.

Reviewer #2 (Public Review):

This study focuses on the association between weight at birth and area, volume and thickness of the cerebral cortex measured at timepoints throughout the lifespan. Overall, the study is well designed, supported by evidence from a large sample drawn from three geographically distinct cohorts with robust analytical and statistical methods.

The authors test the hypotheses: that higher birth weight is associated with greater cortical area in later life; that associations are robust across samples and age; and that associations are stable across the lifespan. Analyses are performed separately in three cohorts: ABCD, UKBB and LCBC and the pattern of associations compared by means of spatial correlations. They find that BW is positively associated with cortical area (and, as a consequence, cortical volume) across most of the cortex, with effect sizes greatest in frontal and temporal regions. These associations remain largely unchanged when accounting for age, sex, length of gestation and (in one cohort) ethnicity. Variations due to MRI scanner and site are accounted for statistically. Measures are taken to determine within sample replicability through split-half analyses.

The authors conclude that BW, as a marker of early development, is associated with brain characteristics throughout the lifespan.

Reviewer #1 (Public Review):

Wang and colleagues recently demonstrated the essential role of RBM24 (RNA-binding motif protein 24a) in the development of mouse hair cells (source: https://doi.org/10.1002/jcp.31003). In this study, they further expand on their findings by revealing that Rbm24 expression is absent in Pou4f3 mutant mice but not in Gfi1 mutant mice. This observation suggests that POU4F3 acts as an upstream regulator of Rbm24. The researchers effectively demonstrate that POU4F3 can bind to and regulate Rbm24 through three distant enhancers, which are located in open chromatin regions and are bound by POU4F3. Lastly, Wang and colleagues discovered that ectopic expression of Rbm24 was unable to prevent the degeneration of POU4F3 null hair cells.

The findings in this manuscript hold great significance as they provide additional insights into the transcriptional cascades crucial for hair cell development. The discovery of enhancers capable of driving transgene expression specifically in hair cells holds promising therapeutic implications. The figures presented in the study are of excellent quality, the employed techniques are state-of-the-art, the data are accurately represented without exaggeration, and the study demonstrates a high level of rigor.

Reviewer #2 (Public Review):

Previous studies have shown that two hair cell transcription factors, Pou4f3 and Gfi1 are both necessary for the survival of cochlear hair cells, and that Gfi1 is regulated by Pou4f3. The authors have previously also shown that mosaic inactivation of the RNA-binding protein RBM24 leads to outer hair cell death.

In the present study, the authors show that hair cells dies in Pou4f3 and Gfi1 mutant mice. They show that Gfi1 is regulated by Pou4f3. Both these observations have been published before. They then show that RBM24 is absent in Pou4f3 knockouts, but not Gfi1 knockouts. They ectopically activate RMB24 in the hair cells of Poui4f3 knockouts, but this does not rescue the hair cell death. Finally the authors validate three RMB24 enhancers that are active in young hair cells and which have been previously shown to bind Pou4f3.

The experiments are well-executed and the data are clear. The results support the conclusions of the paper. The authors have revised the paper slightly, mostly to modify the red/green staining in the figures, and to perform additional analyses of the RBM24 and Ikzf2 mutants, now shown in Supplementary Figure 3.

Much of the work in the paper has been reported before. The result that hair cell transcription factors operate in a network, with some transcription factors activating only a subset of hair cell genes, is an expected result. Since RBM24 is only one of many genes regulated directly by Pou4f3, it is not surprising that it cannot rescue the Pou4f3 knockout hair cell degeneration, and indeed the rationale for attempting such a rescue experiment is not provided by the authors.

The identification of new hair cell enhancers may be of use to investigators wishing to express genes in hair cells.

In sum, this work, although carefully performed, does not shed significant new light on our understanding of hair cell development or survival.

Reviewer #1 (Public Review):

Summary: This is a very meticulous and precise anatomical description of the external sensory organs in Drosophila larvae. It generates an integral and accurate map. The authors revise all the data for the abdominal and thoracic segments and describe in detail, for the first time, the head and tail segments.

Strengths: It is a very thorough anatomical description of the external sensory organs of the genetically amenable fruitfly. This study represents a very useful tool for the research community that will definitely be used it as a reference paper. It will allow us to investigate sensory processing in depth. The discussion places the anatomical data into a functional and developmental frame.

Reviewer #2 (Public Review):

Summary: This study is a superbly written and illustrated documentation of the external sensilla of the Drosophila larva. Serial electron microscopy and digital modeling is used to the fullest to provide a definitive and clear picture of the sensory organs, which is dearly needed in the field.

Strengths: Serial electron microscopy and digital modeling is used to the fullest to provide a comprehensive, definitive and clear picture of the sensory organs, which is dearly needed in the field.

Weaknesses: none detected.

Reviewer #3 (Public Review):

Summary: Richter et al. present a comprehensive anatomical analysis of the external sensory organs of the D. melanogaster larva. Extending on their previous study (Rist and Thum 2017) that analyzed the anatomy of the terminal organ, a major external taste organ of fruit fly larva, the authors examined the anatomy of the remaining head sensory organs - the dorsal organ, the ventral organ, and the labial organ-also described the sensory organs of the thoracic and abdominal segments. Using improved electron microscopy, the authors performed a three-dimensional anatomical analysis of the sensilla and adjacent ganglia to construct a complete structural and neuronal map of the external larval sensilla.

Strengths: Though the manuscript is lengthy, it is written clearly, and the presented data supports the conclusion. In addition to the classification and nomenclature of the different types of sensilla throughout the larval body, the wealth of data presented here will be valuable to the scientific community. The study offers fundamental anatomical insights, which will be helpful for future functional studies and to understand the sensory strategies of Drosophila larvae in response to the external environment. By analyzing different larval stages (L1 and L3), this work offers some insights into the developmental aspects of the larval sense organs and their corresponding sensory cells.

Weaknesses: There are no apparent weaknesses. The repetitiveness of some data and prior studies may be avoided for easy readability.

Reviewer #1 (Public Review):

This study by Paoli et al. used a resonant scanning multiphoton microscope to examine olfactory representation in the projection neurons (PNs) of the honeybee with improved temporal resolution. PNs were classified into 9 groups based on their response patterns. Authors found that excitatory repose in the PNs precedes the inhibitory responses for ~40ms, and ~50% of PN responses contain inhibitory components. They built the neural circuit model of the mushroom body (MB) with evolutionally conserved features such as sparse representation, global inhibition, and a plasticity rule. This MB model fed with the experimental data could reproduce a number of phenomena observed in experiments using bees and other insects, including dynamical representations of odor onset and offset by different populations of Kenyon cells, prolonged representations of after-smell, different levels of odor-specificity for early/delay conditioning, and shift of behavioral timing in delay conditioning. The trace conditioning was not modeled and tested experimentally. Also, the experimental result itself is largely confirmatory to preceding studies using other organisms. Nonetheless, the experimental data and the model provide a solid basis for future studies.

Reviewer #2 (Public Review):

The present manuscript investigates the implication of locus coeruleus-noradrenaline system in the stress-induced transcriptional changes of dorsal and ventral hippocampus, combining pharmacological, chemogenetic, and optogenetic techniques. Authors have revealed that stress-induced release of noradrenaline from locus coeruleus plays a modulatory role in the expression of a large scale of genes in both ventral and dorsal hippocampus through activation of β-adrenoreceptors. Similar transcriptional responses were observed after optogenetic and chemogenetic stimulation of locus coeruleus. Among all the genes analysed, authors identified the most affected ones in response to locus coeruleus-noradrenaline stimulation as being Dio2, Ppp1r3c, Ppp1r3g, Sik1, and Nr4a1. By comparing their transcriptomic data with publicly available datasets, authors revealed that these genes were upregulated upon exposure to different stressors. Additionally, authors found that upregulation of Ppp1r3c, Ppp1r3g, and Dio2 genes following swim stress was sustained from 90 min up to 2-4 hours after stress and that it was predominantly restricted to hippocampal astrocytes, while Sik1 and Nr4a1 genes showed a broader cellular expression and a sharp rise and fall in expression, within 90 min of stress onset.

The paper is well written and provides a useful inventory of dorsal and ventral hippocampal gene expression upregulated by activation of LC-NA system, which can be used as starting point for more functional studies related to the effects of stress-induced physiological and pathological changes. Sex-differences were also explored which represents a strength of the study.

Reviewer #1 (Public Review):

Summary: This paper suggests to apply intrinsically-motivated exploration for the discovery of robust goal states in gene regulatory networks.

Strengths:<br /> The paper is well written. The biological motivation and the need for such methods are formulated extraordinarily well. The battery of experimental models is impressive.

Weaknesses:<br /> (1) The proposed method is compared to the random search. That says little about the performance with regard to the true steady-state goal sets. The latter could be calculated at least for a few simple ODE (e.g., BIOMD0000000454, `Metabolic Control Analysis: Rereading Reder'). The experiment with 'oscillator circuits' may not be directly interpolated to the other models.

The lack of comparison to the ground truth goal set (attractors of ODE) from arbitrary initial conditions makes it hard to evaluate the true performance/contribution of the method. A part of the used models can be analyzed numerically using JAX, while there are models that can be analyzed analytically.

"...The true versatility of the GRN is unknown and can only be inferred through empirical exploration and proxy metrics....": one could perform a sensitivity analysis of the ODEs, identifying stable equilibria. That could provide a proxy for the ground truth 'versatility'.

(2) The proposed method is based on `Intrinsically Motivated Goal Exploration Processes with Automatic Curriculum Learning', which assumes state action trajectories [s_{t_0:t}, a_{t_0:t}], (2.1 Notations and Assumptions' in the IMGEP paper). However, the models used in the current work do not include external control actions, but rather only the initial conditions can be set. It is not clear from the methods whether IMGEP was adapted to this setting, and how the exploration policy was designed w/o actual time-dependent actions. What does "...generates candidate intervention parameters to achieve the current goal...."<br /> mean considering that interventions 'Sets the initial state...' as explained in Table 2?

(3) Fig 2 shows the phase space for (ERK, RKIPP_RP) without mentioning the typical full scale of ERK, RKIPP_RP. It is unclear whether the path from (0, 0) to (~0.575, ~3.75) at t=1000 is significant on the typical scale of this phase space. is it significant on the typical scale of this phase space?

(4) Table 2:<br /> a. Where is 'effective intervention' used in the method?<br /> b. in my opinion 'controllability', 'trainability', and 'versatility' are different<br /> terms. If their correspondence is important I would suggest to extend/enhance the column "Proposed Isomorphism". otherwise, it may be confusing. I don't see how this table generalizes generalizes "concepts from dynamical complex systems and behavioral sciences under a common navigation task perspective".

Reviewer #2 (Public Review):

Summary:<br /> Etcheverry et al. present two computational frameworks for exploring the functional capabilities of gene regulatory networks (GRNs). The first is a framework based on intrinsically-motivated exploration, here used to reveal the set of steady states achievable by a given gene regulatory network as a function of initial conditions. The second is a behaviorist framework, here used to assess the robustness of steady states to dynamical perturbations experienced along typical trajectories to those steady states. In Figs. 1-5, the authors convincingly show how these frameworks can explore and quantify the diversity of behaviors that can be displayed by GRNs. In Figs. 6-9, the authors present applications of their framework to the analysis and control of GRNs, but the support presented for their case studies is often incomplete.

Strengths:<br /> Overall, the paper presents an important development for exploring and understanding GRNs/dynamical systems broadly, with solid evidence supporting the first half of their paper in a narratively clear way.

The behaviorist point of view for robustness is potentially of interest to a broad community, and to my knowledge introduces novel considerations for defining robustness in the GRN context.

Some specific weaknesses, mostly concerning incomplete analyses in the second half of the paper:

(1) The analysis presented in Fig. 6 is exciting but preliminary. Are there other appropriate methods for constructing energy landscapes from dynamical trajectories in gene regulatory networks? How do the results in this particular case study compare to other GRNs studied in the paper?

Additionally, it is unclear whether the analysis presented in Fig. 6C is appropriate. In particular, if the pseudopotential landscapes are constructed from statistics of visited states along trajectories to the steady state, then the trajectories derived from dynamical perturbations do not only reflect the underlying pseudo-landscape of the GRN. Instead, they also include contributions from the perturbations themselves.

(2) In Fig. 7, I'm not sure how much is possible to take away from the results as given here, as they depend sensitively on the cohort of 432 (GRN, Z) pairs used. The comparison against random networks is well-motivated. However, as the authors note, comparison between organismal categories is more difficult due to low sample size; for instance, the "plant" and "slime mold" categories each only have 1 associated GRN. Additionally, the "n/a" category is difficult to interpret.

(3) In Fig. 8, it is unclear whether the behavioral catalog generated is important to the intervention design problem of moving a system from one attractor basin to another. The authors note that evolutionary searches or SGD could also be used to solve the problem. Is the analysis somehow enabled by the behavioral catalog in a way that is complementary to those methods? If not, comparison against those methods (or others e.g. optimal control) would strengthen the paper.

(4) The analysis presented in Fig. 9 also is preliminary. The authors note that there exist many algorithms for choosing/identifying the parameter values of a dynamical system that give rise to a desired time-series. It would be a stronger result to compare their approach to more sophisticated methods, as opposed to random search and SGD. Other options from the recent literature include Bayesian techniques, sparse nonlinear regression techniques (e.g. SINDy), and evolutionary searches. The authors note that some methods require fine-tuning in order to be successful, but even so, it would be good to know the degree of fine-tuning which is necessary compared to their method.

Reviewer #1 (Public Review):

Jiang et al. demonstrated that ablating Neurexins results in alterations to glycinergic transmission and its calcium sensitivity, utilizing a robust experimental system. Specifically, the authors employed rAAV-Cre-EGFP injection around the MNTB in Nrxn1/2/3 triple conditional mice at P0, measuring Glycine receptor-dependent IPSCs from postsynaptic LSO neurons at P13-14. Notably, the authors presented a clear reduction of 60% and 30% in the amplitudes of opto- and electric stimulation-evoked IPSCs, respectively. Additionally, they observed changes in kinetics, alterations in PPR, and sensitivity to lower calcium and the calcium chelator, EGTA, indicating solid evidence for changes in presynaptic properties of glycinergic transmission.

Furthermore, the authors uncovered an unexpected increase in sIPSC frequency without altering amplitude. Despite the reduction in evoked IPSC, immunostaining revealed an increase in GlyT2 and VGAT in TKO mice, supporting the notion of an increase in synapse number. However, the reviewer expresses caution regarding the authors' conclusion that "glycinergic neurotransmission likely by promoting the synapse formation/maintenance, which is distinct from the phenotypes observed in glutamatergic and GABAergic neurons (Chen et al., 2017; Luo et al., 2021)", as outlined in lines 173-175. The reviewer suggests that this statement may be overstated, pointing out the authors' own discussion in lines 254-265, which acknowledges multiple possibilities, including the potential that the increase in synapses is a consequence rather than a causal effect of Nrxn deletion.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Jiang et al., explore the role of neurexins at glycinergic MNTB-LSO synapses. The authors utilize elegant and compelling ex vivo slice electrophysiology to assess how the genetic conditional deletion of Nrxns1-3 impacts inhibitory glycinergic synaptic transmission and found that TKO of neurexins reduced electrically and optically evoked IPSC amplitudes, slowed optically evoked IPSC kinetics and reduced presynaptic release probability. The authors use classic approaches including reduced [Ca2+] in ACSF and EGTA chelation to propose that changes in these evoked properties are likely driven by the loss of calcium channel coupling. Intriguingly, while evoked transmission was impaired, the authors reported that spontaneous IPSC frequency was increased, potentially due to an increased number of synapses in LSO. Overall, this manuscript provides important insight into the role of neurexins at the glycinergic MNTP-LSO synapse and further emphasizes the need for continued study of both the non-redundant and redundant roles of neurexins.

Strengths:<br /> This well-written manuscript seamlessly incorporates mouse genetics and elegant ex vivo electrophysiology to identify a role for neurexins in glycinergic transmission at MNTB-LSO synapses. Triple KO of all neurexins reduced the amplitude and timing of evoked glycinergic synaptic transmission. Further, spontaneous IPSC frequency was increased. The evoked synaptic phenotype is likely a result of reduced presynaptic calcium coupling while the spontaneous synaptic phenotype is likely due to increased synapse numbers. While neuroligin-4 has been identified at glycinergic synapses, this study, to the best of my knowledge, is the first to study Nrxn function at these synapses.

Weaknesses:<br /> The data are compelling and report an intriguing functional phenotype. The role of Neurexins redundantly controls calcium channel coupling has been previously reported. Mechanistic insight would significantly strengthen this study.<br /> The claim that triple KO of Nrxns from MNTB increases the number of synapses in LSO is not strongly supported.<br /> Despite the stated caveats of measuring electrically evoked currents and the more robust synaptic phenotypes observed using optically evoked transmission, the authors rely heavily on electrical stimulation for most measurements.<br /> The differential expression of individual neurexins might indicate that specific neurexins may dominantly regulate synaptic transmission, however, this possibility is not discussed in detail.

Reviewer #3 (Public Review):

Summary:<br /> The authors investigate the hypothesis that neurexins serve a crucial role as regulators of the synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body (MNTB) and the lateral superior olivary complex (LSO). It is worth mentioning that LSO neurons are an integration station of the auditory brainstem circuit displaying high reliability and temporal precision. These features are necessary for computing interaural cues to derive sound source location from comparing the intensities of sounds arriving at the two ears. In this context, the authors' findings build up according to the hypothesis first by displaying that neurexins were expressed in the MNTB at varying levels. They followed this up with the deletion of all neurexins in the MNTB through the employment of a triple knock-out (TKO). Using electrophysiological recordings in acute brainstem slices of these TKO mice, they gathered solid evidence for the role of neurexins in synaptic transmission at this glycinergic synapse primarily by ensuring tight coupling of Ca2+ channels and vesicular release sites. Additionally, the authors uncovered a connection between the deletion of neurexins and a higher number of glycinergic synapses in TKO mice, for which they provided evidence in the form of immunostainings and related it to electrophysiological data on spontaneous release. Consequently, this investigation expands our knowledge on the molecular regulation of synaptic transmission at glycinergic synapses, as well as on the auditory processing at the level of the brainstem.

Strengths:<br /> The authors demonstrate substantial results in support of the hypothesis of a critical role of neurexins for regulating glycinergic transmission in the LSO using various techniques. They provide evidence for the expression of neurexins in the MNTB and consecutively successfully generate and characterize the neurexin TKO. For their study on LSO IPSCs the authors transduced MNTB neurons by co-injection of virus-carrying Cre and ChR2 and subsequently optogenetically evoke release of glycine. As a result, they observed a significant reduction in amplitude and significantly slower rise and decay times of the IPSCs of the TKO in comparison with control mice in which MNTB neurons were only transduced with ChR2. Furthermore, they observed an increased paired pulse ratio (PPR) of LSO IPSCs in the TKO mice, indicating lower release probability. Elaborating on the hypothesis that neurexins are essential for the coupling of synaptic vesicles to Ca2+ channels, the authors show lowered Ca2+ sensitivity in the TKO mice. Additionally, they reveal convincing evidence for the connection between the increased frequency of spontaneous IPSC and the higher number of glycinergic synapses of the LSO in the TKO mice, revealed by immunolabeling against the glycinergic presynaptic markers GlyT2 or VGAT.

Weaknesses:<br /> The major concern is novelty as this work on the effects of pan-neurexin deletion in a glycinergic synapse is quite consistent with the authors' prior work on glutamatergic synapses (Luo et al., 2020). The authors might want to further work out novel aspects and strengthen the comparative perspective. Conceptually, the authors might want to be more clear about interpreting the results on the altered dependence of release on voltage-gated Ca2+ influx (Ca2+ sensitivity, coupling).

Reviewer #1 (Public Review):

Summary:<br /> Compared with conventional SQUID-MEG, OPM-MEG offers theoretical advantages of sensor configurability (that is, sizing to suit the head size) and motion tolerance (the sensors are intrinsically in the head reference frame). This study purports to be the first to experimentally demonstrate these advantages in a developmental study from age 2 to age 34.

In short, while the theoretical advantages of OPM-MEG are attractive - both in terms of young child sensitivity and in terms of motion tolerance - neither was in fact demonstrated in this manuscript. We are left with a replication of SQUID-MEG observations, which certainly establishes OPM-MEG as "substantially equivalent" to conventional technology but misses the opportunity to empirically demonstrate the much-discussed theoretical advantages/opportunities.

Strengths:<br /> A replication of SQUID-MEG observations, which certainly establishes OPM-MEG as "substantially equivalent" to conventional technology but misses the opportunity to empirically demonstrate the much-discussed theoretical advantages/opportunities.

Weaknesses:<br /> The authors describe 64 tri-axial detectors, which they refer to as 192 channels. This is in keeping with some of the SQUID-MEG description, but possibly somewhat disingenuous. For the scientific literature, perhaps "64 tri-axial detectors" is a more parsimonious description.

A small fraction (<20%) of trials were eliminated for analysis because of "excess interference" - this warrants further elaboration.

Figure 3 shows a reduced beta ERD in the youngest children. Although the authors claim that OPM-MEG would be similarly sensitive for all ages and that SQUID-MEG would be relatively insensitive to young children, one trivial counterargument that needs to be addressed is that OPM has NOT in fact increased the sensitivity to young child ERD. This can possibly be addressed by analogous experiments using a SQUID-based system. An alternative would be to demonstrate similar sensitivity across ages using OPM to a brain measure such as evoked response amplitude. In short, how does Figure 3 demonstrate the (theoretical) sensitivity advantage of OPM MEG in small heads ?

The data do not make a compelling case for the motion tolerance of OPM-MEG. Although an apparent advantage of a wearable system, an empirical demonstration is still lacking. How was motion tracked in these participants?

Furthermore, while the introduction discusses at some length the phenomenon of PMBR, there is no demonstration of the recording of PMBR (or post-sensory beta rebound). This is a shame because there is literature suggesting an age-sensitivity to this, that the optimal sensitivity of OPM-MEG might confirm/refute. There is little evidence in Figure 3 for adult beta rebound. Is there an explanation for the lack of sensitivity to this phenomenon in children/adolescents ? Could a more robust paradigm (button-press) have shed light on this?

Data on functional connectivity are valuable but do not rely on OPM recording. They further do not add strength to the argument that OPM MEG is more sensitive to brain activity in smaller heads - in fact, the OPM recordings seem plagued by the same insensitivity observed using conventional systems.

The discussion of burst vs oscillations, while highly relevant in the field, is somewhat independent of the OPM recording approach and does not add weight to the OPM claims.

In short, while the theoretical advantages of OPM-MEG are attractive - both in terms of young child sensitivity and in terms of motion tolerance, neither was in fact demonstrated in this manuscript. We are left with a replication of SQUID-MEG observations, which certainly establishes OPM-MEG as "substantially equivalent" to conventional technology but misses the opportunity to empirically demonstrate the much-discussed theoretical advantages/opportunities.

Reviewer #2 (Public Review):

Summary:<br /> The authors introduce a new 192-channel OPM system that can be configured using different helmets to fit individuals from 2 to 34 years old. To demonstrate the veracity of the system, they conduct a sensorimotor task aimed at mapping developmental changes in beta oscillations across this age range. Many past studies have mapped the trajectory of beta (and gamma) oscillations in the sensorimotor cortices, but these studies have focused on older children and adolescents (e.g., 9-15 years old) and used motor tasks. Thus, given the study goals, the choice of a somatosensory task was surprising and not justified. The authors recorded a final sample of 27 children (2-13 years old) and 24 adults (21-34 years) and performed a time-frequency analysis to identify oscillatory activity. This revealed strong beta oscillations (decreases from baseline) following the somatosensory stimulation, which the authors imaged to discern generators in the sensorimotor cortices. They then computed the power difference between 0.3-0.8 period and 1.0-1.5 s post-stimulation period and showed that the beta response became stronger with age (more negative relative to the stimulation period). Using these same time windows, they computed the beta burst probability and showed that this probability increased as a function of age. They also showed that the spectral composition of the bursts varied with age. Finally, they conducted a whole-brain connectivity analysis. The goals of the connectivity analysis were not as clear as prior studies of sensorimotor development have not conducted such analyses and typically such whole-brain connectivity analyses are performed on resting-state data, whereas here the authors performed the analysis on task-based data. In sum, the authors demonstrate that they can image beta oscillations in young children using OPM and discern developmental effects.

Strengths:<br /> Major strengths of the study include the novel OPM system and the unique participant population going down to 2-year-olds. The analyses are also innovative in many respects.

Weaknesses:<br /> Several weaknesses currently limit the impact of the study. First, the choice of a somatosensory stimulation task over a motor task was not justified. The authors discuss the developmental motor literature throughout the introduction, but then present data from a somatosensory task, which is confusing. Of note, there is considerable literature on the development of somatosensory responses so the study could be framed with that. Second, the primary somatosensory response actually occurs well before the time window of interest in all of the key analyses. There is an established literature showing mechanical stimulation activates the somatosensory cortex within the first 100 ms following stimulation, with the M50 being the most robust response. The authors focus on a beta decrease (desynchronization) from 0.3-0.8 s which is obviously much later, despite the primary somatosensory response being clear in some of their spectrograms (e.g., Figure 3 in older children and adults). This response appears to exhibit a robust developmental effect in these spectrograms so it is unclear why the authors did not examine it. This raises a second point; to my knowledge, the beta decrease following stimulation has not been widely studied and its function is unknown. The maps in Figure 3 suggest that the response is anterior to the somatosensory cortex and perhaps even anterior to the motor cortex. Since the goal of the study is to demonstrate the developmental trajectory of well-known neural responses using an OPM system, should the authors not focus on the best-understood responses (i.e., the primary somatosensory response that occurs from 0.0-0.3 s)?

Regarding the developmental effects, the authors appear to compute a modulation index that contrasts the peak beta window (.3 to .8) to a later 1.0-1.5 s window where a rebound is present in older adults. This is problematic for several reasons. First, it prevents the origin of the developmental effect from being discerned, as a difference in the beta decrease following stimulation is confounded with the beta rebound that occurs later. A developmental effect in either of these responses could be driving the effect. From Figure 3, it visually appears that the much later rebound response is driving the developmental effect and not the beta decrease that is the primary focus of the study. Second, these time windows are a concern because a different time window was used to derive the peak voxel used in these analyses. From the methods, it appears the image was derived using the .3-.8 window versus a baseline of 2.5-3.0 s. How do the authors know that the peak would be the same in this other time window (0.3-0.8 vs. 1.0-1.5)? Given the confound mentioned above, I would recommend that the authors contrast each of their windows (0.3-0.8 and 1.0-1.5) with the 2.5-3.0 window to compute independent modulation indices. This would enable them to identify which of the two windows (beta decrease from 0.3-0.8 s or the increase from 1.0-1.5 s) exhibited a developmental effect. Also, for clarity, the authors should write out the equation that they used to compute the modulation index. The direction of the difference (positive vs. negative) is not always clear.

Another complication of using a somatosensory task is that the literature on bursting is much more limited and it is unclear what the expectations would be. Overall, the burst probability appears to be relatively flat across the trial, except that there is a sharp decrease during the beta decrease (.3-.8 s). This matches the conventional trial-averaging analysis, which is good to see. However, how the bursting observed here relates to the motor literature and the PMBR versus beta ERD is unclear.

Another weakness is that all participants completed 42 trials, but 19% of the trials were excluded in children and 9% were excluded in adults. The number of trials is proportional to the signal-to-noise ratio. Thus, the developmental differences observed in response amplitude could reflect differences in the number of trials that went into the final analyses.

Finally, the discussion could be improved to focus on the somatosensory literature and how this contributes to that. Currently, the discussion includes very little from the somatosensory literature.

Reviewer #3 (Public Review):

This study demonstrated the application of OPM-MEG in neurodevelopment studies of somatosensory beta oscillations and connections with children as young as 2 years old. It provides a new functional neuroimaging method that has a high spatial-temporal resolution as well wearable which makes it a new useful tool for studies in young children. They have constructed a 192-channel wearable OPM-MEG system that includes field compensation coils which allow free head movement scanning with a relatively high ratio of usable trials. Beta band oscillations during somatosensory tasks are well localized and the modulation with age is found in the amplitude, connectivity, and pan-spectral burst probability. It is demonstrated that the wearable OPM-MEG could be used in children as a quite practical and easy-to-deploy neuroimaging method with performance as good as conventional MEG. With both good spatial (several millimeters) and temporal (milliseconds) resolution, it provides a novel and powerful technology for neurodevelopment research and clinical applications not limited to somatosensory areas.

The conclusions of this paper are mostly well supported by data acquired under the proper method. However, some aspects of data analysis need to be improved and extended.

(1) The colour bars selected for the pseudo-T-static pictures of beta modulation in Figures 2 and 3, which are blue/black and red/black, are not easily distinguished from the anatomical images which are grey-scale. A colour bar without black/white would make these figures better. The peak point locations are also suggested to be marked in Figure 2 and averaged locations in Figure 3 with an error bar.

(2) The data points in plots are not constant across figures. In Figures 3 and 5, they are classified into triangles and circles for children and adults, but all are circles in Figures 4 and 6.

(3) Although MEG is much less susceptible to conductivity inhomogeneity of the head than EEG, the forward modulating may still be impacted by the small head profile. Add more information about source localization accuracy and stability across ages or head size.

Reviewer #1 (Public Review):

Summary:<br /> In the manuscript titled "Coevolution due to physical interactions is not a major driving force behind evolutionary rate covariation" by Little et al., explores the potential contribution of physical interaction between correlated evolutionary rates among gene pairs. They find that physical interaction is not the main driving of evolutionary rate covariation (ECR). This finding is similar to a previous report by Clark et al. (2012), Genome Research, wherein the authors stated that "direct physical interaction is not required to produce ERC." The previous study used 18 Saccharomycotina yeast species, whereas the present study used 332 Saccharomycotina yeast species and 11 outgroup taxa. As a result, the present study is better positioned to evaluate the interplay between physical interaction and ECR more robustly.

Strengths & Weaknesses:<br /> Various analyses nicely support the authors' claims.

Reviewer #2 (Public Review):

Summary:<br /> The authors address an important outstanding question: what forces are the primary drivers of evolutionary rate covariation? Exploration of this topic is important because it is currently difficult to interpret the functional/mechanistic implications of evolutionary covariation. These analyses also speak to the predictive power (and limits) of evolutionary rate covariation. This study reinforces the existing paradigm that covariation is driven by a varied/mixed set of interaction-types that all fall under the umbrella explanation of 'co-functional interactions'.

Strengths:<br /> Very smart experimental design that leverages individual protein domains for increased resolution.

Weaknesses:<br /> Nuanced and sometimes inconclusive results that are difficult to capture in a short title/abstract statement.

EDIT: The authors have done a satisfactory job of honing their language to get the nuanced ideas across clearly. The added scholarship and theoretical discussion they added strengthen the impact of the manuscript. The revised edition addresses my concerns.

Reviewer #1 (Public Review):

The manuscript by Park et. al. examines the interaction of macrophages with SARS-CoV-2 spike protein and subsequent inflammatory reactions. The authors demonstrate that following intranasal delivery of spike it rapidly accumulates in alveolar macrophages. Inflammation associated with internalized spike recruits neutrophils to the lung, where they undergo a cell death process consistent with NETosis. The authors demonstrate that modifications spike to contain high mannose reduces uptake of spike protein and limits the inflammation induced. This finding could have implications for vaccine development, as vaccines containing modified spike could be safer and better tolerated.

The authors use a number of different techniques, including in vivo modeling, imaging, human and murine systems to interrogate their hypotheses. These systems provide robust supporting information for their conclusions. There are two key aspects from the current manuscript which would add key evidence. The authors suggest that neutrophils exposed to spike protein undergo a process of NETosis. To confirm this hypothesis inhibitors of NETosis should be used to demonstrate that the cell death is prevented. Additionally, vaccination of a murine model with the modified spike protein would add additional support to the conclusion that modified spike protein would be less inflammatory while maintaining its utility as a vaccine antigen.

Reviewer #3 (Public Review):

The study focuses on in vivo and in vitro cellular responses intranasal instillation of glycoforms and mutants of SARS-CoV2 spike trimer or spike bearing VLP in mice. Collectively, the experiments suggest that SARS-CoV2 spike has pro-inflammatory roles through increase M1 macrophage associated cytokines and induction of neutrophil netosis/necrosis, a proinflammatory cell death pathway. These effects seem largely independent of hACE2 interaction and partly depend upon interactions with SIGLECs on macrophages and neutrophils. A strength of the study is that a number sophisticated methods are used, including intravital microscopy in the cramaster and liver as well as acute lung slice models, to look at uptake of the spike proteins and immune cell dynamics. The weakness is that some of the reagents maybe contaminated with uncharacterized glycoforms and some important controls, such as control spike protein and control VLP are unevenly applied or not included. The authors have revised the manuscript through some improvements in the writing, but the survey nature and suggestive level of evidence is still a weakness. The study calls attention to sources of proinflammatory activity in the SARS CoV2 spike that may involve some carbohydrate interactions.

Joint Public Review:

Using Ts65Dn - the most commonly used mouse model of Down syndrome (DS) - the goal of this study is two-pronged: 1) to conduct a thorough assessment of DS-related genotypic, physiological, behavioral, and phenotypic measures in a longitudinal manner; and 2) to measure the effects of chronic GTE-EGCG on these measures in the Ts65Dn mouse model. Corroborating results from several previous studies on Ts65Dn mice, findings of this study show confirm the Ts65Dn mouse model exhibits the suite of traits associated with DS. The findings also suggest that the mouse model might have experienced drift, given the milder phenotypes than those reported by earlier studies. Results of the GTE-EGCG treatment do not support its therapeutic use and instead show that the treatment exacerbated certain DS-related phenotypes.

Strengths:

The authors performed a rigorous assessment of treatment and examined treatment and genotypic alterations at multiple time points during growth and aging. Detailed analysis shows differences in genotype during aging as well as genotype with treatment. This study is solid in the overarching methodological approach (with the exception of RNAseq, described below). The biggest strength of the study is its approach and dataset, which corroborate results from a multitude of past studies on Ts65Dn mice, albeit on adult specimens.

Comments on revised submission:

The authors have made numerous changes to address the concerns of the reviewers. The strengths remain: a large, longitudinal data set for the Ts65Dn mouse model across multiple organ systems. The results also clearly show the impact of GTE-EGCG treatment and do not support its therapeutic use.

The authors should report their a priori power calculations that they used when designing their experiment. This should be added to either the Animals or Statistics subsections of the Methods.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Yan et al. investigate the molecular bases underlying mating type recognition in Tetrahymena thermophila. This model protist possesses a total of 7 mating types/sexes and mating occurs only between individuals expressing different mating types. The authors aimed to characterize the function of mating type proteins (MTA and MTB) in the process of self- and non-self recognition, using a combination of elegant phenotypic assays, protein studies, and imaging. They showed that the presence of MTA and MTB in the same cell is required for the expression of concavalin-A receptors and for tip transformation - two processes that are characteristic of the costimulation phase that precedes cell fusion. Using protein studies, the authors identify a set of additional proteins of varied functions that interact with MTA and MTB and are likely responsible for the downstream signaling processes required for mating. This is a description of a fascinating self- and non-self-recognition system and, as the authors point out, it is a rare example of a system with numerous mating types/sexes. This work opens the door for the further understanding of the molecular bases and evolution of these complex recognition systems within and outside protists.

The results shown in this study point to the unequivocal requirement of MTA and MTB proteins for mating. Nevertheless, some of the conclusions regarding the mode of functioning of these proteins are not fully supported and require additional investigation.

Strengths:<br /> (1) The authors have established a set of very useful knock-out and reporter lines for MT proteins and extensively used them in sophisticated and well-designed phenotypic assays that allowed them to test the role of these proteins in vivo.

(2) Despite their apparent low abundance, the authors took advantage of a varied set of protein isolation and characterization techniques to pinpoint the localization of MT proteins to the cell membrane, and their interaction with multiple other proteins that could be downstream effectors. This opens the door for the future characterization of these proteins and further elucidation of the mating type recognition cascade.

Weaknesses:<br /> The manuscript is structured and written in a very clear and easy-to-follow manner. However, several conclusions and discussion points fall short of highlighting possible models and mechanisms through which MT proteins control mating type recognition:

(1) The authors dismiss the possibility of a "simple receptor-ligand system", even though the data does not exclude this possibility. The model presented in Figure 2 S1, and on which the authors based their hypothesis, assumes the independence of MTA and MTB proteins in the generation of the intracellular cascade. However, the results presented in Figure 2 show that both proteins are required to be active in the same cell. Coupled with the fact that MTA and MTB proteins interact, this is compatible with a model where MTA would be a ligand and MTB a receptor (or vice-versa), and could thus form a receptor-ligand complex that could potentially be activated by a non-cognate MTA-MTB receptor-ligand complex, leading to an intracellular cascade mediated by the identified MRC proteins. As it stands, it is not clear what is the proposed working model, and it would be very beneficial for the reader for this to be clarified by having the point of view of the authors on this or other types of models.

(2) The presence of MTA/MTB proteins is required for costimulation (Figure 2), and supplementation with non-cognate extracellular fragments of these proteins (MTAxc, or MTBxc) is a positive stimulator of pairing. However, alone, these fragments do not have the ability to induce costimulation (Figure 5). Based on the results in Figures 5 and 6 the authors suggest that MT proteins mediate both self and non-self recognition. Why do MTAxc and MTBxc not induce costimulation alone? Are any other components required? How to reconcile this with the results of Figure 2? A more in-depth interpretation of these results would be very helpful, since these questions remain unanswered, making it difficult for the reader to extract a clear hypothesis on how MT proteins mediate self- and non-self-recognition.

Reviewer #2 (Public Review):

This manuscript reports the discovery and analysis of a large protein complex that controls mating type and sexual reproduction of the model ciliate Tetrahymena thermophila. In contrast to many organisms that have two mating types or two sexes, Tetrahymena is multi-sexual with 7 distinct mating types. Previous studies identified the mating type locus, which encodes two transmembrane proteins called MTA and MTB that determine the specificity of mating type interactions. In this study, mutants are generated in the MTA and MTB genes and mutant isolates are studied for mating properties. Cells missing either MTA or MTB failed to co-stimulate wild-type cells of different mating types. Moreover, a mixture of mutants lacking MTA or MTB also failed to stimulate. These observations support the conclusion that MTA and MTB may form a complex that directs mating-type identity. To address this, the proteins were epitope-tagged and subjected to IP-MS analysis. This revealed that MTA and MTB are in a physical complex, and also revealed a series of 6 other proteins (MRC1-6) that together with MTA/B form the mating type recognition complex (MTRC). All 8 proteins feature predicted transmembrane domains, three feature GFR domains, and two are predicted to function as calcium transporters. The authors went on to demonstrate that components of the MTRC are localized on the cell surface but not in the cilia. They also presented findings that support the conclusion that the mating type-specific region of the MTA and MTB genes can influence both self- and non-self-recognition in mating.

Taken together, the findings presented are interesting and extend our understanding of how organisms with more than two mating types/sexes may be specified. The identification of the six-protein MRC complex is quite intriguing. It would seem important that the function of at least one of these subunits be analyzed by gene deletion and phenotyping, similar to the findings presented here for the MTA and MTB mutants. A straightforward prediction might be that a deletion of any subunit of the MRC complex would result in a sterile phenotype. The manuscript was very well written and a pleasure to read.

Reviewer #3 (Public Review):

The authors describe the role, location, and function of the MTA and MTB mating type genes in the multi-mating-type species T. thermophila. The ciliate is an important group of organisms to study the evolution of mating types, as it is one of the few groups in which more than two mating types evolved independently. In the study, the authors use deletion strains of the species to show that both mating types genes located in each allele are required in both mating individuals for successful matings to occur. They show that the proteins are localized in the cell membrane, not the cilia, and that they interact in a complex (MTRC) with a set of 6 associated (non-mating type-allelic) genes. This complex is furthermore likely to interact with a cyclin-dependent kinase complex. It is intriguing that T. thermophila has two genes that are allelic and that are both required for successful mating. This coevolved double recognition has to my knowledge not been described for any other mating-type recognition system. I am not familiar with experimental research on ciliates, but as far as I can judge, the experiments appear well performed and mostly support the interpretation of the authors with appropriate controls and statistical analyses.

The results show clearly that the mating type genes regulate non-self-recognition, however, I am not convinced that self-recognition occurs leading to the suppression of mating. An alternative explanation could be that the MTA and MTB proteins form a complex and that the two extracellular regions together interact with the MTA+MTB proteins from different mating types. This alternative hypothesis fits with the coevolution of MTA and MTB genes observed in the phylogenetic subgroups as described by Yan et al. (2021 iScience). Adding MTAxc and/or MTBxc to the cells can lead to the occupation of the external parts of the full proteins thereby inhibiting the formation of the complex, which in turn reduces non-self interactions. Self-recognition as explained in Figure 2S1 suggests an active response, which should be measurable in expression data for example. This is in my opinion not essential, but a claim of self-recognition through the MTA and MTB should not be made.

The authors discuss that T. thermophila has special mating-type proteins that are large, while those of other groups are generally small (lines 157-160 and discussion). The complex formed is very large and in the discussion, they argue that this might be due to the "highly complex process, given that there are seven mating types in all". There is no argument given why large is more complex, if this is complex, and whether more mating types require more complexity. In basidiomycete fungi, many more mating types than 7 exist, and the homeodomain genes involved in mating types are relatively small but highly diverse (Luo et al. 1994 PMID: 7914671). The mating types associated with GPCR receptors in fungi are arguably larger, but again their function is not that complex, and mating-type specific variations appear to evolve easily (Fowler et al 2004 PMID: 14643262; Seike et al. 2015 PMID: 25831518). The large protein complex formed is reminiscent of the fusion patches that develop in budding or fission yeasts. In these species, the mating type receptors are activated by ligand pheromones from the opposite mating type that induce polarity patch formation (see Sieber et al. 2023 PMID: 35148940 for a recent review). At these patches, growth (shmooing) and fusion occur, which is reminiscent (in a different order) of the tip transformation in T. thermophilia. The fusion of two cells is in all taxa a dangerous and complex event that requires the evolution of very strict regulation and the existence of a system like the MTRC and cyclin-dependent complex to regulate this process is therefore not unexpected. The existence of multiple mating types should not greatly complicate the process, as most of the machinery (except for the MTA and MTB) is identical among all mating types.

The Tetrahymena/ciliate genetics and lifecycle could be better explained. For a general audience, the system is not easy to follow. For example, the ploidy of the somatic nucleus with regards to the mating type is not clear to me. The MAC is generally considered "polyploid", but how does this work for the mating type? I assume only a single copy of the mating type locus is available in the MAC to avoid self-recognition in the cells. Is it known how the diploid origin reduces to a single mating type? This does not become apparent from Cervantes et al. 2013. Also, the explanation of co-stimulation is not completely clear (lines 49-60). Initially, direct cell-cell contact is mentioned, but later it is mentioned that "all cells become fully stimulated", even when unequal ratios are used. Is physical contact necessary? Or is this due to the "secrete mating-essential factors" (line 601)? These details are essential, for interpretation of the results and need to be explained better.

Abstract and introduction: Sexes are not mating types. In general, mating types refer to systems in which there is no obvious asymmetry between the gametes, beyond the compatibility system. When there is a physiological difference such as size or motility, sexes are used. This distinction is of importance because in many species mating types and sexes can occur together, with each sex being able to have either (when two) or multiple mating types. An example are SI in angiosperms as used as an example by the authors or mating types in filamentous fungi. See Billiard et al. 2011 [PMID: 21489122] for a good explanation and argumentation for the importance of making this distinction.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript, titled "Allosteric Modulation of the CXCR4:CXCL12 Axis by Targeting Receptor Nanoclustering via the TMV-TMVI Domain," presents a compelling investigation into the development of a potential anti-cancer therapeutic agent. The study focuses on targeting specific CXCR4 intermolecular interactions via an allosteric antagonist which binds proximal to the orthosteric ligand binding site. The novel compounds developed aim to mitigate tumor dissemination, proliferation, and metastasis in transgenic Zebrafish models implanted with HeLa cells.

Strengths:<br /> The study holds significant promise, offering a novel approach to addressing the targeted modulation of CXCR4. The multidisciplinary methodology employed is commendable, providing a comprehensive understanding of the underlying molecular interactions. The proposed workflow, although requiring some adjustments, is reasonable and has the potential to make a substantial impact in the field.

Weaknesses:<br /> Despite the brilliance of the concept and its potential impact, the computational approach appears somewhat superficial and lacks essential considerations. A comprehensive revision of the computational methodology is strongly recommended, with a focus on addressing key points. Additionally, the experimental section should be modified accordingly to align with the refined results. While the study's foundations are promising, its current state warrants a thorough revision to enhance its scientific rigor and overall robustness.

Reviewer #2 (Public Review):

Summary:<br /> This work describes a new pharmacological targeting approach to inhibit selective functions of the ubiquitously expressed chemokine receptor CXCR4, a potential target of immunomodulatory or anti-cancer treatments. Overall, the results build a strong case for the potential of this new compound to target specific functions of CXCR4, particularly linked to tumorigenesis. However, a more thorough evaluation of the function of the compound as well as future studies in mammalian model systems are needed to better assess the promise of the compound.

Strengths:<br /> The work elegantly utilizes in silico drug modelling to propose new small molecule compounds with specific features. This way, the authors designed compound AGR1.137, which abolishes ligand-induced CXCR4 receptor nanoclustering and the subsequent directed cell migration without affecting ligand binding itself or some other ligand-induced signaling pathways. The authors have used a relatively broad set of experiments to validate and demonstrate the effects of the drug. Importantly, the authors also test AGR1.137 in vivo, using a zebrafish model of tumorigenesis and metastasis. A relatively strong inhibitory effect of the compound is reported.

Weaknesses:<br /> The data would be significantly strengthened by adding kinetics and titration of concentrations. This is particularly important as it is the first description of these particular compounds and would help to evaluate the potency and possible side effects of the drug.

The authors carry out single-molecule tracking experiments to analyze nanoclustering of CXCR4 upon ligand binding. This complex data is presented in a sub-optimal manner. Representative images of the data should be included together with more thorough analysis tools like autocorrelation function or mean square displacement to get a more conclusive view of receptor clustering and the effects of the compound.

In the in vivo tumorigenesis experiments, again more kinetics and different concentrations of the drug would generate more convincing data. Also, the individual data points should be visualized to allow full evaluation of the data, throughout the experiments.

Reviewer #1 (Public Review):

Summary:<br /> Jin et al. investigated how the bacterial DNA damage (SOS) response and its regulator protein RecA affect the development of drug resistance under short-term exposure to beta-lactam antibiotics. Canonically, the SOS response is triggered by DNA damage, which results in the induction of error-prone DNA repair mechanisms. These error-prone repair pathways can increase mutagenesis in the cell, leading to the evolution of drug resistance. Thus, inhibiting the SOS regulator RecA has been proposed as a means to delay the rise of resistance.

In this paper, the authors deleted the RecA protein from E. coli and exposed this ∆recA strain to selective levels of the beta-lactam antibiotic, ampicillin. After an 8-hour treatment, they washed the antibiotic away and allowed the surviving cells to recover in regular media. They then measured the minimum inhibitory concentration (MIC) of ampicillin against these treated strains. They note that after just 8-hour treatment with ampicillin, the ∆recA had developed higher MICs towards ampicillin, while by contrast, wild-type cells exhibited unchanged MICs. This MIC increase was also observed in subsequent generations of bacteria, suggesting that the phenotype is driven by a genetic change.

The authors then used whole genome sequencing (WGS) to identify mutations that accounted for the resistance phenotype. Within resistant populations, they discovered key mutations in the promoter region of the beta-lactamase gene, ampC; in the penicillin-binding protein PBP3 which is the target of ampicillin; and in the AcrB subunit of the AcrAB-TolC efflux machinery. Importantly, mutations in the efflux machinery can impact the resistance towards other antibiotics, not just beta-lactams. To test this, they repeated the MIC experiments with other classes of antibiotics, including kanamycin, chloramphenicol, and rifampicin. Interestingly, they observed that the ∆recA strains pre-treated with ampicillin showed higher MICs towards all other antibiotics tested. This suggests that the mutations conferring resistance to ampicillin are also increasing resistance to other antibiotics.

The authors then performed an impressive series of genetic, microscopy, and transcriptomic experiments to show that this increase in resistance is not driven by the SOS response, but by independent DNA repair and stress response pathways. Specifically, they show that deletion of the recA reduces the bacterium's ability to process reactive oxygen species (ROS) and repair its DNA. These factors drive the accumulation of mutations that can confer resistance to different classes of antibiotics. The conclusions are reasonably well-supported by the data, but some aspects of the data and the model need to be clarified and extended.

Strengths:<br /> A major strength of the paper is the detailed bacterial genetics and transcriptomics that the authors performed to elucidate the molecular pathways responsible for this increased resistance. They systemically deleted or inactivated genes involved in the SOS response in E. coli. They then subjected these mutants to the same MIC assays as described previously. Surprisingly, none of the other SOS gene deletions resulted in an increase in drug resistance, suggesting that the SOS response is not involved in this phenotype. This led the authors to focus on the localization of DNA PolI, which also participates in DNA damage repair. Using microscopy, they discovered that in the RecA deletion background, PolI co-localizes with the bacterial chromosome at much lower rates than wild-type. This led the authors to conclude that deletion of RecA hinders PolI and DNA repair. Although the authors do not provide a mechanism, this observation is nonetheless valuable for the field and can stimulate further investigations in the future.

In order to understand how RecA deletion affects cellular physiology, the authors performed RNA-seq on ampicillin-treated strains. Crucially, they discovered that in the RecA deletion strain, genes associated with antioxidative activity (cysJ, cysI, cysH, soda, sufD) and Base Excision Repair repair (mutH, mutY, mutM), which repairs oxidized forms of guanine, were all downregulated. The authors conclude that down-regulation of these genes might result in elevated levels of reactive oxygen species in the cells, which in turn, might drive the rise of resistance. Experimentally, they further demonstrated that treating the ∆recA strain with an antioxidant GSH prevents the rise of MICs. These observations will be useful for more detailed mechanistic follow-ups in the future.

Weaknesses:<br /> Throughout the paper, the authors use language suggesting that ampicillin treatment of the ∆recA strain induces higher levels of mutagenesis inside the cells, leading to the rapid rise of resistance mutations. However, as the authors note, the mutants enriched by ampicillin selection can play a role in efflux and can thus change a bacterium's sensitivity to a wide range of antibiotics, in what is known as cross-resistance. The current data is not clear on whether the elevated "mutagenesis" is driven ampicillin selection or by a bona fide increase in mutation rate.

Furthermore, on a technical level, the authors employed WGS to identify resistance mutations in the treated ampicillin-treated wild-type and ∆recA strains. However, the WGS methodology described in the paper is inconsistent. Notably, wild-type WGS samples were picked from non-selective plates, while ΔrecA WGS isolates were picked from selective plates with 50 μg/mL ampicillin. Such an approach biases the frequency and identity of the mutations seen in the WGS and cannot be used to support the idea that ampicillin treatment induces higher levels of mutagenesis.

Finally, it is important to establish what the basal mutation rates of both the WT and ∆recA strains are. Currently, only the ampicillin-treated populations were reported. It is possible that the ∆recA strain has inherently higher mutagenesis than WT, with a larger subpopulation of resistant clones. Thus, ampicillin treatment might not in fact induce higher mutagenesis in ∆recA.

Reviewer #2 (Public Review):

Summary:<br /> This study aims to demonstrate that E. coli can acquire rapid antibiotic resistance mutations in the absence of a DNA damage response. To investigate this, the authors employed a sophisticated experimental framework based on a modified Adaptive Laboratory Evolution (ALE) workflow. This workflow involves numerous steps culminating in the measurement of antibiotic resistance. The study presents evidence that a recA strain develops ampicillin resistance mutations more quickly than the wild-type, as shown by measuring the Minimum Inhibitory Concentration (MIC) and mutation frequency. Whole-genome sequencing of 15 recA- colonies resistant to ampicillin revealed predominantly inactivation of genes involved in the multi-drug efflux pump system, whereas, in the wild-type, mutations appear to enhance the activity of the chromosomal ampC cryptic promoter. By analyzing mutants involved in the SOS response, including a lexA3 mutant incapable of inducing the SOS response, the authors conclude that the rapid evolution of antibiotic resistance occurs in an SOS-independent manner when recA is absent.

Furthermore, RNA sequencing (RNA-seq) of the four experimental conditions suggests that genes related to antioxidative responses drive the swift evolution of antibiotic resistance in the recA- strain.

Weaknesses:<br /> However, a potential limitation of this study is the experimental design used to determine the 'rapid' evolution of antibiotic resistance. It may introduce a significant bottleneck in selecting ampicillin-resistant mutants early on. A recA mutant could be more susceptible to ampicillin than the wild-type, and only resistant mutants might survive after 8 hours, potentially leading to their enrichment in subsequent steps. To address this concern, it would be critical to perform a survival analysis at various time points (0h, 2h, 4h, 6h, and 8h) during ampicillin treatment for both recA and wild-type strains, ensuring there is no difference in viability.

The observation that promoter mutations are absent in recA strains could be explained by previous research indicating that amplification of the AmpC genes is a mechanism for E. coli resistance to ampicillin, which does not occur in a recA-deficient background (PMID# 19474201).

The section describing Figure 3 is poorly articulated, and the conclusions drawn are apparent. The inability of a recA strain to induce the SOS response is well-documented (lines 210 and 278). The data suggest that merely blocking SOS induction is insufficient to cause 'rapid' evolution in their experimental conditions. To investigate whether SOS response can be induced independently of lexA cleavage by recA, alternative experiments, such as those using a sulA-GFP fusion, might be more informative.

In Figure 4E, the lack of increased SulA gene expression in the wild-type strain treated with ampicillin is unexpected, given that SulA is an SOS-regulated gene. The fact that polA (Pol I) is going down should be taken into account in the interpretation of Figures 2D and 2E.

The connection between compromised DNA repair, the accumulation of Reactive Oxygen Species (ROS) based on RNA-seq data, and accelerated evolution is merely speculative at this point and not experimentally established.

Reviewer #3 (Public Review):

Summary:<br /> In the present work, Zhang et al investigate the involvement of the bacterial DNA damage repair SOS response in the evolution of beta-lactam drug resistance evolution in Escherichia coli. Using a combination of microbiological, bacterial genetics, laboratory evolution, next-generation, and live-cell imaging approaches, the authors propose short-term drug resistance evolution that can take place in RecA-deficient cells in an SOS response-independent manner. They propose the evolvability of drug resistance is alternatively driven by the oxidative stress imposed by the accumulation of reactive oxygen species and inhibition of DNA repair. Overall, this is a nice study that addresses a growing and fundamental global health challenge (antimicrobial resistance). However, although the authors perform several multi-disciplinary experiments, there are several caveats to the authors' proposal that ultimately do not fully support their interpretation that the observed antimicrobial resistance evolution phenotype is due to compromised DNA repair.

Strengths:<br /> The authors introduce new concepts to antimicrobial resistance evolution mechanisms. They show short-term exposure to beta-lactams can induce durably fixed antimicrobial resistance mutations. They propose this is due to comprised DNA repair and oxidative stress. This is primarily supported by their observations that resistance evolution phenotypes only exist for recA deletion mutants and not other genes in the SOS response

Weaknesses:<br /> The authors do not show any direct evidence (1) that these phenotypes exist in strains harboring deletions in other DNA repair genes outside of the SOS response, (2) that DNA damage is increased, (3) that reactive oxygen species accumulate, (4) that accelerated resistance evolution can be reversed by anything other than recA complementation. The authors do not directly test alternative hypotheses. The conclusions drawn are therefore premature.

Reviewer #1 (Public Review):

In this study, the researchers aimed to investigate the cellular landscape and cell-cell interactions in cavernous tissues under diabetic conditions, specifically focusing on erectile dysfunction (ED). They employed single-cell RNA sequencing to analyze gene expression patterns in various cell types within the cavernous tissues of diabetic individuals. The researchers identified decreased expression of genes associated with collagen or extracellular matrix organization and angiogenesis in several cell types, including fibroblasts, chondrocytes, myofibroblasts, valve-related lymphatic endothelial cells, and pericytes. They also discovered a newly identified marker, LBH, that distinguishes pericytes from smooth muscle cells in mouse and human cavernous tissues. Furthermore, the study revealed that pericytes play a role in angiogenesis, adhesion, and migration by communicating with other cell types within the corpus cavernosum. However, these interactions were found to be significantly reduced under diabetic conditions. The study also investigated the role of LBH and its interactions with other proteins (CRYAB and VIM) in maintaining pericyte function and highlighted their potential involvement in regulating neurovascular regeneration. Overall, the manuscript is well-written and the study provides novel insights into the pathogenesis of ED in patients with diabetes and identifies potential therapeutic targets for further investigation.

Comments on revised version:

For Figure 4, immunofluorecent staining of LBH following intracavernous injections with lentiviruses is required to justify overexpression and tissue specificity.

Reviewer #3 (Public Review):

Bae et al. described the key roles of pericytes in cavernous tissues in diabetic erectile dysfunction using both mouse and human single-cell transcriptomic analysis. Erectile dysfunction (ED) is caused by dysfunction of the cavernous tissue and affects a significant proportion of men aged 40-70. The most common treatment for ED is phosphodiesterase 5 inhibitors; however, these are less effective in patients with diabetic ED. Therefore, there is an unmet need for a better understanding of the cavernous microenvironment, cell-cell communications in patients with diabetic ED, and the development of new therapeutic treatments to improve the quality of life.

Pericytes are mesenchymal-derived mural cells that directly interact with capillary endothelial cells (ECs). They play a vital role in the pathogenesis of erectile function as their interactions with ECs are essential for penile erection. Loss of pericytes has been associated with diabetic retinopathy, cancer, and Alzheimer's disease and has been investigated in relation to the permeability of cavernous blood vessels and neurovascular regeneration in the authors' previous studies. This manuscript explores the mechanisms underlying the effect of diabetes on pericyte dysfunction in ED. Additionally, the cellular landscape of cavernous tissues and cell type-specific transcriptional changes were carefully examined using both mouse and human single-cell RNA sequencing in diabetic ED. The novelty of this work lies in the identification of a newly identified pericyte (PC)-specific marker, LBH, in mouse and human cavernous tissues, which distinguishes pericytes from smooth muscle cells. LBH not only serves as a cavernous pericyte marker, but its expression level is also reduced in diabetic conditions. The LBH-interacting proteins (Cryab and Vim) were further identified in mouse cavernous pericytes, indicating that these signaling interactions are critical for maintaining normal pericyte function. Overall, this study demonstrates the novel marker of pericytes and highlights the critical role of pericytes in diabetic ED.

Comments on revised version:

Bae and colleagues substantially improved the data quality and revised their manuscript "Pericytes contribute to pulmonary vascular remodeling via HIF2a signaling". While these revisions clarify some of the concerns raised, others remain. In my view, the following question must be addressed.

In my prior question on #3, I completely disagree with the statement that "identified cells with pericyte-like characteristics in the walls of large blood vessels". The staining that authors provided for LBH, was clearly stained for SMCs, not pericytes. Per Fig 2E, the authors are correct that LBH is colocalized with SMA+ cells( SMCs). However, the red signal from LBH clearly stains endothelial cells. In the rest of 2E and 2D, LBH is CD31- and their location suggests LBH stained for SMCs in the Aorta, Kidney vasculature, Dorsal vein, and Dorsal Artery.

Reviewer #1 (Public Review):

Summary:<br /> The authors utilize fluid-structure interaction analyses to simulation fluid flow within and around the Cambrian cnidarian Quadrapyrgites to reconstruct feeding/respiration dynamics. Based on vorticity and velocity flow patterns, the authors suggest that the polyp expansion and contraction ultimately develop vortices around the organism that are like what modern jellyfish employ for movement and feeding. Lastly, the authors suggest that this behavior is likely a prerequisite transitional form to swimming medusae.

Strengths:<br /> While fluid-structure-interaction analyses are common in engineering, physics, and biomedical fields, they are underutilized in the biological and paleobiological sciences. Zhang et al. provide a strong approach to integrating active feeding dynamics into fluid flow simulations of ancient life. Based on their data, it is entirely likely the described vortices would have been produced by benthic cnidarians feeding/respiring under similar mechanisms. However, some of the broader conclusions require additional justification.

Weaknesses:

1. The claim that olivooid-type feeding was most likely a prerequisite transitional form to jet-propelled swimming needs much more support or needs to be tailored to olivooids. This suggests that such behavior is absent (or must be convergent) before olivooids, which is at odds with the increasing quantities of pelagic life (whose modes of swimming are admittedly unconstrained) documented from Cambrian and Neoproterozoic deposits. Even among just medusozoans, ancestral state reconstruction suggests that they would have been swimming during the Neoproterozoic (Kayal et al., 2018; BMC Evolutionary Biology) with no knowledge of the mechanics due to absent preservation.<br /> 2. While the lack of ambient flow made these simulations computationally easier, these organisms likely did not live in stagnant waters even within the benthic boundary layer. The absence of ambient unidirectional laminar current or oscillating current (such as would be found naturally) biases the results.<br /> 3. There is no explanation for how this work could be a breakthrough in simulation gregarious feeding as is stated in the manuscript.

Despite these weaknesses the authors dynamic fluid simulations convincingly reconstruct the feeding/respiration dynamics of the Cambrian Quadrapyrgites, though the large claims of transitionary stages for this behavior are not adequately justified. Regardless, the approach the authors use will be informative for future studies attempting to simulate similar feeding and respiration dynamics.

The following text is directly in response to the revised version of the manuscript.<br /> Dynamic simulations of feeding and respiration of the early Cambrian periderm-bearing cnidarian polyps

Revision 1

I think this manuscript has been improved by the authors, and I appreciate their time and effort in considering my earlier comments. While most of my line by line comments have been incorporated, I do feel that some of my larger points have been insufficiently addressed. Those are repeated with additional clarifications below.

Original comment: The claim that olivooid-type feeding was most likely a prerequisite transitional form to jet-propelled swimming needs much more support or needs to be tailored to olivooids. This suggests that such behavior is absent (or must be convergent) before olivooids, which is at odds with the increasing quantities of pelagic life (whose modes of swimming are admittedly unconstrained) documented from Cambrian and Neoproterozoic deposits. Even among just medusozoans, ancestral state reconstruction suggests that they would have been swimming during the Neoproterozoic (Kayal et al., 2018; BMC Evolutionary Biology) with no knowledge of the mechanics due to absent preservation.

Author response: Thanks for your suggestions. Yes, we agree with you that the ancestral swimming medusae may appear before the early Cambrian, even at the Neoproterozoic deposits. However, discussions on the affinities of Ediacaran cnidarians are severely limited because of the lack of information concerning their soft anatomy. So, it is hard to detect the mechanics due to absent preservation. Olivooids found from the basal Cambrian Kuanchuanpu Formation can be reasonably considered as cnidarians based on their radial symmetry, external features, and especially the internal anatomies (Bengtson and Yue 1997; Dong et al. 2013; 2016; Han et al. 2013; 2016; Liu et al. 2014; Wang et al. 2017; 2020; 2022). The valid simulation experiment here was based on the soft tissue preserved in olivooids.

Reviewer response: This response does not sufficiently address my earlier comment. While the authors are correct that individual Ediacaran affinities are an area of active research and that Olivooids can reasonably be considered cnidarians, this doesn't address the actual critique in my comment. Most (not all) Ediacaran soft-bodied fossils are considered to have been benthic, but pelagic cnidarian life is widely acknowledged to at least be present during later White Sea and Nama assemblages (and earlier depending on molecular clock interpretations). The authors have certainly provided support for the mechanics of this type of feeding being co-opted for eventual jet-propulsion swimming in Olivooids. They have not provided sufficient justifications within the manuscript for this to be broadened beyond this group.

Original comment: There is no explanation for how this work could be a breakthrough in simulation gregarious feeding as is stated in the manuscript.

Author response: Thanks for your suggestion. We revised the section "Perspectives for future work and improvements" (lines 396-404 in our revised version of MS). Conducting simulations of gregarious active feeding behavior generally need to model multi (or clustered) organisms, which is beyond the present computational capability. However, exploiting the simulation result and thus building a simplified model can be possible to realize that, as we may apply an inlet or outlet boundary condition to the peridermal aperture of Quadrapyrgites with corresponding exhale or inhale flow velocity profiles collected in this work. By doing this we can obtain a simplified version of an active feeding Quadrapyrgites model without using computational expensive moving mesh feature. Such a model can be used solely or in cluster to investigate gregarious feeding behavior incorporated with ambient current. Those above are explicit explanations for how this work could be a "breakthrough" in simulation gregarious feeding. However, we modified the corresponding description in section "Perspectives for future work and improvements" to make it more appropriate.

Reviewer response: I think I understand where the authors are trying to take this next step. If the authors were to follow up on this study with the proposed implementation of inhalant/exhalent velocities profiles (or more preferably velocity/pressure fields), then that study would be a breakthrough in simulating such gregarious feeding. Based on what has been done within the present study, I think the term "breakthrough" is instead overly emphatic.<br /> An additional note on this. The authors are correct that incorporating additional models could be used to simulation a population (as has been successfully done for several Ediacaran taxa despite computational limitations), but it's not the only way. The authors might explore using periodic boundary conditions on the external faces of the flow domain. This could require only a single Olivooid model to assess gregarious impacts - see the abundant literature of modeling flow through solar array fields.

Original comment: L446: two layers of hexahedral elements is a very low number for meshing boundary layer flow

Author response: Many thanks for your question. We agree that an appropriate hexahedral elements mesh for boundary layer is essential to recover boundary flow, especially in cases where turbulence model incorporated with wall function is adopted such as the standard k-epsilon model. In this case, the boundary flow is not the main point since the velocity profile was collected above periderm aperture rather than near no-slip wall region. What else, we do not need drag (related to sheer stress and pressure difference) computations in this case, which requires a more accurate flow velocity reconstruction near no-slip walls as what previous palaeontological CFD simulations have done. Thus, we think two layers of hexahedral elements are enough. What else, hexahedral elements added to periderm aperture domain, as illustrated in figure below, can let the velocity near wall vary smoothly and thus can benefit the convergency of simulations.

Reviewer response: As the authors point out in the main text, these organisms are small (millimeters in scale) and certainly lived within the boundary layer range of the ocean. While the boundary layer is not the main point, it still needs to be accurately resolved as it should certainly affect the flow further towards the far field at this scale. I'm not suggesting the authors need to perfectly resolve the boundary layer or focus on using turbulence models more tailored to boundary layer flows (such as k-w), but the flow field still needs sufficient realism for a boundary bounded flow. The authors really should consider quantitatively assessing the number of hexahedral elements within their mesh refinement study.

Reviewer #2 (Public Review):

Summary: The authors seek to elucidate the early evolution of cnidarians through computer modeling of fluid flow in the oral region of very small, putative medusozoan polyps. They propose that the evolutionary advent of the free-swimming medusoid life stage was preceded by a sessile benthic life stage equipped with circular muscles that originally functioned to facilitate feeding and that later became co-opted for locomotion through jet propulsion.

Strengths: Assumptions of the modeling exercise laid out clearly; interpretations of the results of the model runs in terms of functional morphology plausible. An intriguing investigation that should stimulate further discussion and research.

Weaknesses: Speculation on the origin of the medusoid life stage in cnidarians heavily dependent on prior assumptions concerning the soft part anatomy and material properties of the skeleton of the modeled fossil organism that may be open to alternative interpretations. Logically, of course, the hypothesis that cnidarian medusae originated from benthic polyps must be evaluated along with the alternative hypotheses that the medusa came first and that the ancestral cnidarian exhibited both life stages.