Reviewer #2 (Public review):

In this study, Nartker et al. examine how much observers are conscious of using variations of classic inattentional blindness studies. The key idea is that rather than simply asking observers if they noticed a critical object with one yes/no question, the authors also ask follow-up questions to determine if observers are aware of more than the yes/no questions suggest. Specifically, by having observers make forced choice guesses about the critical object, the authors find that many observers who initially said "no" they did not see the object can still "guess" above chance about the critical object's location, color, etc. Thus, the authors claim, that prior claims of inattentional blindness are mistaken and that using such simple methods has led numerous researchers to overestimate how little observers see in the world. To quote the authors themselves, these results imply that "inattentionally blind subjects consciously perceive these stimuli after all... they show sensitivity to IB stimuli because they can see them."

Before getting to a few issues I have with the paper, I do want to make sure to explicitly compliment the researchers for many aspects of their work. Getting massive amounts of data, using signal detection measures, and the novel use of a "super subject" are all important contributions to the literature that I hope are employed more in the future.

Main point 1: My primary issue with this work is that I believe the authors are misrepresenting the way people often perform inattentional blindness studies. In effect, the authors are saying, "People do the studies 'incorrectly' and report that people see very little. We perform the studies 'correctly' and report that people see much more than previously thought." But the way previous studies are conducted is not accurately described in this paper. The authors describe previous studies as follows on page 3:

"Crucially, however, this interpretation of IB and the many implications that follow from it rest on a measure that psychophysics has long recognized to be problematic: simply asking participants whether they noticed anything unusual. In IB studies, awareness of the unexpected stimulus (the novel shape, the parading gorilla, etc.) is retroactively probed with a yes/no question, standardly, "Did you notice anything unusual on the last trial which wasn't there on previous trials?". Any subject who answers "no" is assumed not to have any awareness of the unexpected stimulus.

If this quote were true, the authors would have a point. Unfortunately, I do not believe it is true. This is simply not how many inattentional blindness studies are run. Some of the most famous studies in the inattentional blindness literature do not simply as observes a yes/no question (e.g., the invisible gorilla (Simons et al. 1999), the classic door study where the person changes (Simons and Levin, 1998), the study where observers do not notice a fight happening a few feet from them (Chabris et al., 2011). Instead, these papers consistently ask a series of follow-up questions and even tell the observers what just occurred to confirm that observers did not notice that critical event (e.g., "If I were to tell you we just did XYZ, did you notice that?"). In fact, after a brief search on Google Scholar, I was able to relatively quickly find over a dozen papers that do not just use a yes/no procedure, and instead as a series of multiple questions to determine if someone is inattentionally blind. In no particular order some papers (full disclosure: including my own):

(1) Most et al. (2005) Psych Review<br /> (2) Drew et al. (2013) Psych Science<br /> (3) Drew et al. (2016) Journal of Vision<br /> (4) Simons et al. (1999) Perception<br /> (5) Simons and Levin (1998) Perception<br /> (6) Chabris et al. (2011) iPerception<br /> (7) Ward & Scholl (2015) Psych Bulletin and Review<br /> (8) Most et al. (2001) Psych Science<br /> (9) Todd & Marois (2005) Psych Science<br /> (10) Fougnie & Marois (2007) Psych Bulletin and Review<br /> (11) New and German (2015) Evolution and Human Behaviour<br /> (12) Jackson-Nielsen (2017) Consciousness and cognition<br /> (13) Mack et al. (2016) Consciousness and cognition<br /> (14) Devue et al. (2009) Perception<br /> (15) Memmert (2014) Cognitive Development<br /> (16) Moore & Egeth (1997) JEP:HPP<br /> (17) Cohen et al. (2020) Proc Natl Acad Sci<br /> (18). Cohen et al. (2011) Psych Science

This is a critical point. The authors' key idea is that when you ask more than just a simple yes/no question, you find that other studies have overestimated the effects of inattentional blindness. But none of the studies listed above only asked simple yes/no questions. Thus, I believe the authors are mis-representing the field. Moreover, many of the studies that do much more than ask a simple yes/no question are cited by the authors themselves! Furthermore, as far as I can tell, the authors believe that if researchers do these extra steps and ask more follow-ups, then the results are valid. But since so many of these prior studies do those extra steps, I am not exactly sure what is being criticized.

To make sure this point is clear, I'd like to use a paper of mine as an example. In this study (Cohen et al., 2020, Proc Natl Acad Sci USA) we used gaze-contingent virtual reality to examine how much color people see in the world. On the critical trial, the part of the scene they fixated on was in color, but the periphery was entirely in black and white. As soon as the trial ended, we asked participants a series of questions to determine what they noticed. The list of questions included:

(1) "Did you notice anything strange or different about that last trial?"<br /> (2) "If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?"<br /> (3) "If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?"<br /> (4) "Did you notice anything different about the colors in the last scene?"<br /> (5) We then showed observers the previous trial again and drew their attention to the effect and confirmed that they did not notice that previously.<br /> In a situation like this, when the observers are asked so many questions, do the authors believe that "the inattentionally blind can see after all?" I believe they would not say that and the reason they would not say that is because of the follow-up questions after the initial yes/no question. But since so many previous studies use similar follow-up questions, I do not think you can state that the field is broadly overestimating inattentional blindness. This is why it seems to me to be a bit of a straw-man: most people do not just use the yes/no method.

Main point 2: Let's imagine for a second that every study did just ask a yes/no question and then would stop. So, the criticism the authors are bringing up is valid (even though I believe it is not). I am not entirely sure that above chance performance on a forced choice task proves that the inattentionally blind can see after all. Could it just be a form of subliminal priming? Could there be a significant number of participants who basically would say something like, "No I did not see anything, and I feel like I am just guessing, but if you want me to say whether the thing was to the left or right, I will just 100% guess"? I know the literature on priming from things like change and inattentional blindness is a bit unclear, but this seems like maybe what is going on. In fact, maybe the authors are getting some of the best priming from inattentional blindness because of their large sample size, which previous studies do not use.<br /> I'm curious how the authors would relate their studies to masked priming. In masked priming studies, observers say the did not see the target (like in this study) but still are above chance when forced to guess (like in this study). Do the researchers here think that that is evidence of "masked stimuli are truly seen" even if a participant openly says they are guessing?

Main point 3: My last question is about how the authors interpret a variety of inattentional blindness findings. Previous work has found that observers fail to notice a gorilla in a CT scan (Drew et al., 2013), a fight occurring right in front of them (Chabris et al., 2011), a plane on a runway that pilots crash into (Haines, 1991), and so forth. In a situation like this, do the authors believe that many participants are truly aware of these items but simply failed to answer a yes/no question correctly? For example, imagine the researchers made participants choose if the gorilla was in the left or right lung and some participants who initially said they did not notice the gorilla were still able to correctly say if it was in the left or right lung. Would the authors claim "that participant actually did see the gorilla in the lung"? I ask because it is difficult to understand what it means to be aware of something as salient as a gorilla in a CT scan, but say "no" you didn't notice it when asked a yes/no question. What does it mean to be aware of such important, ecologically relevant stimuli, but not act in response to them and openly say "no" you did not notice them?

Overall: I believe there are many aspects of this set of studies that are innovative and I hope the methods will be used more broadly in the literature. However, I believe the authors misrepresent the field and overstate what can be interpreted from their results. While I am sure there are cases where more nuanced questions might reveal inattentional blindness is somewhat overestimated, claims like "the inattentionally blind can see after all" or "Inattentionally blind subjects consciously perceive thest stimuli after all" seem to be incorrect (or at least not at all proven by this data).

Reviewer #3 (Public review):

Summary:

Authors try to challenge the mainstream scientific as well as popularly held view that Inattentional Blindness (IB) signifies subjects having no conscious awareness of what they report not seeing (after being exposed to unexpected stimuli). They show that even when subjects indicate NOT having seen the unexpected stimulus, they are at above chance level for reporting features such as location, color or movement of these stimuli. Also, they show that 'not seen' responses are in part due to a conservative bias of subjects, i.e. they tend to say no more than yes, regardless of actual visibility. Their conclusion is that IB may not (always) be blindness, but possibly amnesia, uncertainty etc.

Strengths:

A huge pool of (25.000) subjects is used. They perform several versions of the IB experiments, both with briefly presented stimuli (as the classic Mack and Rock paradigm), as well as with prolonged stimuli moving over the screen for 5 seconds (a bit like the famous gorilla version), and all these versions show similar results, pointing in the same direction: above chance detection of unseen features, as well as conservative bias towards saying not seen.

Weaknesses:

Results are all significant but effects are not very strong, typically a bit above chance. Also, it is unclear what to compare these effects to, as there are no control experiments showing what performance would have been in a dual task version where subjects have to also report features etc for stimuli that they know will appear in some trials

There are quite some studies showing that during IB, neural processing of visual stimuli continues up to high visual levels, for example, Vandenbroucke et al 2014 doi:10.1162/jocn_a_00530 showed preserved processing of perceptual inference (i.e. seeing a kanizsa illusion) during IB. Scholte et al 2006 doi: 10.1016/j.brainres.2005.10.051 showed preserved scene segmentation signals during IB. Compared to the strength of these neural signatures, the reported effects may be considered not all that surprising, or even weak.

Reviewer #3 (Public review):

Summary:

The authors first tested whether EAA supplementation increases olfactory preference for bacterial food for a variety of bacterial strains. Of the EAAs, they found only leucine supplementation increased olfactory preference (within a bacterial strain), and only for 3 of the bacterial strains tested. Leucine itself was not found to be intrinsically attractive.

They determined that leucine supplementation increases isoamyl alcohol (IAA) production in the 3 preferred bacterial strains. They identify the biochemical pathway that catabolizes leucine to IAA, showing that a required enzyme for this pathway is upregulated upon supplementation.

Consistent with earlier studies, they find that AWC olfactory neuron is primarily responsible for increased preference for IAA-producing bacteria.

Testing volatile compounds produced by bacteria and identified by GC/MS, and identified several as attractive, most of them require AWC for the full effect. Adaptation assays were used to show that odorant levels produced by bacterial lawns were sufficient to induce olfactory adaptation, and adaptation to IAA reduced chemotaxis to leucine-supplemented lawns. They then showed that IAA attractiveness is conserved across wild strains, while other compounds are more variable, suggesting IAA is a principal foraging cue.

Finally, using the CeNGEN database, they developed a list of candidate IAA receptors. Using behavioral tests, they show that mutation of srd-12 greatly impairs IAA chemotaxis without affecting locomotion or attraction to another AWC-sensed odor, PEA.

Comments

This study will be of great interest in the field of C. elegans behavior, chemical senses and chemical ecology, and understanding of the sensory biology of foraging.

Strengths:

The identification of a receptor for IAA is an excellent finding. The combination of microbial metabolic chemistry and the use of natural bacteria and nematode strains makes an extremely compelling case for the ecological and adaptive relevance of the findings.

Weaknesses:

AWC receives synaptic input from other chemosensory neurons, and thus could potentially mediate navigation behaviors to compounds detected in whole or in part by those neurons. Language concluding detection by AWC should be moderated (e.g. p9 "worms sense an extensive repertoire...predominantly using AWC") unless it has been demonstrated.

srd-12 is not exclusively expressed in AWC. Normally, cell-specific rescue or knockdown would be used to demonstrate function in a specific cell. The authors should provide such a demonstration or explain why they are confident srd-12 acts in AWC.

A comparison of AWC's physiological responses between WT and srd-12, preferably in an unc-13 background, would be nice. Even further, the expression of srd-12 in a different neuron type and showing that it confers responsiveness to IAA (in this case, inhibition) would be very convincing.

Reviewer #1 (Public review):

Summary:

Rossi et al. asked whether gait adaptation is solely a matter of slow perceptual realignment or if it also involves fast/flexible stimulus-response mapping mechanisms. To test this, they conducted a series of split-belt treadmill experiments with ramped perturbations, revealing behavior indicative of a flexible, automatic stimulus-response mapping mechanism.

Strengths:

(1) The study includes a perceptual test of leg speed, which correlates with the perceptual realignment component of motor aftereffects. This indicates that there are motor performances that are not accounted for by perceptual re-alignment.

(2) They study incorporates qualitatively distinct, hypothesis-driven models of adaptation and proposes a new framework that integrates these various mechanisms.

Weaknesses:

(1) The study could benefit from considering other alternative models. As the authors noted in their discussion, while the descriptive models explain some patterns of behaviour/aftereffects, they don't currently account for how these mechanisms influence the initial learning process itself.

a. For example, the pattern of gait asymmetric might differ for perceptual realignment (a smooth, gradual process), structural learning (more erratic, involving hypothesis testing/reasoning to understand the perturbation, see (Tsay et al. 2024) for a recent review on Reasoning), and stimulus-response mapping (possibly through a reinforcement based trial-and-error approach). If not formally doing a model comparison, the manuscript might benefit from clearly laying out the behavioural predictions for how these different processes shape initial learning.

b. Related to the above, the authors noted that the absence of difference during initial learning suggests that the differences in Experiment 2 in the ramp-up phase are driven by two distinct processes: structural learning and memory-based processes. If the assumptions about initial learning are not clear, this logic of this conclusion is hard to follow.

c. The authors could also test a variant of the dual-rate state-space model with two perceptual realignment processes where the constraints on retention and learning rate are relaxed. This model would be a stronger test for two perceptual re-alignment processes: one that is flexible and another that is rigid, without mandating that one be fast learning and fast forgetting, and the other be slow learning and slow forgetting.

(2) The authors claim that stimulus-response mapping operates outside of explicit/deliberate control. While this could be true, the survey questions may have limitations that could be more clearly acknowledged.

a. Specifically, asking participants at the end of the experiments to recall their strategies may suffer from memory biases (e.g., participants may be biased by recent events, and forget about the explicit strategies early in the experiment), be susceptible to the framing of the questions (e.g., participants not being sure what the experimenter is asking and how to verbalize their own strategy), and moreover, not clear what is the category of explicit strategies one might enact here which dictates what might be considered "relevant" and "accurate".

b. The concept of perceptual realignment also suggests that participants are somewhat aware of the treadmill's changing conditions; therefore, as a thought experiment, if the authors have asked participants throughout/during the experiment whether they are trying different strategies, would they predict that some behaviour is under deliberate control?

(3) The distinction between structural and memory-based differences in the two subgroups was based on the notion that memory-based strategies increase asymmetry. However, an alternative explanation could be that unfamiliar perturbations, due to the ramping up, trigger a surprise signal that leads to greater asymmetry due to reactive corrections to prevent one's fall - not because participants are generalizing from previously learned representations (e.g., (Iturralde & Torres-Oviedo, 2019)).

Further contextualization:

Recognizing the differences in dependent variables (reaching position vs. leg speed/symmetry in walking), could the Proprioceptive/Perceptual Re-alignment model also apply to gait adaptation (Tsay et al., 2022; Zhang et al., 2024)? Recent reaching studies show a similar link between perception and action during motor adaptation (Tsay et al., 2021) and have proposed a model aligning with the authors' correlations between perception and action. The core signal driving implicit adaptation is the discrepancy between perceived and desired limb position, integrating forward model predictions with proprioceptive/visual feedback.

References

Iturralde, P. A., & Torres-Oviedo, G. (2019). Corrective Muscle Activity Reveals Subject-Specific Sensorimotor Recalibration. eNeuro, 6(2). https://doi.org/10.1523/ENEURO.0358-18.2019

Tsay, Jonathan S., Hyosub E. Kim, Samuel D. McDougle, Jordan A. Taylor, Adrian Haith, Guy Avraham, John W. Krakauer, Anne G. E. Collins, and Richard B. Ivry. 2024. "Fundamental Processes in Sensorimotor Learning: Reasoning, Refinement, and Retrieval." ELife 13 (August). https://doi.org/10.7554/eLife.91839.

Tsay, Jonathan S., Hyosub E. Kim, Darius E. Parvin, Alissa R. Stover, and Richard B. Ivry. 2021. "Individual Differences in Proprioception Predict the Extent of Implicit Sensorimotor Adaptation." Journal of Neurophysiology, March. https://doi.org/10.1152/jn.00585.2020.

Tsay, Jonathan S., Hyosub Kim, Adrian M. Haith, and Richard B. Ivry. 2022. "Understanding Implicit Sensorimotor Adaptation as a Process of Proprioceptive Re-Alignment." ELife 11 (August). https://doi.org/10.7554/eLife.76639.

Zhang, Zhaoran, Huijun Wang, Tianyang Zhang, Zixuan Nie, and Kunlin Wei. 2024. "Perceptual Error Based on Bayesian Cue Combination Drives Implicit Motor Adaptation." ELife. https://doi.org/10.7554/elife.94608.1.

Reviewer #2 (Public review):

Recent findings in the field of motor learning have pointed to the combined action of multiple mechanisms that potentially contribute to changes in motor output during adaptation. A nearly ubiquitous motor learning process occurs via the trial-by-trial compensation of motor errors, often attributed to cerebellar-dependent updating. This error-based learning process is slow and largely unconscious. Additional learning processes that are rapid (e.g., explicit strategy-based compensation) have been described in discrete movements like goal-directed reaching adaptation. However, the role of rapid motor updating during continuous movements such as walking has been either under-explored or inconsistent with those found during the adaptation of discrete movements. Indeed, previous results have largely discounted the role of explicit strategy-based mechanisms for locomotor learning. In the current manuscript, Rossi et al. provide convincing evidence for a previously unknown rapid updating mechanism for locomotor adaptation. Unlike the now well-studied explicit strategies employed during reaching movements, the authors demonstrate that this stimulus-response mapping process is largely unconscious. The authors show that in approximately half of subjects, the mapping process appears to be memory-based while the remainder of subjects appear to perform structural learning of the task design. The participants that learned using a structural approach had the capability to rapidly generalize to previously unexplored regions of the perturbation space.

One result that will likely be particularly important to the field of motor learning is the authors' quite convincing correlation between the magnitude of proprioceptive recalibration and the magnitude error-based updating. This result beautifully parallels results in other motor learning tasks and appears to provide a robust marker for the magnitude of the mapping process (by means of subtracting off the contribution of error-based motor learning). This is a fascinating result with implications for the motor learning field well beyond the current study.

A major strength of this manuscript is the large sample size across experiments and the extent of replication performed by the authors in multiple control experiments.

Finally, I commend the authors on extending their original observations via Experiment 2. While it seems that participants use a range of mapping mechanisms (or indeed a combination of multiple mapping mechanisms), future experiments may be able to tease apart why some subjects use memory versus structural mapping. A future ability to push subjects to learn structurally-based mapping rules has the potential to inform rehabilitation strategies.

Overall, the manuscript is well written, the results are clear, and the data and analyses are convincing. The manuscript's weaknesses are minor, mostly related to the presentation of the results and modeling.

Weaknesses:

The overall weaknesses in the manuscript are minor and can likely be addressed with textual changes.

(1) A key aspect of the experimental design is the speed of the "ramp down" following the adaptation period. If the ramp-down is too slow, then no after-effects would be expected even in the alternative recalibration-only/error-based only hypothesis. How did the authors determine the appropriate rate of ramp-down? Do alternative choices of ramp-down rates result in step length asymmetry measures that are consistent with the mapping hypothesis?

(2) Overall, the modeling as presented in Figure 3 (Equation 1-3) is a bit convoluted. To my mind, it would be far more useful if the authors reworked Equations 1-3 and Figure 3 (with potential changes to Figure 2) so that the motor output (u) is related to the stride rather than the magnitude of the perturbation. There should be an equation relating the forward model recalibration (i.e., Equation 1) to the fraction of the motor error on a given stride, something akin to u(k+1) = r * (u(k) - p(k)). This formulation is easier to understand and commonplace in other motor learning tasks (and likely what the authors actually fit given the Smith & Shadmehr citation and the derivations in the Supplemental Materials). Such a change would require that Figure 3's independent axes be changed to "stride," but this has the benefit of complementing the presentation that is already in Figure 5.

Reviewer #3 (Public review):

Summary:

In this work, Rossi et al. use a novel split-belt treadmill learning task to reveal distinct sub-components of gait adaptation. The task involved following a standard adaptation phase with a "ramp-down" phase that helped them dissociate implicit recalibration and more deliberate SR map learning. Combined with modeling and re-analysis of previous studies, the authors show multiple lines of evidence that both processes run simultaneously, with implicit learning saturating based on intrinsic learning constraints and SR learning showing sensitivity to a "perceptual" error. These results offer a parallel with work in reaching adaptation showing both explicit and implicit processes contributing to behavior; however, in the case of gait adaptation the deliberate learning component does not appear to be strategic but is instead a more implicit SR learning processes.

Strengths:

(1) The task design is very clever and the "ramp down" phase offers a novel way to attempt to dissociate competing models of multiple processes in gait adaptation.

(2) The analyses are thorough, as is the re-analysis of multiple previous data sets.

(3) The querying of perception of the different relative belt speeds is a very nice addition, allowing the authors to connect different learning components with error perception.

(4) The conceptual framework is compelling, highlighting parallels with work in reaching but also emphasizing differences, especially w/r/t SR learning versus strategic behaviors. Thus the discovery of an SR learning process in gait adaptation would be both novel and also help conjoin different siloed subfields of motor learning research.

Weaknesses:

(1) The behavior in the ramp-down phase does indeed appear to support multiple learning processes. However, I may have missed something, but I have a fundamental worry about the specific modeling and framing of the "SR" learning process. If I correctly understand, the SR process learns by adjusting to perceived L/R belt speed differences (Figure 7). What is bugging me is why that process would not cause the SR system to still learn something in the later parts of the ramp-down phase when the perceived speed differences flip (Figure 4). I do believe this "blunted learning" is what the SR component is actually modeled with, given this quote in the caption to Figure 7: "When the perturbation is perceived to be opposite than adaptation, even if it is not, mapping is zero and the Δ motor output is constant, reflecting recalibration adjustments only." It seems a priori odd and perhaps a little arbitrary to me that a SR learning system would just stop working (go to zero) just because the perception flipped sign. Or for that matter "generalize" to a ramp-up (i.e., just learn a new SR mapping just like the system did at the beginning of the first perturbation). What am I missing that justifies this key assumption? Or is the model doing something else? (if so that should be more clearly described).

(2) A more minor point, but given the sample size it is hard to be convinced about the individual difference analysis for structure learning (Figure 5). How clear is it that these two groups of subjects are fully separable and not on a continuum? The lack of clusters in another data set seems like a somewhat less than convincing control here.

Reviewer #1 (Public Review):

In this work, the authors aimed to understand how titins derived from different nuclei within the syncytium are organized and integrated after cell fusion during skeletal muscle development and remodeling. The authors developed mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The results suggested that titin exhibited homogenous distribution after cell fusion. The authors also probed on how titin behaves during muscle injury by implantation of titin-eGFP myoblasts into adult mCherry-titin mice. Interestingly, titin is retained at the proximal nucleus and does not diffuse across the whole syncytium in this system. The findings of the study are novel and interesting. The experimental approaches are appropriate. The results are described well. However, the manuscript needs revisions to enhance its clarity.

(1) In this work, the authors have not described the statistical analysis appropriately. In most of the figures, significance levels are not described. The information on the biological and technical replicates is missing in almost all the figures. This information is critical for understanding the strength of the experimentation.<br /> (2) The in vivo experiments are underpowered. The authors have used only 3 animals in the cardiotoxin injury experiment and eliminated another 3 animals from the analysis. How did they determine insufficient myoblast integration?<br /> (3) Similarly, the in vitro imaging experiments, especially the in vitro titin mobility assays used only 3 cells (Fig 2b) or 6-9 cells (Fig 2c-2e). The number of cells imaged is insufficient to derive a valid conclusion. What is the variability in the results between cells? Whether all the cells behave similarly in titin mobility assays?<br /> (4) Figure 1c-e, Figure 2a, Figure 3, Figure 4, Figure 5, Figure 6- please describe the replicates and also if possible, quantify the data and present them as separate figures.<br /> (5) Figure 2- the authors excluded samples with an obvious decrease in cell quality during imaging from the analysis. How do the authors assess the cell quality? Simply by visual examination? Or were the samples that did not show fluorescence recovery eliminated? I am wondering what percentage of cells showed poor cell quality. How do they avoid the bias? I recommend that the authors include these cells also for the analysis of data presented in Figures 2b, 2c, and 2f.<br /> (6) It is unclear how the authors identified the different stages of cell fusion in the microscopy images i.e. early fusion, distribution, and complete distribution.

Reviewer #2 (Public Review):

The titin protein, a large component of striated muscle, plays a crucial role in the formation of the sarcomere during muscle development. As myocytes merge, titin integrates into the sarcomere structure, creating a stable myofilament system. The authors of the present study have shed light on the intricate process of myofilament assembly and disassembly, which is made possible by tracking labeled sarcomere components. In this study, they introduced the mCherry marker into titin's Z-disk to investigate its role in skeletal muscle development and remodeling. Their findings demonstrate that the integration of titin into the sarcomere is tightly regulated, with its unexpected mobility aiding in the uniform distribution of titin post-cell fusion. This distribution is crucial for the formation and maturation of skeletal muscle syncytium. In adult mice with mCherry-labeled titin, treating muscle injuries by introducing titin-eGFP myoblasts illustrates how myocytes integrate, fuse, and contribute to a seamless myofilament system across cell boundaries. The manuscript is well written, and the study is very novel.

Reviewer #3 (Public Review):

Hüttemeister et. al. describe a study where researchers utilized a genetic modification technique to knockin a red fluorescence protein variant mCherry into titin, a giant muscle protein, at the Z-disk in order to investigate skeletal muscle development and remodeling. The study revealed that titin's integration into the sarcomere is tightly regulated during muscle development, and its mobility allows for a homogeneous distribution of titin after cell fusion, which is crucial for syncytium formation and skeletal muscle maturation. Furthermore, in adult mice with mCherry-tagged titin, the researchers observed the process of muscle injury treatment by implanting myoblasts containing titin tagged with another fluorescent protein, eGFP. This experiment provided insights into how myocytes integrate, fuse, and contribute to the continuous myofilament system across cell boundaries during muscle regeneration. Interestingly, the behavior of titin proteins differed between immature primary cells and adult muscle tissue. The manuscripts point our interesting observation that develop treatment protocols that target the early postnatal patient or consider in utero cell therapy approaches based on controlling the ratio of therapeutic to diseased cells. though the approach is very interesting, the paper is very qualitative in its approaches. Community will benefit from better quantification of data as most of them are microscopic data that requires quantification.

Reviewer #1 (Public Review):

Lactobacillus plantarum is a beneficial bacterium renowned for its positive physiological effects and probiotic functions. Fu et al. conducted an investigation into the involvement of this bacterium in host purine metabolism. Initially, they employed microbiomics to analyze changes in L. plantarum within a hyperuricemia model, followed by isolation of the bacterium from this model. The gene map associated with purine nucleoside metabolism was determined through whole-genome analysis. Metabolic shifts in L. plantarum under nucleoside-enriched conditions were assessed using HPLC and metabolomics, while underlying mechanisms were explored through gene knockout experiments. Finally, the efficacy of L. plantarum was validated in hyperuricemia models involving goslings and mice. The authors presented their findings coherently and logically, addressing key questions using appropriate methodologies and yielding significant and innovative results. The authors demonstrated that host-derived Lactobacillus plantarum alleviates host hyperuricemia by influencing purine metabolism. However, their study primarily focused on this bacterium without delving deeper into the mechanisms underlying hyperuricemia beyond verification through two models. Nevertheless, these findings are sufficient to support their conclusion effectively. Additionally, further research is warranted to investigate the metabolites of Lactobacillus plantarum.

Reviewer #2 (Public Review):

Summary:<br /> Purine nucleoside metabolism in intestinal flora is integral to the purine nucleoside metabolism in the host. This study identified the iunH gene in Lactobacillus plantarum that regulates its purine nucleoside metabolism. Oral gavage of Lactobacillus plantarum and subsequent analysis showed it maintains homeostasis of purine nucleoside metabolism in the host.

Strengths:<br /> This study presents sufficient evidence for the role of Lactobacillus plantarum in alleviating hyperuricaemia, combining microbiomics, whole genomics, in vitro bacterial culture, and metabolomics. These results suggest the iunH gene of Lactobacillus plantarum is crucial in host purine nucleoside metabolism. The experimental design is robust, and the data are of high quality. This study makes significant contributions to the fields of hyperuricaemia, purine nucleoside metabolism, and Lactobacillus plantarum investigation.

Weaknesses:<br /> A key limitation of this manuscript is the absence of an in-depth study on the alleviation metabolism of Lactobacillus plantarum. Notable questions include: What overall metabolic changes occur in a purine nucleoside-enriched environment? How do the metabolites of Lactobacillus plantarum vary? Do these metabolites influence host purine nucleoside metabolism? These areas merit further investigation.

Reviewer #3 (Public Review):

Fu et al. present a multi-model study using goose and mouse that investigates the protective effects of Lactobacillus plantarum against hyperuricaemia. They highlight this strain's significance and clarify its role in responding to intestinal nucleoside levels and affecting uric acid metabolism through modulation of host signaling pathways.

Strengths:<br /> (1) Fu et al. created two animal models for validation, yielding more reliable and extensive data. In addition, the in vitro tests were repeatedly tested by a multitude of methods, proving to be convincing.<br /> (2) This study integrates microbiomics, whole genomics, in vitro bacterial culture, and metabolomics, providing a wealth of data and valuable insights for future research.

Weakness:<br /> Fu et al. clearly described the role of Lactobacillus plantarum, but it is also important to explore its other mechanisms influencing uric acid metabolism in the host. While changes in hepatic and renal uric acid metabolism were confirmed, the gut's role in this process deserves investigation, particularly regarding whether Lactobacillus plantarum or its metabolites act within the gut. The authors have effectively conveyed the story outlined in the article's title, and the remainder can be explored later. In addition, further discussion is needed to highlight how this strain of Lactobacillus plantarum differs from other Lactobacillus strains or how it innovatively functions differ from some literature reported.

Reviewer #1 (Public review):

Theoretical principles of viscous fluid mechanics are used here to assess likely mechanisms of transport in the ER. A set of candidate mechanisms are evaluated, making good use of imaging to represent ER network geometries. Evidence is provided that contraction of peripheral sheets provides a much more credible mechanism than contraction of individual tubules, junctions or perinuclear sheets.

The work has been conducted carefully and comprehensively, making good use of underlying physical principles. There is good discussion of the role of slip; sensible approximations (low volume fraction, small particle size, slender geometries, pragmatic treatment of boundary conditions) allow tractable and transparent calculations; clear physical arguments, including an analysis of energy budgets, provide useful bounds; stochastic and deterministic features of the problem are well integrated.

Reviewer #1 (Public review):

Summary:

This paper by Watanabe et al described an expression system that can express the paired heavy and light chains of IgG antibodies from single cell B cells. In addition, they used FACS sorting for specific antigen to screen/select the specific populations for more targeted cloning of mAb genes. By staining with multiple antigens, they were able to zoom in to cross-reactive antibodies.

Strengths:

A highly efficient process which combines selection/screening with dua expression of both antibody chains. It is particularly suitable for isolation of cross-reactive antibodies against conserved epitopes of different antigens, such as surface proteins of related viruses.

Weaknesses:

(1) The overall writing is very difficult to follow and the authors need to work on significant re-writing<br /> (2) The paper in its current form really lacks detail and it is not possible for readers to repeat or follow their methods. For example: a) It is not clear whether the authors checked the serum to see if the mice were producing antibodies before they sacrificed them to harvest spleen/blood i.e. using ELISA? b) How long after administration of the second dose were the mice sacrificed? c) What cell types are taken for single B cell sorting? Splenocytes or PBMC? These are just some of the questions which need to be addressed.<br /> (3) According to the authors, 77 clones were sorted from the PR8+ and H2+ double positive quadrant. It is surprising that after transfection and re-analysing of bulk antibody presenting EXPI cells on FACS from, only 13 clones (or 8 clones? - unclear) seemed to be truly cross reactive. If that is the case, the approach is not as efficient as the authors claimed.

The authors have adequately addressed the issues raised

Reviewer #2 (Public review):

Summary:

Watanabe, Takashi et al. investigated the use of the Golden Gate dual-expression vector system to enhance the modern standard for rapid screening of recombinant monoclonal antibodies. The presented data builds upon modern techniques that currently use multiple expression vectors to express heavy and light chain pairs. In a single vector, they express the linked heavy and light chain variable genes with a membrane-bound Ig which allows for rapid and more affordable cell-based screening. The final validation of H1 and H2 strain influenza screening resulted in 81 "H1+", 48 "H2+", and 9 "cross" reactive clones. The kinetics of some of the soluble antibodies were tested via SPR and validated with a competitive inhibition with classical well-characterized neutralizing clones.

Strengths:

In this study, Watanabe, Takashi et al. further develop and refine the methodologies for the discovery of monoclonal antibodies. They elegantly merge newer technologies to speed up turnaround time and reduce the cost of antibody discovery. Their data supports the feasibility of their technique.

This study will have an impact on pandemic preparedness and antibody-based therapies.

Weaknesses:

Limitations of this new technique are as follows: there is a significant loss of cells during FACs, transfection and cloning efficiency are critical to success, and well-based systems limit the number of possible clones (as the author discussed in the conclusions).

Reviewer #1 (Public review):

Summary:

This work sought to demonstrate that gut microbiota dysbiosis may promote the colonization of mycobacteria, and they tried to prove that Nos2 down-regulation was a key mediator of such gut-lung pathogenesis transition.

Strengths:

They did large-scale analysis of RNAs in lungs to analyze the gene expression of mice upon gut dysbiosis in MS-infected mice. This might help provide overview of gene pathways and critical genes for lung pathology in gut dysbiosis. This data is somewhat useful and important for the TB field.

Weaknesses:

(1) They did not use wide-type Mtb strain (e.g. H37Rv) to develop mouse TB infection models, and this may lead to the failure for establishment of TB granuloma and other TB pathology icons.<br /> (2) The usage of in vitro assays based on A542 to examine the regulation function of Nos2 expression on NO and ROS may not be enough. A542 is not the primary Mtb infection target in the lungs.<br /> (3) They did not examine the lung pathology upon gut dysbiosis to examine the true significance of increased colonization of Mtb.<br /> (4) Most of the studies are based on MS-infected mouse models with lack of clinical significance.

Reviewer #2 (Public Review):

Olszyński et al. claim that they identified a "new-type" ultrasonic vocalization around 44 kHz that occurs in response to prolonged fear conditioning (using foot-shocks of relatively high intensity, i.e. 1 mA) in rats. Typically, negative 22-kHz calls and positive 50-kHz calls are distinguished in rats, commonly by using a frequency threshold of 30 or 32 kHz. Olszyński et al. now observed so-called "44-kHz" calls in a substantial number of subjects exposed to 10 tone-shock pairings, yet call emission rate was low (according to Fig. 1G around 15%, according to the result text around 7.5%). They also performed playback experiments and concluded that "the responses to 44-kHz aversive calls presented from the speaker were either similar to 22-kHz vocalizations or in-between responses to 22-kHz and 50-kHz playbacks".

Strengths: Detailed spectrographic analysis of a substantial data set of ultrasonic vocalizations recorded during prolonged fear conditioning, combined with playback experiments.

Reviewer #1 (Public review):

Devakinandan et al. present a revised version of their manuscript. Their scRNA-seq data is a valuable resource to the community, and they further validate their findings via in situ hybridizations and electron microscopy. Overall, they have addressed my major concerns. I only have two minor comments.

(1) The authors note in Figure 4I, and K that because the number of C2 V2Rs or H2-Mv receptors increased while the normalized expression of Gnao1 remained constant (and likewise for V1Rs and Gnai2 in Figure 4-S4C) that their results are unlikely to be capturing doublets. I'm not sure that this is the case. If the authors added together two V2R cells the total count of every gene might double, but the normalized expression of Gnao1 would remain the same. To address this concern, the authors should also show the raw counts for Gnao1 as well as the total number of UMIs for these cells.

(2) As requested, the authors have now added a colorbar to the pseudocolored images in Figures 7. However, this colorbar still doesn't have any units. Can the authors add some units, or clarify in the methods how the raw data relates to the colors (e.g. is it mapped linearly, at a logscale, with gamma or other adjustments, etc.)? Moreover, it's also unclear what the dots in the backgrounds of plots like Figure 7E mean. Are they pixels? Showing the individual lines, the average for each animal, or omitting them entirely, might make more sense.

Reviewer #2 (Public review):

Summary:

The study focuses on the vomeronasal organ, the peripheral chemosensory organ of the accessory olfactory system, by employing single-cell transcriptomics. The author analyzed the mouse vomeronasal organ, identifying diverse cell types through their unique gene expression patterns. Developmental gene expression analysis revealed that two classes of sensory neurons diverge in their maturation from common progenitors, marked by specific transient and persistent transcription factors. A comparative study between major neuronal subtypes, which differ in their G-protein sensory receptor families and G-protein subunits (Gnai2 and Gnao1, respectively), highlighted a higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons. Moreover, distinct differences in ER content and ultrastructure suggest some intriguing roles of ER in Gnao1-positive vomeronasal neurons. This work is likely to provide useful data for the community and is conceptually novel with the unique role of ER in a subset of vomeronasal neurons.

Strengths:

(1) The study identified diverse cell types based on unique gene expression patterns, using single-cell transcriptomic.

(2) The analysis suggest that two classes of sensory neurons diverge during maturation from common progenitors, characterized by specific transient and persistent transcription factors.

(3) A comparative study highlighted differences in Gnai2- and Gnao1-positive sensory neurons.

(4) Higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons.

(5) Distinct differences in ER content and ultrastructure suggest unique roles of ER in Gnao1-positive vomeronasal neurons.

(6) The research provides conceptually novel on the unique role of ER in a subset of vomeronasal neurons, offering valuable insights to the community.

Comments on latest version:

In the revised manuscript, the authors have thoroughly addressed all of this reviewer's concerns.

Reviewer #3 (Public review):

Summary:

In this manuscript, Devakinandan and colleagues have undertaken a thorough characterization of the cell types of the mouse vomeronasal organ, focusing on the vomeronasal sensory neurons (VSNs). VSNs are known to arise from a common pool of progenitors that differentiate into two distinct populations characterized by the expression of either the G protein subunit Gnao1 or Gnai2. Using single-cell RNA sequencing followed by unsupervised clustering of the transcriptome data, the authors identified three Gnai2+ VSN subtypes and a single Gnao1+ VSN type. To study VSN developmental trajectories, Devakinandan and colleagues took advantage of the constant renewal of the neuronal VSN pool, which allowed them to harvest all maturation states. All neurons were re-clustered and a pseudotime analysis was performed. The analysis revealed the emergence of two pools of Gap43+ clusters from a common lineage, which differentiate into many subclusters of mature Gnao1+ and Gnai2+ VSNs. By comparing the transcriptomes of these two pools of immature VSNs, the authors identified a number of differentially expressed transcription factors in addition to known markers. Next, by comparing the transcriptomes of mature Gnao1+ and Gnai2+ VSNs, the authors report an enrichment of ER-related genes in Gnao1+ VSNs. Using electron microscopy, they found that this enrichment was associated with specific ER morphology in Gnao1+ neurons. Finally, the authors characterized chemosensory receptor expression and co-expression (as well as H2-Mv proteins) in mature VSNs, which recapitulated known patterns.

Strengths:

The data presented here provide new and interesting perspectives on the distinguishing features between Gnao1+ and Gnai2+ VSNs. These features include newly identified markers, such as transcription factors, as well as an unsuspected ER-related peculiarity in Gnao1+ neurons, consisting in a hypertrophic ER and an enrichment in ER-related genes. In addition, the authors provide a comprehensive picture of specific co-expression patterns of V2R chemoreceptors and H2-Mv genes.

Importantly, the authors provide a browser (scVNOexplorer) for anyone to explore the data, including gene expression and co-expression, number and proportion of cells, with a variety of graphical tools (violin plots, feature plots, dot plots, ...).

Reviewer #1 (Public review):

Summary:

In this study, Liu, Jiang, Diao et.al. investigated the role of GSDMD in psoriasis-like skin inflammation in mice. The authors have used full-body GSDMD knock-out mice and Gsdm floxed mice crossed with the S100A8- Cre. In both mice, the deficiency of GSDMD ameliorated the skin phenotype induced by the imiquimod. The authors also analyzed RNA sequencing data from the psoriatic patients to show an elevated expression of GSDMD in the psoriatic skin.

Overall, this is a potentially interesting study, however, the manuscript in its current format is not completely a novel study.

Strengths:

It has the potential to unravel the new role of neutrophils.

Weaknesses:

The main claims are only partially supported and have scope to improve

Reviewer #2 (Public review):

Summary:

The authors describe elevated GSDMD expression in psoriatic skin, and knock-out of GSDMD abrogates psoriasis-like inflammation.

Strengths:

The study is well conducted with transgenic mouse models. Using mouse-models with GSDMD knock-out showing abrogating inflammation, as well as GSDMD fl/fl mice without neutrophils having a reduced phenotype.

I fear that some of the conclusions cannot be drawn by the suggested experiments. My major concern would be the involvement of other inflammasome and GSDMD bearing cell types, esp. Keratinocytes (KC), which could be an explanation why the experiments in Fig 4 still show inflammation.

Weaknesses:

The experiments do not entirely support the conclusions towards neutrophils.

Specific questions/comments:

Fig 1b: mainly in KC and Neutrophils?

Fig 2a: PASI includes erythema, scaling, thickness and area. Guess area could be trick, esp. in an artificial induced IMQ model (WT) vs. the knock-out mice.

Fig 2d: interesting finding. I thought that CASP-1 is cleaving GSDMD. Why would it be downregulated?

Line 313: as mentioned before (see Fig 1b). KC also show a stron GSDMD staining positivity and are known producers of IL-1b and inflammasome activation. Guess here the relevance of KC in the whole model needs to be evaluated.

Fig 4i - guess here the conclusion would be that neutrophils are important for the pathogenesis in the IMQ model, which is true. This experiment does not support that this is done by pyroptosis.

Joint Public Reviews:

De Waele et al. framed the mass-spectrum-based prediction of antimicrobial resistance (AMR) prediction as a drug recommendation task. Neural networks were trained on the recently available DRIAMS database of MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry data and their associated antibiotic susceptibility profiles (Weis et al. 2022). Weis et al. (2022) also introduced the benchmark models which take as the input a single species and are trained to predict resistance to a single drug. Instead here, a pair of drugs and spectrum are fed to two neural network models to predict a resistance probability. In this manner, knowledge from different drugs and species can be shared through the model parameters. Questions asked: What is the best way to encode the drugs? Does the dual neural network outperform the single spectrum-drug network?

The authors showed consistent performance of their strategy to predict antibiotic susceptibility for different spectrum and antibiotic representations (i.e., embedders). Remarkably, the authors showed how small datasets collected at one location can improve the performance of a model trained with limited data collected at a second location. The authors also showed that species-specific models (trained in multiple antibiotic resistance profiles) outperformed both the single recommender model and the individual species-antibiotic combination models.

Strengths:

• A single antimicrobial resistance recommender system could potentially facilitate the adoption of MALDI-TOF based antibiotic susceptibility profiling into clinical practices by reducing the number of models to be considered, and the efforts that may be required to periodically update them.<br /> • The authors tested multiple combinations of embedders for the mass spectra and antibiotics while using different metrics to evaluate the performance of the resulting models. Models trained using different spectrum embedder-antibiotic embedder combinations had remarkably good performance for all tested metrics. The average ROC AUC scores for global and species-specific evaluations were above 0.8.<br /> • Authors developed species-specific recommenders as an intermediate layer between the single recommender system and single species-antibiotic models. This intermediate approach achieved maximum performance (with one type of the species-specific recommender achieving a 0.9 ROC AUC), outlining the potential of this type of recommenders for frequent pathogens.<br /> • Authors showed that data collected in one location can be leveraged to improve the performance of models generated using a smaller number of samples collected at a different location. This result may encourage researchers to optimize data integration to reduce the burden of data generation for institutions interested in testing this method.

Weaknesses:

• Authors do not offer information about the model features associated with resistance. While reviewers understand that it is difficult to map mass spectra to specific pathways or metabolites, mechanistic insights are much more important in the context of AMR than in the context of bacterial identification. For example, this information may offer additional antimicrobial targets. Thus, authors should at least identify mass spectra peaks highly associated with resistance profiles. Are those peaks consistent across species? This would be a key step towards a proteomic survey of mechanisms of AMR. See previous work on this topic (Hrabak et al. 2013, Torres-Sangiao et al. 2022).

References:

Hrabak et al. (2013). Clin Microbiol Rev 26. doi: 10.1128/CMR.00058-12.<br /> Torres-Sangiao et al. (2022). Front Med 9. doi: 10.3389/fmed.2022.850374.<br /> Weis et al. (2022). Nat Med 28. doi: 10.1038/s41591-021-01619-9.

Reviewer #2 (Public review):

Most neurodegenerative diseases are characterized by the self-templated misfolding of a particular protein in a manner that enables progressive spread throughout the central nervous system. In diseases including Parkinson's disease (PD) and multiple system atrophy (MSA), the protein a-synuclein misfolds into unique strains, which use this self-replicating mechanism to encode disease-specific information. Previous research suggests that a major contributor to the lack of successful clinical trials across neurodegenerative diseases is the lack of disease-relevant strains used in preclinical testing. While MSA patient samples are known to replicate efficiently in cell and mouse models of disease, Lewy body disease (LBD) patient samples do not. To overcome this obstacle, the seeding amplification assay (SAA) uses recombinant a-synuclein to amplify the misfolded protein structure present in a human patient sample. The resulting fibrils are then widely used by many laboratories as a model of PD. In this manuscript, Lee et al., set out to compare the strain properties of a-synuclein fibrils isolated from LBD and MSA patient samples with the resulting amplified fibrils following SAA. Using orthogonal biochemical and structural approaches to strengthen their analyses, the authors report that the SAA-amplified fibrils do not recapitulate the disease-relevant strains present in the patient samples. Moreover, their data suggest that regardless of which strain is used to seed the SAA reaction, the same strain is generated. These results clearly demonstrate that the SAA-amplified material is likely not disease-relevant. SAA fibrils are broadly used throughout academic and pharmaceutical laboratories. They are used in ongoing drug discovery efforts and recombinant fibrils broadly inform much of what is known about a-synuclein strain biology in LBD patients. The implications of the reported work are, therefore, expansive. These findings add to the growing ledger of reasons that the use of SAA fibrils in research should be halted until improved methods for amplification with high fidelity are developed.

Reviewer #1 (Public review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group

Weaknesses:

Too few cells included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more a collection of preliminary transcriptomic data than a true scientific report to help advance the field.

Reviewer #2 (Public review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Weaknesses:

(1) Language expression must be improved. Many incomplete sentences exist throughout the manuscript. A few examples: Line 70-71: In order to further elucidate the effects and regulatory mechanisms of the lymphatic vessels in the repair process of myocardial injury following MI. Line 71-73. This study, integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points after MI from publicly available data (E-MTAB-7895, GSE214611) in the ArrayExpress and gene expression omnibus (GEO) databases. Line 88-89: Since the membrane protein LYVE1 can present lymphatic vessel morphology more clearly than PROX1.<br /> (2) The type of animal models (i.e., permeant MI or MI plus reperfusion) included in ArrayExpress and gene expression omnibus (GEO) databases must be clearly defined as these two models may have completely different effects on lymphatic vessel development during post-MI remodeling.<br /> (3) Line 119-120: Caution must be taken regarding Cav1 as a lymphocyte marker because Cav1 is expressed in all endothelial cells, not limited to LEC.<br /> (4) Figure 1 legend needs to be improved. RZ, BZ, and IZ need to be labeled in all IF images. Day 0 images suggest that RZ is the tissue section from the right ventricle. Was RZ for all other time points sampled from the right ventricular tissue section?<br /> (5) The discussion section needs to be improved and better focused on the findings from the current study.

Reviewer #1 (Public Review):

Tsai and Seymen et al. investigate associations between RTE expression and methylation and age and inflammation, using multiple public datasets. The text of the manuscript has been polished and the phrasing of several findings has been made clearer and more precise. The authors also provided ample discussion to the prior reviewer comments in their rebuttal, including new analyses.

Reviewer #2 (Public Review):

Summary:

Yi-Ting Tsai and colleagues conducted a systematic analysis of the correlation between the expression of retrotransposable elements (RTEs) and aging, using publicly available transcriptional and methylome microarray datasets of blood cells from large human cohorts, as well as single-cell transcriptomics. Although DNA hypomethylation was associated with chronological age across all RTE biotypes, the authors did not find a correlation between the levels of RTE expression and chronological age. However, expression levels of LINEs and LTRs positively correlated with DNA demethylation, and inflammatory and senescence gene signatures, indicative of "biological age". Gene set variation analysis showed that the inflammatory response is enriched in the samples expressing high levels of LINEs and LTRs. In summary, the study demonstrates that RTE expression correlates with "biological" rather than "chronological" aging.

Strengths:

The question the authors address is both relevant and important to the fields of aging and transposon biology.

Comments on latest version:

The authors introduced the analysis of RNA-seq data, addressing the key concerns raised by Reviewer #1 and myself. They also adopted more explicit terminology in their latest version, reducing ambiguity. The RNA-seq analysis demonstrating that the expression of different transposon groups is not associated with chronological aging is convincing, though, in my opinion, it still lacks granularity.

I have two minor points:

(1) Previously, I have mentioned the following:

"The authors pool signals from RTEs by class or family, despite the fact that these groups include subfamilies and members with very different properties and harmful potentials. For example, while older subfamilies might be expressed through readthrough transcription, certain members of younger groups could be autonomously reactivated and cause inflammation... The aggregation of signals from different RTE biotypes may obscure potential reactivation of smaller groups or specific subfamilies."

The authors responded that they would lose statistical power by studying RTE subfamilies with limited microarray probes, which is a fair point. However, the suggested analysis could have been conducted using the RNA-seq data they explored in the second round of revision. Choosing not to leverage RNA-seq to increase the granularity of their analysis is a matter of choice. In my opinion, however, the authors could have acknowledged in the discussion that some smaller yet potentially influential RTE species may be masked by their global approach.

(2) Previously, I mentioned that 10x scRNA-seq is not ideal for analysing RTEs and requested a classical UMAP plot to visualize RTE expression across cell populations. The authors argued that they could only achieve sufficient statistical power by quantifying RTE classes through cumulative read counts for each cell type, which I accept. However, they divided cells into "high" and "low" BAR gene signature groups. I am surprised that the comparison of BAR signature expression between these groups was not presented using standard visualization methods commonly applied in scRNA-seq data analysis.

Reviewer #2 (Public Review):

Summary:

The authors aimed to identify which regions of DVL2 contribute to its endogenous/basal clustering, as well as the relevance of such domains to condensate/phase separation and WNT activation.

Strengths:

A strength of the study is the focus on endogenous DVL2 to set up the research questions, as well as the incorporation of various techniques to tackle it. I found also quite interesting that DVL2-CFR addition to DVL1 increased its MW in density gradients.

Weaknesses:

The authors have addressed important drawbacks regarding the overexpression experiments, most notably by including DVL tKO cells in collaboration with Vita. I think that this part has clearly improved.

Unfortunately, I still stand with my key concern: at this stage in the field, with many papers on DVL over expression, there is a clear need to address how endogenous DVL behaves. While the attempts to o/e low levels of DVL mutants in tKO cells are useful for validation experiments, the manuscript does not -in my opinion - address the requirements of DVL2 condensation for WNT signalling. Of note, several of the described effects are partial, including in tKO cells, often indicating that the targeted domains contribute, but are not required, for these processes. I understand that generating endogenous tagged lines or targeting specific endogenous domains is not trivial. But, as indicated in both initial reviews, I think that monitoring endogenous proteins is required to fully address the proposed research question.

In my opinion, the current manuscript A) shows that endogenous DVL2 forms large complexes in a higher proportion as DVL1/3, B) identifies and describes a couple of motifs that contribute to clustering and signalling in overexpressed DVL, including in tKO cells* C) shows that one of those motifs (CFR) rewires o/e DVL1 into behaving similarly as DVL2.

*On a minor note, I am not sure how DVL tKO cells partially react to Wnt3a in Figure 7G

Reviewer #1 (Public review):

Summary:

In the manuscript the authors describe a new pipeline to measure changes in vasculature diameter upon opt-genetic stimulation of neurons.<br /> The work is useful to better understand the hemodynamic response on a network /graph level.

Strengths:

The manuscript provides a pipeline that allows to detect changes in the vessel diameter as well as simultaneously allows to locate the neurons driven by stimulation.<br /> The resulting data could provide interesting insights into the graph level mechanisms of regulating activity dependent blood flow.

Weaknesses:

(1) The manuscript contains (new) wrong statements and (still) wrong mathematical formulas.<br /> (2) The manuscript does not compare results to existing pipelines for vasculature segmentation (opensource or commercial).<br /> Comparing performance of the pipeline to a random forest classifier (illastik) on images that are not preprocessed (i.e. corrected for background etc.) seems not a particularly useful comparison.<br /> (3) The manuscript does not clearly visualize performance of the segmentation pipeline (e.g. via 2d sections, highlighting also errors etc.). Thus, it is unclear how good the pipeline is, under what conditions it fails or what kind of errors to expect.<br /> (4) The pipline is not fully open-source due to use of matlab. Also, the pipeline code was not made available during review contrary to the authors claims (the provided link did not lead to a repository). Thus, the utility of the pipeline was difficult to judge.

Detailed remarks to the revision and new manuscript:

- Generalizability: The authors addressed the point of generalizability by applying the pipeline to other data sets. This demonstrates that their pipeline can be applied to other data sets and makes it more useful.<br /> However, from the visualizations it's unclear to see the performance of the pipeline, where the pipelines fails etc. The 3d visualizations are not particularly helpful in this respect .<br /> In addition, the dice measure seems quite low, indicating roughly 20-40% of voxels do not overlap between inferred and ground truth. I did not notice this high discrepancy earlier. A through discussion of the errors appearing in the segmentation pipeline would be necessary in my view to better asses the quality of the pipeline.

Reviewer #2 (Public review):

The authors have addressed most of my concerns sufficiently. There are still a few serious concerns I have. Primarily, the temporal resolution of the technique still makes me dubious about nearly all of the biological results. It is good that the authors have added some vessel diameter time courses generated by their model. But I still maintain that data sampling every 42 seconds - or even 21 seconds - is problematic. First, the evidence for long vascular responses is lacking. The authors cite several papers:

Alarcon-Martinez et al. 2020 show and explicitly state that their responses (stimulus-evoked) returned to baseline within 30 seconds. The responses to ischemia are long lasting but this is irrelevant to the current study using activated local neurons to drive vessel signals.<br /> Mester et al. 2019 show responses that all seem to return to baseline by around 50 seconds post-stimulus.<br /> O'Herron et al. 2022 and Hartmann et al. 2021 use opsins expressed in vessel walls (not neurons as in the current study) and directly constrict vessels with light. So this is unrelated to neuronal activity-induced vascular signals in the current study.

There are other papers including Vazquez et al 2014 (PMID: 23761666) and Uhlirova et al 2016 (PMID: 27244241) and many others showing optogenetically-evoked neural activity drives vascular responses that return back to baseline within 30 seconds. The stimulation time and the cell types labeled may be different across these studies which can make a difference. But vascular responses lasting 300 seconds or more after a stimulus of a few seconds are just not common in the literature and so are very suspect - likely at least in part due to the limitations of the algorithm.

Another major issue is that the time courses provided show that the same vessel constricts at certain points and dilates later. So where in the time course the data is sampled will have a major effect on the direction and amplitude of the vascular response. In fact, I could not find how the "response" window is calculated. Is it from the first volume collected after the stimulation - or an average of some number of volumes? But clearly down-sampling the provided data to 42 or even 21 second sampling will lead to problems. If the major benefit to the field is the full volume over large regions that the model can capture and describe, there needs to be a better way to capture the vessel diameter in a meaningful way.

It still seems possible that if responses are bi-phasic, then depth dependencies of constrictors vs dilators may just be due to where in the response the data are being captured - maybe the constriction phase is captured in deeper planes of the volume and the dilation phase more superficially. This may also explain why nearly a third of vessels are not consistent across trials - if the direction the volume was acquired is different across trials, different phases of the response might be captured.

I still have concerns about other aspects of the responses but these are less strong. Particularly, these bi-phasic responses are not something typically seen and I still maintain that constrictions are not common. The authors are right that some papers do show constriction. Leaving out the direct optogenetic constriction of vessels (O'Herron 2022 & Hartmann 2021), the Alarcon-Martinez et al. 2020 paper and others such as Gonzales et al 2020 (PMID: 33051294) show different capillary branches dilating and constricting. However, these are typically found either with spontaneous fluctuations or due to highly localized application of vasoactive compounds. I am not familiar with data showing activation of a large region of tissue - as in the current study - coupled with vessel constrictions in the same region. But as the authors point out, typically only a few vessels at a time are monitored so it is possible - even if this reviewer thinks it unlikely - that this effect is real and just hasn't been seen.

I also have concerns about the spatial resolution of the data. It looks like the data in Figure 7 and Supplementary Figure 7 have a resolution of about 1 micron/pixel. It isn't stated so I may be wrong. But detecting changes of less than 1 micron, especially given the noise of an in vivo prep (brain movement and so on), might just be noise in the model. This could also explain constrictions as just spurious outputs in the model's diameter estimation. The high variability in adjacent vessel segments seen in Figure 6C could also be explained the same way, since these also seem biologically and even physically unlikely.

I still think the difference in distance-to-nearest-neuron between dilators and constrictors is insignificant. These points were not addressed - the difference in neuronal density between cortical layers and the ~ 5 micron difference in this parameter between dilators and constrictors. Given the concerns raised above, there is very little confidence in even knowing which vessels constricted and which dilated.

All-in-all, I think this is potentially a very useful pipeline for automating numerous tasks which are very time consuming and vulnerable to subjective judgements (which leads to reproducibility problems and others). However, I think the challenge of capturing large volumes at high speed and with high resolution is very real and hasn't been adequately accomplished for the claims the authors want to make about their data. It is encouraging that with the right technology, such data could be captured and this pipeline would be excellent for processing it. But given the limitations in the data collection here, I think that many of the biological claims are hard to fully accept.

Reviewer #1 (Public review):

Summary:

This is a detailed description of the role of PKCδ in Drosophila learning and memory. The work is based on a previous study (Placais et al. 2017) that has already shown that for the establishment of long-term memory, the repetitive activity of MP1 dopaminergic neurons via the dopamine receptor DAMB is essential to increase mitochondrial energy flux in the mushroom body. In this paper, the role of PKCδ is now introduced. PKCδ is a molecular link between the dopaminergic system and the mitochondrial pyruvate metabolism of mushroom body Kenyon cells. For this purpose, the authors establish a genetically encoded FRET-based fluorescent reporter of PKCδ-specific activity, δCKAR.

Strengths:

This is a thorough study on the long-term memory of Drosophila. The work is based on the extensive, high-quality experience of the senior authors. This is particularly evident in the convincing use of behavioral assays and imaging techniques to differentiate and explore various memory phases in Drosophila. The study also establishes a new reporter to measure the activity of PKCδ - the focus of this study - in behaving animals. The authors also elucidate how recurrent spaced training sessions initiate a molecular gating mechanism, linking a dopaminergic punishment signal with the regulation of mitochondrial pyruvate metabolism. This advancement will enable a more precise molecular distinction of various memory phases and a deeper comprehension of their formation in the future.

Weaknesses:

The study offers novel insights into the molecular mechanisms underlying long-term memory formation and presents no apparent weaknesses in either content or methodology.

Reviewer #2 (Public review):

Summary

This study deepens the former authors' investigations of the mechanisms involved in gating the long-term consolidation of an associative memory (LTM) in Drosophila melanogaster. After having previously found that LTM consolidation 1. costs energy (Plaçais and Préat, Science 2013) provided through pyruvate metabolism (Plaçais et al., Nature Comm 2017) and 2. is gated by the increased tonic activity in a type of dopaminergic neurons ('MP1 neurons') following only training protocol relevant for LTM, i.e. interspaced in time (Plaçais et al., Nature Neuro 2012), they here dig into the intra-cell signalling triggered by dopamine input and eventually responsible for the increased mitochondria activity in Kenyon Cells. They identify a particular PKC, PKCδ, as a major molecular interface in this process and describe its translocation to mitochondria to promote pyruvate metabolism, specifically after spaced training.

Methodological approach

To that end, they use RNA interference against the isozyme PKCδ, in a time-controlled way and in the whole Kenyon cells populations or in the subpopulation forming the α/β lobe. This knock-down decreased the total PKCδ mRNA level in the brain by ca. 30%, and is enough to observe decreased in flies performances for LTM consolidation. Using Pyronic, a sensor for pyruvate for in vivo imaging, and pharmacological disruption of mitochondrial function, the authors then show that PKCδ knock-down prevents high level of pyruvate from accumulating in the Kenyon cells at the time of LTM consolidation, pointing towards a role of PKCδ in promoting pyruvate metabolism. They further identify the PDH kinase PDK as a likely target for PKCδ since knocking down both PKCδ and PDK led to normal LTM performances, likely counterbalancing PKCδ knock-down alone.

To understand the timeline of PKCδ activation and to visualise its mitochondrial translocation in subpart of Mushroom body lobes they imported in fruitfly the genetically-encoded FRET reporters of PKCδ, δCKAR and mitochondria-δCKAR (Kajimoto et al 2010). They show that PKCδ is activated to the sensor's saturation only after spaced training, and not other types of training that are 'irrelevant' for LTM. Further, adding thermogenetic activation of dopaminergic neurons and RNA interference against Gq-coupled dopamine receptor to FRET imaging, they identify that a dopamine-triggered cascade is sufficient for the elevated PKCδ-activation.

Strengths and weaknesses

The authors use a combination of new fluorescent sensors and behavioral, imaging, and pharmacological protocols they already established to successfully identify the molecular players that bridge the requirement for spaced training/dopaminergic neurons MP1 oscillatory activity and the increased metabolic activity observed during long-term memory consolidation.<br /> The study is dense in new exciting findings and each methodological step is carefully designed. The experiments one could think of to make this link have been done in this study and the results seem solid.<br /> The discussion is well conducted, with interesting parallel with mammals, where the possibility that this process takes place as well is yet unknown.

Impact

Their findings should interest a large audience:<br /> They discover and investigate a new function for PKCδ in regulating memory processes in neurons in conjunction with other physiological functions, making this molecule a potentially valid target for neuropathological conditions. They also provide new tools in drosophila to measure PKCδ activation in cells. They identify the major players for lifting the energetic limitations preventing the formation of a long-term memory.

Reviewer #1 (Public review):

Summary:

The authors use the teleost medaka as an animal model to study the effect of seasonal changes in day-length on feeding behaviour and oocyte production. They report a careful analysis of how day-length affects female medakas and a thorough molecular genetic analysis of genes potentially involved in this process. They show a detailed analysis of two genes and include a mutant analysis of one gene to support their conclusions

Strengths:

The authors pick their animal model well and exploit the possibilities to examine in this laboratory model the effect of a key environmental influence, namely the seasonal changes of day-length. The phenotypic changes are carefully analysed and well-controlled. The mutational analysis of the agrp1 by a ko-mutant provides important evidence to support the conclusions. Thus this report exceeds previous findings on the function of agrp1 and npyb as regulators of food-intake and shows how in medaka these genes are involved in regulating the organismal response to an environmental change. It thus furthers our understanding of how animals react to key exogenous stimuli for adaptation.

Weaknesses:

The authors are too modest when it comes to underscoring the importance of their findings. Previous animal models used to study the effect of these neuropeptides on feeding behaviour have either lost or were most likely never sensitive to seasonal changes of day length. Considering the key importance of this parameter on many aspects of plant and animal life it could be better emphasised that a suitable animal model is at hand that permits this.<br /> The molecular characterization of the agrp1 ko-mutant that the authors have generated lacks some details that would help to appreciate the validity of the mutant phenotype. Additional data would help in this respect.

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

While medaka is an appropriate model for studying seasonal breeding, the results presented are insufficient to fully support the authors' conclusions.

Specifically, methods and data analyses are incomplete in justifying the primary claims:<br /> - the procedure for the food intake assay is unclear;<br /> - the sample size is very small;<br /> - the statistical analysis is not always adequate.

Additionally, the discussion fails to consider the possible role of other hormones that may be involved in the feeding mechanism.

Reviewer #3 (Public review):

Summary:

Understanding the mechanisms whereby animals restrict the timing of their reproduction according to day length is a critical challenge given that many of the most relevant species for agriculture are strongly photoperiodic. However, the principal animal models capable of detailed genetic analysis do not respond to photoperiod so this has inevitably limited progress in this field. The fish model medaka occupies a uniquely powerful position since its reproduction is strictly restricted to long days and it also offers a wide range of genetic tools for exploring, in depth, various molecular and cellular control mechanisms.

For these reasons, this manuscript by Tagui and colleagues is particularly valuable. It uses the medaka to explore links bridging photoperiod, feeding behaviour, and reproduction. The authors demonstrate that in female, but not male medaka, photoperiod-induced reproduction is associated with an increase in feeding, presumably explained by the high metabolic cost of producing eggs on a daily basis during the reproductive period. Using RNAseq analysis of the brain, they reveal that the expression of the neuropeptides agrp and npy that have been previously implicated in the regulation of feeding behaviour in mice are upregulated in the medaka brain during exposure to long photoperiod conditions. Unlike the situation in mice, these two neuropeptides are not co-expressed in medaka neurons, and food deprivation in medaka led to increases in agrp but also a decrease in npy expression. Furthermore, the situation in fish may be more complicated than in mice due to the presence of multiple gene paralogs for each neuropeptide. Exposure to long-day conditions increases agrp1 expression in medaka as the result of increases in the number of neurons expressing this neuropeptide, while the increase in npyb levels results from increased levels of expression in the same population of cells. Using ovariectomized medaka and in situ hybridization assays, the authors reveal that the regulation of agrp1 involves estrogen acting via the estrogen receptor esr2a. Finally, a loss of agrp1 function mutant is generated where the female mutants fail to show the characteristic increase in feeding associated with long-day enhanced reproduction as well as yielding reduced numbers of eggs during spawning.

Strengths:

This manuscript provides important foundational work for future investigations aiming to elucidate the coordination of photoperiod sensing, feeding activity, and reproduction function. The authors have used a combination of approaches with a genetic model that is particularly well suited to studying photoperiodic-dependent physiology and behaviour. The data are clear and the results are convincing and support the main conclusions drawn. The findings are relevant not only for understanding photopriodic responses but also provide more general insight into links between reproduction and feeding behaviour control.

Weaknesses:

Some experimental models used in this study, namely ovariectomized female fish and juvenile fish have not been analysed in terms of their feeding behaviour and so do not give a complete view of the position of this feeding regulatory mechanism in the context of reproduction status. Furthermore, the scope of the discussion section should be expanded to speculate on the functional significance of linking feeding behaviour control with reproductive function.

Reviewer #1 (Public review):

Summary:

In this manuscript, Shibata describes a method to assess rapidly fluctuating CpG sites (fCpGs) from single-cell methylation sequencing (sc-MeSeq) data. Assuming that fCpGs are largely consistent over time with changes induced by inheritable events during replication, the author infers lineage relationships in available brain-derived sc-MeSeq. Supplementing current lineage tracing through genomic and mitochondrial mosaic variants is an interesting concept that could supplement current work or allow additional lineage analysis in existing data.

However, the author failed to convincingly show the power of fCpG analysis to determine lineages in the human brain. While the correlation with cellular division and distinction of cell types appears plausible and strong, the application to detect specific lineages is less convincing. Aspects of this might be due to a lack of clarity in presentation and erroneous use of developmental concepts. However, without addressing these problems it is challenging for a reader to come to the same conclusions as the author.

On the flip side, this novel application of fCpGs will allow the re-use of existing sc-MeSeq to infer additional features that were previously unavailable, once the biological relevance has been further elucidated.

Strengths:

(1) Novel re-analysis application of methylation data to infer the status of fCpGs and the use as a lineage marker.

(2) Application of this method to an innovative existing data set to benchmark this framework against existing developmental knowledge.

Weaknesses:

(1) Insufficient clarity when presenting results (this includes an incredible shortness of the methods section making an informed assessment very difficult). This makes it hard to fully grasp and evaluate the presented results.

(2) Inconsistent or erroneous use of neurodevelopmental concepts which hinders appropriate interpretation of the results.

(3) Lack of consideration for alternative explanations for the observed data (i.e., considering fCpGs as a cellular division clock that diverges over 'time').

Reviewer #2 (Public review):

The manuscript by Shibata proposed a potentially interesting idea that variation in methylcytosine across cells can inform cellular lineage in a way similar to single nucleotide variants (SNVs). The work builds on the hypothesis that the "replication" of methylcytosine, presumably by DNMT1, is inaccurate and produces stochastic methylation variants that are inherited in a cellular lineage. Although this notion can be correct to some extent, it does not account for other mechanisms that modulate methylcytosines, such as active gain of methylation mediated by DNMT3A/B activity and activity demethylation mediated by TET activity. In some cases, it is known that the modulation of methylation is targeted by sequence-specific transcription factors. In other words, inaccurate DNMT1 activity is only one of the many potential ways that can lead to methylation variants, which fundamentally weakens the hypothesis that methylation variants can serve as a reliable lineage marker. With that being said (being skeptical of the fundamental hypothesis), I want to be as open-minded as possible and try to propose some specific analyses that might better convince me that the author is correct. However, I suspect that the concept of methylation-based lineage tracing cannot be validated without some kind of lineage tracing experiment, which has been successfully demonstrated for scRNA-seq profiling but not yet for methylation profiling (one example is Delgado et al., nature. 2022).

(1) The manuscript reported that fCpG sites are predominantly intergenic. The author should also score the overlap between fCpG sites and putative regulatory elements and report p-values. If fCpG sites commonly overlap with regulatory elements, that would increase the possibility that these sites being actively regulated by enhancer mechanisms other than maintenance methyltransferase activity.

(2) The overlap between fCpG and regulatory sequence is a major alternative explanation for many of the observations regarding the effectiveness of using fCpG sites to classify cell types correctly. One would expect the methylation level of thousands of enhancers to be quite effective in distinguishing cell types based on the published single-cell brain methylome works.

(3) The methylation level of fCpG sites is higher in hindbrain structures and lower in forebrain regions. This observation was interpreted as the hindbrain being the "root" of the methylation barcodes and, through "progressive demethylation" produced the methylation states in the forebrain. This interpretation does not match what is known about methylation dynamics in mammalian brains, in particular, there is no data supporting the process of "progressive demethylation". In fact, it is known that with the activation of DNMT3A during early postnatal development in mice or humans (Lister et al., 2013. Science), there is a global gain of methylation in both CH and CG contexts. This is part of the broader issue I see in this manuscript, which is that the model might be correct if "inaccurate mC replication" is the only force that drives methylation dynamics. But in reality, active enzymatic processes such as the activation of DNMT3A have a global impact on the methylome, and it is unclear if any signature for "inaccurate mC replication" survives the de novo methylation wave caused by DNMT3A activity.

(3) Perhaps one way the author could address comment 3 is to analyze methylome data across several developmental stages in the same brain region, to first establish that the signal of "inaccurate mC replication" is robust and does not get erased during early postnatal development when DNMT3A deposits a large amount of de novo methylation.

(4) The hypothesis that methylation barcodes are homogeneous among progenitor cells and more polymorphic in derived cells is an interesting one. However, in this study, the observation was likely an artifact caused by the more granular cell types in the brain stem, intermediate granularity in inhibitory cells, and highly continuous cell types in cortical excitatory cells. So, in other words, single-cell studies typically classify hindbrain cell types that are more homogenous, and cortical excitatory cells that are much more heterogeneous. The difference in cell type granularity across brain structures is documented in several whole-brain atlas papers such as Yao et al. 2023 Nature part of the BICCN paper package.

(5) As discussed in comment 2, the author needs to assess whether the successful classification of cell types (brain lineage) using fCpG was, in fact, driven by fCpG sites overlapping with cell-type specific regulatory elements.

(6) In Figure 5E, the author tried to address the question of whether methylation barcodes inform lineage or post-mitotic methylation remodeling. The Y-axis corresponds to distances in tSNE. However, tSNE involves non-linear scaling, and the distances cannot be interpreted as biological distances. PCA distances or other types of distances computed from high-dimensional data would be more appropriate.

Reviewer #3 (Public review):

Summary:

In the manuscript entitled "Human Brain Barcodes", the author sought to use single-cell CpG methylation information to trace cell lineages in the human brain.

Strengths:

Tracing cell lineages in the human brain is important but technically challenging. Lineage tracing with single-cell CpG methylation would be interesting if convincing evidence exists.

Weaknesses:

As the author noted, "DNA methylation patterns are usually copied between cell division, but the replication errors are much higher compared to base replication". This unstable nature of CpG methylation would introduce significant problems in inferring the true cell lineage. The unreliable CpG methylation status also raises the question of what the "Barcodes" refer to in the title and across this study. Barcodes should be stable in principle and not dynamic across cell generations, as defined in Reference#1. It is not convincing that the "dynamic" CpG methylation fits the "barcodes" terminology. This problem is even more concerning in the last section of results, where CpG would fluctuate in post-mitotic cells.

Reviewer #1 (Public review):

Summary:

This theoretical paper addresses how to optimize reward-rate-maximizing decisions in certain foraging-style environments. It presents a series of equations and graphical illustrations for quantities such as reward rates and time-related costs that a decision maker could estimate as a basis for such decisions. One of the main takeaways is that if the hypothetical agent underweights the time spent outside a focal reward pursuit relative to the time spent within it, this can predict a broadly realistic pattern of impatience in two alternative intertemporal choices paired with well-calibrated take-it-or-leave-it decisions. Another takeaway is that if the optimally estimated subjective value of a reward pursuit is plotted as a function of a range of temporal durations, the result resembles a hyperbolic discounting function and is affected in empirically realistic ways by the magnitude and sign of the reward. Thus, the rate-maximization framework might lead to a hypothesis about the basis for the magnitude and sign effects in discounting.

Strengths:

The paper makes a useful contribution by broadening the application of reward-rate maximization to time-related decision scenarios. The paper's breadth of scope includes applying the same framework to accept/reject decisions and multi-alternative discounting decisions. The figures take a creative approach to illustrating the internal quantities in the model. It's particularly useful that the paper gives consideration to internal distortions that could give rise to documented anomalies in decision behavior.

Weaknesses:

(1) Although there are many citations acknowledging relevant previous work, there often isn't a very granular attribution of individual previous findings to their sources. In the results section, it's sometimes ambiguous when the paper is recapping established background and when it is breaking new ground. For example, around equation 8 in the results (sv = r - rho*t), it would be good to refer to previous places where versions of this equation have been presented. Offhand, McNamara 1982 (Theoretical Population Biology) is one early instance and Fawcett et al. 2012 (Behavioural Processes) is a later one. Line 922 of the discussion seems to imply this formulation is novel here.

(2) The choice environments that are considered in detail in the paper are very simple. The simplicity facilitates concrete examples and visualizations, but it would be worth further consideration of whether and how the conclusions generalize to more complex environments. The paper considers "forgo" scenario in which the agent can choose between sequences of pursuits like A-B-A-B (engaging with option B at all opportunities, which are interleaved with a default pursuit A) and A-A-A-A (forgoing option B). It considers "choice" scenarios where the agent can choose between sequences like A-B-A-B and A-C-A-C (where B and C are larger-later and smaller-sooner rewards, either of which can be interleaved with the default pursuit). Several forms of additional complexity would be valuable to consider. One would be a greater number of unique pursuits, not repeated identically in a predictable sequence, akin to a prey-selection paradigm. It seems to me this would cause t_out and r_out (the time and reward outside of the focal prospect) to be policy-dependent, making the 'apportionment cost' more challenging to ascertain. Another relevant form of complexity would be if there were variance or uncertainty in reward magnitudes or temporal durations or if the agent had the ability to discontinue a pursuit such as in patch-departure scenarios.

(3) I had a hard time arriving at a solid conceptual understanding of the 'apportionment cost' around Figure 5. I understand the arithmetic, but it would help if it were possible to formulate a more succinct verbal description of what makes the apportionment cost a useful and meaningful quality to focus on. I think Figure 6C relates to this, but I had difficulty relating the axis labels to the points, lines, and patterned regions in the plot. I also was a bit confused by how the mathematical formulation was presented. As I understood it, the apportionment cost essentially involves scaling the rest of the SV expression by t_out/(t_in + t_out). The way this scaling factor is written in Figure 5C, as 1/(1 + (1/t_out)t_in), seems less clear than it could be. Also, the apportionment cost is described in the text as being subtracted from SV rather than as a multiplicative scaling factor. It could be written as a subtraction, by subtracting a second copy of the rest of the SV expression scaled by t_in/(t_in + t_out). But that shows the apportionment cost to depend on the opportunity cost, which is odd because the original motivation on line 404 was to resolve the lack of independence between terms in the SV expression.

(4) In the analysis of discounting functions (line 664 and beyond), the paper doesn't say much about the fact that many discounting studies take specific measures to distinguish true time preferences from opportunity costs and reward-rate maximization. In many of the human studies, delay time doesn't preclude other activities. In animal studies, rate maximization can serve as a baseline against which to measure additional effects of temporal discounting. This is an important caveat to claims about discounting anomalies being rational under rate maximization (e.g., line 1024).

(5) The paper doesn't feature any very concrete engagement with empirical data sets. This is ok for a theoretical paper, but some of the characterizations of empirical results that the model aims to match seem oversimplified. An example is the contention that real decision-makers are optimal in accept/reject decisions (line 816 and elsewhere). This isn't always true; sometimes there is evidence of overharvesting, for example.

(6) Related to the point above, it would be helpful to discuss more concretely how some of this paper's theoretical proposals could be empirically evaluated in the future. Regarding the magnitude and sign effects of discounting, there is not a very thorough overview of the several other explanations that have been proposed in the literature. It would be helpful to engage more deeply with previous proposals and consider how the present hypothesis might make unique predictions and could be evaluated against them. A similar point applies to the 'malapportionment hypothesis' although in this case there is a very helpful section on comparisons to prior models (line 1163). The idea being proposed here seems to have a lot in common conceptually with Blanchard et al. 2013, so it would be worth saying more about how data could be used to test or reconcile these proposals.

Reviewer #2 (Public review):

Summary:

This paper from Sutlief et al. focuses on an apparent contradiction observed in experimental data from two related types of pursuit-based decision tasks. In "forgo" decisions, where the subject is asked to choose whether or not to accept a presented pursuit, after which they are placed into a common inter-trial interval, subjects have been shown to be nearly optimal in maximizing their overall rate of reward. However, in "choice" decisions, where the subject is asked which of two mutually-exclusive pursuits they will take, before again entering a common inter-trial interval, subjects exhibit behavior that is believed to be sub-optimal. To investigate this contradiction, the authors derive a consistent reward-maximizing strategy for both tasks using a novel and intuitive geometric approach that treats every phase of a decision (pursuit choice and inter-trial interval) as vectors. From this approach, the authors are able to show that previously reported examples of sub-optimal behavior in choice decisions are in fact consistent with a reward-maximizing strategy. Additionally, the authors are able to use their framework to deconstruct the different ways the passage of time impacts decisions, demonstrating that time cost contains both an opportunity cost and an apportionment cost, as well as examining how a subject's misestimation of task parameters impacts behavior.

Strengths:

The main strength of the paper lies in the authors' geometric approach to studying the problem. The authors chose to simplify the decision process by removing the highly technical and often cumbersome details of evidence accumulation that are common in most of the decision-making literature. In doing so, the authors were able to utilize a highly accessible approach that is still able to provide interesting insights into decision behavior and the different components of optimal decision strategies.

Weaknesses:

While the details of the paper are compelling, the authors' presentation of their results is often unclear or incomplete:

(1) The mathematical details of the paper are correct but contain numerous notation errors and are presented as a solid block of subtle equation manipulations. This makes the details of the authors' approach (the main contribution of the paper to the field) highly difficult to understand.

(2) One of the main contributions of the paper is the notion that time cost in decision-making contains an apportionment cost that reflects the allocation of decision time relative to the world. The authors use this cost to pose a hypothesis as to why subjects exhibit sub-optimal behavior in choice decisions. However, the equation for the apportionment cost is never clearly defined in the paper, which is a significant oversight that hampers the effectiveness of the authors' claims.

(3) Many of the paper's figures are visually busy and not clearly detailed in the captions (for example, Figures 6-8). Because of the geometric nature of the authors' approach, the figures should be as clean and intuitive as possible, as in their current state, they undercut the utility of a geometric argument.

(4) The authors motivate their work by focusing on previously-observed behavior in decision experiments and tell the reader that their model is able to qualitatively replicate this data. This claim would be significantly strengthened by the inclusion of experimental data to directly compare to their model's behavior. Given the computational focus of the paper, I do not believe the authors need to conduct their own experiments to obtain this data; reproducing previously accepted data from the papers the authors' reference would be sufficient.

(5) While the authors reference a good portion of the decision-making literature in their paper, they largely ignore the evidence-accumulation portion of the literature, which has been discussing time-based discounting functions for some years. Several papers that are both experimentally-(Cisek et al. 2009, Thurs et al. 2012, Holmes et al. 2016) and theoretically-(Drugowitsch et al. 2012, Tajima et al. 2019, Barendregt et al. 22) driven exist, and I would encourage the authors to discuss how their results relate to those in different areas of the field.

Reviewer #3 (Public review):

Summary:

The goal of the paper is to examine the objective function of total reward rate in an environment to understand the behavior of humans and animals in two types of decision-making tasks: (1) stay/forgo decisions and (2) simultaneous choice decisions. The main aims are to reframe the equation of optimizing this normative objective into forms that are used by other models in the literature like subjective value and temporally discounted reward. One important contribution of the paper is the use of this theoretical analysis to explain apparent behavioral inconsistencies between forgo and choice decisions observed in the literature.

Strengths:

The paper provides a nice way to mathematically derive different theories of human and animal behavior from a normative objective of global reward rate optimization. As such, this work has value in trying to provide a unifying framework for seemingly contradictory empirical observations in literature, such as differentially optimal behaviors in stay-forgo v/s choice decision tasks. The section about temporal discounting is particularly well motivated as it serves as another plank in the bridge between ecological and economic theories of decision-making.

Weaknesses:

One broad issue with the paper is readability. Admittedly, this is a complicated analysis involving many equations that are important to grasp to follow the analyses that subsequently build on top of previous analyses.

But, what's missing is intuitive interpretations behind some of the terms introduced, especially the apportionment cost without referencing the equations in the definition so the reader gets a sense of how the decision-maker thinks of this time cost in contrast with the opportunity cost of time.

Re-analysis of some existing empirical data through the lens of their presented objective functions, especially later when they describe sources of error in behavior.

Reviewer #1 (Public review):

Summary:

This manuscript by Shan, Guo, Zhang, Chen et al., shows a raft of interesting data including the first cryo-EM structures of human PIEZO1. Clearly, the molecular basis of PIEZO channel inactivation is of great interest and as such this manuscript provides some valuable extra information that may help to ultimately build a molecular picture of PIEZO channel inactivation. However, the current manuscript though does not provide any compelling evidence for a detailed mechanism of PIEZO inactivation.

Strengths:

This manuscript documents the first cryo-EM structures of human PIEZO1 and the gain of function mutants associated with hereditary anaemia. It is also the first evidence showing that PIEZO1 gain of function mutants are also regulated by the auxiliary subunit MDFIC.

Weaknesses:

While the structures are interesting and clear differences can be seen in the presence of the auxiliary subunit MDFIC the major conclusions and central tenets of the paper, especially a role for pore lipids in inactivation, lack data to support them. The post-translational modification of PIEZOs auxiliary subunit MDFIC is not modelled as a covalent interaction.

Reviewer #2 (Public review):

Summary:

Mechanically activated ion channels PIEZOs have been widely studied for their role in mechanosensory processes like touch sensation and red blood cell volume regulation. PIEZO in vivo roles are further exemplified by the presence of gain-of-function (GOF) or loss-of-function (LOF) mutations in humans that lead to disease pathologies. Hereditary xerocytosis (HX) is one such disease caused due to GOF mutation in Human PIEZO1, which are characterized by their slow inactivation kinetics, the ability of a channel to close in the presence of stimulus. But how these mutations alter PIEZO1 inactivation or even the underlying mechanisms of channel inactivation remains unknown. Recently, MDFIC (myoblast determination family inhibitor proteins) was shown to directly interact with mouse PIEZO1 as an auxiliary subunit to prolong inactivation and alter gating kinetics. Furthermore, while lipids are known to play a role in the inactivation and gating of other mechanosensitive channels, whether this mechanism is conserved in PIEZO1 is unknown. Thus, the structural basis for PIEZO1 inactivation mechanism, and whether lipids play a role in these mechanisms represent important outstanding questions in the field and have strong implications for human health and disease.

To get at these questions, Shan et al. use cryogenic electron microscopy (Cryo-EM) to investigate the molecular basis underlying differences in inactivation and gating kinetics of PIEZO1 and human disease-causing PIEZO1 mutations. Notably, the authors provide the first structure of human PIEZO1 (hPIEZO1), which will facilitate future studies in the field. They reveal that hPIEZO1 has a more flattened shape than mouse PIEZO1 (mPIEZO1) and has lipids that insert into the hydrophobic pore region. To understand how PIEZO1 GOF mutations might affect this structure and the underlying mechanistic changes, they solve structures of hPIEZO1 as well as two HX-causing mild GOF mutations (A1988V and E756del) and a severe GOF mutation (R2456H). Unable to glean too much information due to poor resolution of the mutant channels, the authors also attempt to resolve MCFIC-bound structures of the mutants. These structures show that MDFIC inserts into the pore region of hPIEZO1, similar to its interaction with mPIEZO1, and results in a more curved and contracted state than hPIEZO1 on its own. The authors use these structures to hypothesize that differences in curvature and pore lipid position underlie the differences in inactivation kinetics between wild-type hPIEZO1, hPIEZO1 GOF mutations, and hPIEZO1 in complex with MDFIC.

Strengths:

This is the first human PIEZO1 structure. Thus, these studies become the stepping stone for future investigations to better understand how disease-causing mutations affect channel gating kinetics.

Weaknesses:

Many of the hypotheses made in this manuscript are not substantiated with data and are extrapolated from mid-resolution structures.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors used structural biology approaches to determine the molecular mechanism underlying the inactivation of the PIEZO1 ion channel. To this end, the authors presented structures of human PIEZO1 and its slow-inactivating mutants. The authors also determined the structures of these PIEZO1 constructs in complexes with the auxiliary subunit MDFIC, which substantially slows down PIEZO1 inactivation. From these structures, the authors suggested an anti-correlation between the inactivation kinetics and the resting curvature of PIEZO1 in detergent. The authors also observed a unique feature of human PIEZO1 in which the lipid molecules plugged the channel pore. The authors proposed that these lipid molecules could stabilize human PIEZO1 in a prolonged inactivated state.

Strengths:

Notedly, this manuscript reported the first structures of a human PIEZO1 channel, its channelopathy mutants, and their complexes with MDFIC. The evidence that lipid molecules could occupy the channel pore of human PIEZO1 is solid. The authors' proposals to correlate PIEZO1 resting curvature and pore-resident lipid molecules with the inactivation kinetics are novel and interesting.

Weaknesses:

However, in my opinion, additional evidence is needed to support the authors' proposals.

(1) The authors determined the apo structure of human PIEZO1, which showed a more flattened architecture than that of the mouse PIEZO1. Functionally, the inactivation kinetics of human PIEZO1 is faster than its mouse counterpart. From this observation (and some subsequent observations such as the complex with MDFIC), the authors proposed the anti-correlation between curvature and inactivation kinetics. However, the comparison between human and mouse PIEZO1 structure might not be justified. For example, the human and mouse structures were determined in different detergent environments, and the choice of detergent could influence the resting curvature of the PIEZO structures.

(2) Related to point 1), the 3.7 Å structure of the A1988V mutant presented by the authors showed a similar curvature as the WT but has a slower inactivating kinetics.

(3) Related to point 1), the authors stated that human PIEZO1 might not share the same mechanism as mouse PIEZO1 due to its unique properties. For example, MDFIC only modifies the curvature of human PIEZO1, and lipid molecules were only observed in the pore of the human PIEZO1. Therefore, it may not be justified to draw any conclusions by comparing the structures of PIEZO1 from humans and mice.

(4) Related to point 1), it is well established that PIEZO1 opening is associated with a flattened structure. If the authors' proposal were true, in which a more flattened structure led to faster inactivation, we would have the following prediction: more opening is associated with faster inactivation. In this case, we would expect a pressure-dependent increase in the inactivation kinetics. Could the authors provide such evidence, or provide other evidence along this direction?

(5) In Figure S2, the authors showed representative experiments of the inactivation kinetics of PIEZO1 using whole-cell poking. However, poking experiments have high cell-to-cell variability. The authors should also show statics of experiments obtained from multiple cells.

(6) In Figure 2 and Figure 5, when the authors show the pore diameter, it could be helpful to also show the side chain densities of the pore lining residues.

(7) The authors observed pore-plugging lipids in slow inactivating conditions such as channelopathy mutations or in complex with MDFIC. The authors propose that these lipid molecules stabilize a "deep resting state" of PIEZO1, making it harder to open and harder to inactivate once opened. This will lead to the prediction that the slow-inactivating conditions will lead to a higher activation threshold, such as the mid-point pressure in the activation curve. Is this true?

Reviewer #1 (Public review):

Summary:

The concept that trained immunity, as defined, can be beneficial to subsequent immune challenges is important in the broad context of health and disease. The significance of this manuscript is the finding that trained immunity is actually a two-edged sword, herein, detrimental in the context of LPS-induced Acute Lung Injury that is mediated by AMs.

Strengths:

Several lines of evidence in different mouse models support this conclusion. The postulation that differences in immune responses in individuals are linked to differences in the mycobiome and consequent B-glucan makeup is provocative.

Weaknesses:

The findings that the authors state are relevant to sepsis, are actually confined to a specific lung injury model and not classically-defined sepsis. In addition, the ontogeny of the reprogrammed AMs is uncertain. Links in the proposed signaling pathways need to be strengthened.

Reviewer #2 (Public review):

Summary:

Prével et al. present an in vivo study in which they reveal an interesting aspect of β-glucan, a known inducer of enhanced immune responses termed trained immunity in sterile inflammation. The authors can show, that β-glucan's can reprogram alveolar macrophages (AMs) in the lungs through neutrophils and IFNγ signaling and independent of Dectin1. This reprogramming occurs at both transcriptional and metabolic levels. After β-glucan training, LPS-induced sterile inflammation exacerbated acute lung injury via enhanced immunopathology. These findings highlight a new aspect of β-glucan's role in trained immunity and its potential detrimental effects when enhanced pathogen clearance is not required.

Strengths:

(1) This manuscript is well-written and effectively conveys its message.

(2) The authors provide important evidence that β-glucan training is not solely beneficial, but depending on the context can also enhance immunopathology. This will be important to the field for two reasons. It shows again, that trained immunity can also be harmful. Jentho et al. 2021 have already provided further evidence for this aspect. And it highlights anew that LPS application is an insufficient infection model.

Weaknesses:

(1) Only a little physiological data is provided by the in vivo models.

(2) The effects in histology appear to be rather weak.

Reviewer #1 (Public Review):

Summary:

This study investigates the hypoxia rescue mechanisms of neurons by non-neuronal cells in the brain from the perspective of exosomal communication between brain cells. Through multi-omics combined analysis, the authors revealed this phenomenon and logically validated this intercellular rescue mechanism under hypoxic conditions through experiments. The study proposed a novel finding that hemoglobin maintains mitochondrial function, expanding the conventional understanding of hemoglobin. This research is highly innovative, providing new insights for the treatment of hypoxic encephalopathy.

Overall, the manuscript is well organized and written, however, there are some minor/major points that need to be revised before this manuscript is accepted.

Major points:

(1) Hypoxia can induce endothelial cells to release exosomes carrying hemoglobin, however, how neurons are able to actively take up these exosomes? It is possible for other cells to take up these exosomes also? This point needs to be clarified in this study.

(2) The expression of hemoglobin in neurons is important for mitochondrial homeostasis, but its relationship with mitochondrial homeostasis needs to be further elucidated in the study.

Reviewer #2 (Public Review):

Summary:

This is an interesting study with a lot of data. Some of these ideas are intriguing. But a few major points require further consideration.

Major points:

(1) What disease is this model of whole animal hypoxia supposed to mimic? If one is focused on the brain, can one just use a model of focal or global cerebral ischemia?

(2) If this model subjects the entire animal to hypoxia, then other organs will also be hypoxic. Should one also detect endothelial upregulation and release of extracellular vesicles containing hemoglobin mRNA in non-CNS organs? Where do these vesicles go? Into blood?

(3) What other mRNA are contained in the vesicles released from brain endothelial cells?

(4) Where do the endothelial vesicles go? Only to neurons? Or to other cells as well?

(5) Neurons can express endogenous hemoglobin. Is it useful to subject neurons to hypoxia and then see how much the endogenous mRNA goes up? How large is the magnitude of endogenous hemoglobin gene upregulation compared to the hypothesized exogenous mRNA that is supposed to be donated from endothelial vesicles?

(6) Finally, it may be useful to provide more information and data to explain how the expression of this exogenous endothelial-derived hemoglobin binds to neuronal mitochondria to alter function.

Reviewer #1 (Public review):

Summary:

Shrestha et al report an investigation of mechanisms underlying gustatory preference for carboxylic acids in Drosophila. They begin with a screen of selected IR mutants, identifying 5 candidates - 2 IR co-receptors and 3 other IRs - whose loss of function causes defects in feeding preference for one or more of the three tested carboxylic acids. The requirement for IR51b, IR94a, and IR94h in carboxylic acid responses is evaluated in more detail using behavior, electrophysiology (labellar sensilla), and calcium imaging (pharyngeal neurons). The behavioral valence of IR94a and IR94h neurons is assessed using optogenetics. Overall the study uses a variety of approaches to test and validate the requirement of IRs in pharyngeal carboxylic acid taste.

Strengths:

The involvement of the identified IRs in gustatory responses to carboxylic acids is very clear from this study. The authors use mutants and transgenic rescue experiments and evaluate outcomes using electrophysiology, behavior, and imaging. Complementary approaches of loss-of-function and artificial activation support the main conclusion that the identified pharyngeal neurons sense carboxylic acids and convey a positive behavioral valence.

Weaknesses:

Some aspects of expression analysis and calcium imaging need to be clarified to better support the conclusions.

(1) The conclusion of two parallel IR-mediated pathways rests on expression analysis of Ir94a-GAL4 and Ir94h-GAL4 lines and the observation that Ir51b expression driven by either can rescue the Ir51b mutant phenotype. However, the expression analysis is not as rigorous as it needs to be for such a conclusion. Prior work found co-expression of Ir94a and Ir94h in the LSO. Here, the co-expression of the two drivers has not been examined, and Ir94a-GAL4 does not appear to be expressed in the LSO. Given the challenges in validating expression patterns in pharyngeal organs, the possibility that the drivers do not entirely capture endogenous expression cannot be ruled out. Rescue experiments using feeding preference or single-cell imaging don't suffice as validation. Plus, the expression of Ir51b could not be defined.

(2) The description of methods and results for the ex vivo calcium imaging is not satisfactory. Details about which cells are being analyzed, and in which organs are not included. No solvent stimulus is tested. The temporal dynamics of the responses are not presented. Movies of the imaging are not included as supplementary information - it would be important to visualize those with what was considered modest movement.

(3) The observed differences in phenotypes of Ir25a and Ir76b mutants are intriguing, as are those between the co-receptor mutants and Ir51b, Ir94a, and Ir94h, but have not been sufficiently considered. Prior studies have also found roles for other response modes (OFF response), other IRs and GRs, and other organs (labellum, tarsi) in behavioral responses to carboxylic acids. Overall, the authors' model may be overly simplistic, and the discussion does not do justice to how their model reconciles with the body of work that already exists.

Reviewer #2 (Public review):

Shrestha et al investigated the role of IR receptors in the detection of 3 carboxylic acids in adult Drosophila. A low concentration of either of these carboxylic acids added to 2 mM sucrose (1% lactic acid (LA), citric acid (CA), or glycolic acid (GA)) stimulates the consumption of adult flies in choice conditions. The authors use this behavioral test to screen the impact of mutations within 33 receptors belonging to the IR family, a large family of receptors derived from glutamate receptors and expressed both in the olfactory and gustatory sensilla of insects. Within the panel of mutants tested, they observed that 3 receptors (IR25a, IR51b, and IR76b) impaired the detection of LA, CA, and GA, and that 2 others impacted the detection of CA and GA (IR94a and IR94h). Interestingly, impairing IR51b, IR94a, and IR94h did not affect the electrophysiological responses of external gustatory sensilla to LA, CA, and GA. Thanks to the use of GAL4 strains associated with these receptors and thanks to the use of poxn mutants (which do not develop external gustatory sensilla but still have functional internal receptors), they show evidence that IR94a and IR94h are only expressed in two clusters of gustatory neurons of the pharynx, respectively in the VCSO (ventral cibarial sense organ) and in the VCSO + LSO (labral sense organ). As for IR51b, the GAL4 approach was not successful but RT-PCR made on different parts of the insect showed an expression both in the pharyngeal organs and in peripheral receptors. These main findings are then complemented by a host of additional experiments meant to better understand the respective roles of IR94a and IR94h, by using optogenetics and brain calcium imaging using GCamp6. They also report a failed attempt to co-express IR51b, IR94a, and IR94h into external receptors, a co-expression which did not confer the capability of bitter-sensitive cells (expressing GR33a-GAL4) to detect either of the carboxylic acids. These data complete and expand previous observations made on this group and others, and dot to 2 new IR receptors which show an unsuspected specific expression, into organs that still remain difficult to study.

The conclusions of this paper are supported by the data presented, but it remains difficult to make general conclusions as concerns the mechanisms by which carboxylic acids are detected.

(1) All experiments were done with 1% of carboxylic acids. What is the dose dependency of the behavioral responses to these acids, and is it conceivable that other receptors are involved at other concentrations?

(2) One result needs to be better discussed and hypotheses proposed - which is why the mutations of most receptors lead to a loss of detection (mutant flies become incapable of detecting the acid) while mutations in IR94a and IR94h make CA and GA potent deterrents. Does it mean that CA and GA are detected by another set of receptors that, when activated, make flies actively avoid CA and GA? In that case, do the authors think that testing receptors one by one is enough to uncover all the receptors participating in the detection of these substances?

(3) The paper needs to be updated with a recent paper published by Guillemin et al (2024), indicating that LA is detected externally by a combination of IR94e, IR76b and IR25a. IR25a might help to form a fully functional receptor in GR33a neurons (a former study from Chen et al (2017) indicate that IR25a is expressed in all gustatory neurons of the pharynx).

(4) Although it was not the main focus of the paper, it would have been most interesting if the cells expressing IR94a and IR94h were identified, and placed on the functional map proposed by the group of Dahanukar (Chen et al 2017 Cell Reports, Chen et al 2019 Cell Reports).

Reviewer #3 (Public review):

Summary:

In this work, the authors investigated the molecular and cellular basis of sour taste perception in Drosophila melanogaster, focusing on identifying receptors that mediate attractive responses to certain carboxylic acids. It builds on previous work from the same group that had identified the IR co-receptors IR25a and IR76b for this sensory process, screening a set of mutants in IRs to identify three, IR51b, IR94a, and IR94h, required for feeding preference responses to some or all of the tested acids.

Strengths:

The work is of interest because it assigns sensory roles to IRs of previously unknown function, in particular IR94a and IR94h, and points to pharyngeal neurons in which these receptors are expressed as the relevant sensory neurons (potentially with different roles for IR94a- and IR94h-expressing neurons). The work combines elegant genetics, simple but effective feeding and taste assays, chemo-/opto-genetic activation, and some calcium imaging. Overall the presented data look solid and well-controlled.

Weaknesses:

The in situ expression analysis relies entirely on transgenic driver lines for IR94a and IR94h (which had been previously described, though not fully cited in this work). Importantly, given that many of the behavioral experiments (genetic rescue, physiology, artificial activation) use the IR94a and IR94h GAL4 driver lines, it would be helpful to validate that these faithfully reflect IR94a and IR94h expression (as far as I can tell, such validation wasn't done in the original papers describing these lines as part of a large collection of IR drivers). For IR51b, pharyngeal expression is concluded indirectly from non-quantitative RT-PCR analysis (genetic reporters did not work). The lack of direct detection of gene/protein expression (for example, through RNA FISH, immunofluorescence, or protein tagging) would have made for a more complete characterization of these receptors (for example, there is no direct evidence that they also express IR25a and IR76b, as one might expect). Finally, the relationship of IR94a and IR94h neurons to other types of pharyngeal neurons remains unclear, as are their projection patterns in the SEZ.

Conceptually, the work is of interest mostly to those in the immediate field; there have been a very large number of studies in the past decade (several from this lab) characterizing the contributions of different IRs to various chemosensory processes. The current work doesn't lend much insight into the nature of the minimal functional unit of gustatory IRs (reconstitution of a functional IR in a heterologous neuron/cell has not been achieved here, but this is a limitation of many other previous studies), nor to how different pharyngeal sensory pathways might collaborate to control behavior. Nevertheless, the findings provide a useful contribution to the literature.

Reviewer #1 (Public review):

The authors examine how probabilistic reversal learning is affected by dopamine by studying the effects of methamphetamine (MA) administration. Based on prior evidence that the effects of pharmacological manipulation depend on baseline neurotransmitter levels, they hypothesized that MA would improve learning in people with low baseline performance. They found this effect, and specifically found that MA administration improved learning in noisy blocks, by reducing learning from misleading performance, in participants with lower baseline performance. The authors then fit participants' behavior to a computational learning model and found that an eta parameter, responsible for scaling learning rate based on previously surprising outcomes, differed in participants with low baseline performance on and off MA.

Questions:

(1) It would be helpful to confirm that the observed effect of MA on the eta parameter is responsible for better performance in low baseline performers. If performance on the task is simulated for parameters estimated for high and low baseline performers on and off MA, does the simulated behavior capture the main behavioral differences shown in Figure 3?

(2) In Figure 4C, it appears that the main parameter difference between low and high baseline performance is inverse temperature, not eta. If MA is effective in people with lower baseline DA, why is the effect of MA on eta and not IT?

Also, this parameter is noted as temperature but appears to be inverse temperature as higher values are related to better performance. The exact model for the choice function is not described in the methods.

Reviewer #2 (Public review):

Summary:

Kirschner and colleagues test whether methamphetamine (MA) alters learning rate dynamics in a validated reversal learning task. They find evidence that MA can enhance performance for low-performers and that the enhancement reflects a reduction in the degree to which these low-performers dynamically up-regulate their learning rates when they encounter unexpected outcomes. The net effect is that poor performers show more volatile learning rates (e.g. jumping up when they receive misleading feedback), when the environment is actually stable, undermining their performance over trials.

Strengths:

The study has multiple strengths including large sample size, placebo control, double-blind randomized design, and rigorous computational modeling of a validated task.

Weaknesses:

The limitations, which are acknowledged, include that the drug they use, methamphetamine, can influence multiple neuromodulatory systems including catecholamines and acetylcholine, all of which have been implicated in learning rate dynamics. They also do not have any independent measures of any of these systems, so it is impossible to know which is having an effect.

Another limitation that the authors should acknowledge is that the fact that participants were aware of having different experiences in the drug sessions means that their blinding was effectively single-blind (to the experimenters) and not double-blind. Relatedly, it is difficult to know whether subjective effects of drugs (e.g. arousal, mood, etc.) might have driven differences in attention, causing performance enhancements in the low-performing group. Do the authors have measures of these subjective effects that they could include as covariates of no interest in their analyses?

Reviewing Editor (Public Review):

Summary:

In this well-written paper, a pharmacological experiment is described in which a large group of volunteers is tested on a novel probabilistic reversal learning task with different levels of noise, once after intake of methamphetamine and once after intake of placebo. The design includes a separate baseline session, during which performance is measured. The key result is that drug effects on learning rate variability depend on performance in this separate baseline session.

The approach and research question are important, the results will have an impact, and the study is executed according to current standards in the field. Strengths include the interventional pharmacological design, the large sample size, the computational modeling, and the use of a reversal-learning task with different levels of noise.

(i) One novel and valuable feature of the task is the variation of noise (having 70-30 and 80-20 conditions). This nice feature is currently not fully exploited in the modeling of the task and the data. For example, recently reported new modeling approaches for disentangling two types of uncertainty (stochasticity vs volatility) could be usefully leveraged here (by Piray and Daw, 2021, Nat Comm). The current 'signal to noise ratio' analysis that is targeting this issue relies on separately assessing learning rates on true reversals and learning rates after misleading feedback, in a way that is experimenter-driven. As a result, this analysis cannot capture a latent characteristic of the subject's computational capacity.

(ii) An important caveat is that all the drug x baseline performance interactions, including for the key computational eta parameter did not reach the statistical threshold, and only tended towards significance.

(iii) Both the overlap and the differences between the current study and previous relevant work (that is, how this goes beyond prior studies in particular Rostami Kandroodi et al, which also assessed effects of catecholaminergic drug administration as a function of baseline task performance using a probabilistic reversal learning task) are not made explicit, particularly in the introduction.

(iv) In the discussion, it is stated that the existing literature has, to date, overlooked baseline performance effects, but this is not true in the general sense, given that an accumulating number of studies have shown that the effects of drugs like MA depend on baseline performance on working memory tasks, which often but certainly not always correlates positively with performance on the task under study.

Reviewer #1 (Public review):

Summary:

The current work explored the link between the pulvinar intrinsic organisation and its functional and structural connectivity patterns of the cortex using different dimensional reduction techniques. Overall they find relationships between pulvinar-cortical organization and cortico-cortical organization, and little evidence for clustered organization. Moreover, they investigate PET maps to understand how neurotransmitter/receptor distributions vary within the pulvinar and along its structural and functional connectivity axes.

Strengths:

There is a replication dataset and different modalities are compared against each other to understand the structural and functional organisation of the pulvinar complex.

Weaknesses:

(1) What is the motivation of the study and how does this work extend previous assessments of the organization of the complete thalamus within the gradient framework?

(2) Why is the current atlas chosen for the delineation of the pulvinar and individualised maps not considered? Given the size of the pulvinar, more validation of the correctness of the atlas may be helpful.

(3) Overall the study feels a little incremental and a repetition of what others have done already in the thalamus. It would be good to know how focussing only on the pulvinar changes interpretation, for example by comparing thalamic and pulvinar gradients?

(4) Could it be that the gradient patterns stem from lacking anatomical and functional resolutions (or low SNR) therefore generating no sharp boundaries?

Reviewer #2 (Public review):

Summary:

The authors aimed to explore and better understand the complex topographical organization of the human pulvinar, a brain region crucial for various high-order functions such as perception and attention. They sought to move beyond traditional histological subdivisions by investigating continuous 'gradients' of cortical connections along the dorsoventral and mediolateral axes. Using advanced imaging techniques and a comprehensive PET atlas of neurotransmitter receptors, the study aimed to identify and characterize these gradients in terms of structural connections, functional coactivation, and molecular binding patterns. Ultimately, the authors targeted to provide a more nuanced understanding of pulvinar anatomy and its implications for brain function in both healthy and diseased states.

Strengths:

A key strength of this study lies in the authors' effort to comprehensively combine multimodal data, encompassing both functional and structural connectomics, alongside the analysis of major neurotransmitter distributions. This approach enabled a more nuanced understanding of the overarching organizational principles of the pulvinar nucleus within the broader context of whole-brain connectivity. By employing cortex-wide correlation analyses of multimodal embedding patterns derived from 'gradients,' which provide spatial maps reflecting the underlying connectomic and molecular similarities across voxels, the study offers a thorough characterization of the functional neuroanatomy of the pulvinar.

Weaknesses:

Despite its strengths, the current manuscript falls short in presenting the authors' unique perspectives on integrating the diverse biological principles derived from the various neuroimaging modalities. The findings are predominantly reported as correlations between different gradient maps, without providing the in-depth interpretations that would allow for a more comprehensive understanding of the pulvinar's role as a central hub in the brain's network. Another limitation of the study is the lack of clarity regarding the application of pulvinar and its subnuclei segmentation maps to individual brains prior to BOLD signal extraction and gradient reconstruction. This omission raises concerns about the precision and reproducibility of the findings, leaving their robustness less transparently evaluable.

Reviewer #3 (Public review):

Summary of the Study:

The authors investigate the organization of the human pulvinar by analyzing DWI, fMRI, and PET data. The authors explore the hypothesis of the "replication principle" in the pulvinar.

Strengths and Weaknesses of the Methods and Results:

The study effectively integrates diverse imaging modalities to provide a view of the pulvinar's organization. The use of analysis techniques, such as diffusion embedding-driven gradients combined with detailed interpretations of the pulvinar, is a strength.

Even though the study uses the best publicly available resolution possible with current MR-technology, the pulvinar is densely packed with many cell bodies, requiring even higher spatial resolution. In addition, the model order selection of gradients may vary with the acquired data quality. Therefore, the pulvinar's intricate organization needs further exploration with even higher spatial resolution to capture gradients closer to the biological organization of the pulvinar.

Appraisal of the Study's Aims and Conclusions:

The authors delineate the gradient organization of the pulvinar. The study provides a basis for understanding the pulvinar's role in mediating brain network communication.

Impact and Utility of the Work:

This work contributes to the field by offering insights into pulvinar organization.

Reviewer #1 (Public review):

Summary:

The overall analysis and discovery of the common motif is important and exciting. Very few human/primate ribozymes have been published and this manuscript presents a detailed analysis of two of them. The minimized domains appear to be some of the smallest known self-cleaving ribozymes.

Strengths:

The manuscript is rooted in deep mutational analysis of the human OR4K15 and LINE1 ribozymes and subsequently in modeling of their active site based on the closely-related core of the TS ribozyme. The experiments support the HTS findings and provide convincing evidence that the ribozymes are structurally related to the core of the TS ribozyme, which has not been found in primates prior to this work.

Reviewer #1 (Public review):

Summary:

The aim of the present work is to evaluate the role of BMP9 and BMP10 in liver by depleting Bmp9 and Bmp10 from the main liver cell types (endothelial cells (EC), hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes (H)) using cell-specific cre recombinases. They show that HSCs are the main source of BMP9 and BMP10 in the liver. Using transgenic ALK1 reporter mice, they show that ALK1, the high affinity type 1 receptor for BMP9 and BMP10, is expressed on KC and EC. They have also performed bulk RNAseq analyses on whole liver, and cell-sorted EC and KC, and showed that loss of Bmp9 and Bmp10 decreased KC signature and that KC are replaced by monocyte-derived macrophages. EC derived from these Bmp9fl/flBmp10fl/flLratCre mice also lost their identity and transdifferentiated into continuous ECs. Liver iron metabolism and metabolic zonation were also affected in these mice. In conclusion, this work supports that BMP9 and BMP10 produced by HSC play a central role in mediating liver cell-cell crosstalk and liver homeostasis.

Strengths:

This work further supports the role of BMP9 and BMP10 in liver homeostasis. Using a specific HSC-Cre recombinase, the authors show for the first time that it is the BMP9 and BMP10 produced by HSC that play a central role in mediating liver cell-cell crosstalk to maintain a healthy liver. Although the overall message of the key role of BMP9 in liver homeostasis has been described by several groups, the role of hepatic BMP10 has not been studied before. Thus, one of the novelties of this work is to have used liver cell specific Cre recombinase to delete hepatic Bmp9 and Bmp10. The second novelty is the demonstration of the role of BMP9 and BMP10 in KC Differentiation/homeostasis which has already been slightly addressed by this group by knocking out ALK1, the high affinity receptor of BMP9 and BMP10 (Zhao et al. JCI, 2022).

Weaknesses:

This work remains rather descriptive and the molecular mechanisms are barely touched upon and could have been more explored.

Reviewer #1 (Public review):

The study by Korona and colleagues presents a rigorous experimental strategy for generating and maintaining a nearly complete set of monosomic yeast lines, thereby establishing a new standard for studying monosomes. Their careful approach in generating and handling monosome yeast lines, coupled with their use of high-throughput DNA sequencing and RNA sequencing, addresses concerns related to genomic instability and is commendable. However, I would like to express my concerns regarding the second part of the study, particularly the calculation of epistasis and the conclusion that vast positive epistatic effects have been observed. I believe that the conclusion of positive epistasis for fitness might be premature due to potential errors in estimating the expected fitness.

The method used to calculate fitness expectation (1 + sum(di), where di = rDRi - 1) may be inappropriate. The logarithm transformation mentioned by the authors is designed to transform the exponential growth curve into a linear relation for estimating doubling rate, and thus the fitness expectation should be calculated as the product of rDRi values. As an illustration, if gene A exhibits a 20% reduction in fitness when halved (A/-) and gene B exhibits a 30% reduction (B/-), the expected fitness of A/- B/- should be 56%, rather than the 50% estimated in the study. In other words, the formula used by the authors could underestimate the fitness expectation.

This issue is evident in Figure 2b, where negative values were obtained due to the use of an incorrect formula for estimating fitness expectations. It is worth noting that Figure 2a shows rDR values around one, indicating that no further logarithmic transformation was applied.

While widespread positive epistasis in yeast has been reported by other studies (e.g., doi: 10.1038/ng.524, but not to the extent reported in this study), the conclusion of the current study might not be sufficiently supported. I recommend that the authors revisit their calculation methods to provide a more convincing conclusion on the presence of positive epistasis for fitness in their dataset. Overall, I appreciate the authors' efforts in this study but believe that addressing these concerns is essential for strengthening the validity of their findings.

Comments on revised version:

The authors have adequately addressed all my previous concerns during revision.

Reviewer #2 (Public review):

This study examines monosomies in yeast in comparison to synthetic lethals resulting from combinations of heterozygous gene deletions that individually have a detrimental effect. The survival of monosomies, albeit with detrimental growth defects, is interpreted as positive epistasis for fitness. Gene expression was examined in monosomies in an attempt to gain insight into why monosomies can survive when multiple heterozygous deletions on the respective chromosome do not. In the RNAseq experiments, many genes were interpreted to be increased in expression and some were interpreted as reduced. Those with the apparent strongest increase were the subunits of the ribosome and those with the apparent strongest decreases were subunits of the proteasome.

The initiation and interpretation of the results were apparently performed in a vacuum of a century of work on genomic balance. Classical work in the flowering plant Datura and in Drosophila found that changes in chromosomal dosage would modulate phenotypes in a dosage sensitive manner (for references see Birchler and Veitia, 2021, Cytogenetics and Genome Research 161: 529-550). In terms of molecular studies, the most common modulation across the genome for monosomies is an upregulation (Guo and Birchler, Science 266: 1999-2002; Shi et al. 2021, The Plant Cell 33: 917-939).

It was also apparently performed in a vacuum of results of evolutionary genomics that indicate the classes of genes for which dosage causes fitness consequences. It was from yeast genomics that it was realized that there is a difference in the fate of duplicate genes that are members of molecular complexes following whole genome duplications (WGD) versus small segmental duplications (SSD) with longer retention times from WGD than other genes and an underrepresentation in small scale duplications (e.g. Papp et al. 2003, Nature 424: 194-197; Hakes et al 2007, Genome Biol 8: R209). This pattern arises from negative fitness consequences of deletion of some but not all members of a complex after WGD or the overexpression of individual subunits after SSD (Defoort et al., 2019 Genome Biol Evol 11: 2292-2305; Shi et al., 2020, Mol Biol Evol 37: 2394-2413). In order for this pattern to occur, there must be a reasonably close relationship between mRNA and the respective protein levels. This pattern of retention and underrepresentation has been found throughout eukaryotes (e.g. Tasdighian et al 2017, Plant Cell 29: 2766-2785) indicating that yeast is not an outlier in its behavior.

In the present yeast study, not only are there apparent increases for ribosomal subunits but also for many genes in the GAAC pathway, the NCR pathway, and Msn2p. The word "apparent" is used because RNAseq studies can only determine relative changes in gene expression (Loven et al., 2012, Cell 151: 476-482). Because aneuploidy can change the transcriptome size in general (Yang et al., 2021, The Plant Cell 33: 1016-1041), it is possible and maybe probable that this occurs in yeast monosomies as well. If there is an increase in the general transcriptome size, then there might not be as much reduction of the proteosome subunits as claimed and the increases might be somewhat less than indicated.

Indeed, the authors claim that there is an increased cell volume in the monosomies. Given that cell volume correlates very well with the total transcriptome size (Loven et al., 2012, Cell 151: 476-482; Sun et al 2020, Current Biol 30: 1217-1230; Swaffer et al., 2023, Cell 186: 5254-5268), it could well be that there is an increased transcriptome size in the monosomies. Thus, the interpretation of the relative changes from RNAseq is compromised.

It should be noted that contrary to the claims of the cited paper of Torres et al 2007 (Science 317: 916-924), a reanalysis of the data indicated that yeast disomies have many modulated genes in trans with downregulated genes being more common (Hou et al, 2018, PNAS 115: E11321-E11330). The claim of Torres et al that there are no global modulations in trans is counter to the knowledge that transcription factors are typically dosage sensitive and have multiple targets across the genome. The inverse effect trend is also true of maize disomies (Yang et al., 2021, The Plant Cell 33: 1016-1041), maize trisomies (Shi et al., 2021), Arabidopsis trisomies (Hou et al. 2018), Drosophila trisomies (Sun et al. 2013, PNAS 110: 7383-7388; Sun et al., 2013, PNAS 110: 16514-16519; Zhang et al., 2021, Scientific Reports 11: 19679; Zhang et al., genes 12: 1606) and human trisomies (Zhang et al., 2024, genes 15: 637). Taken as a whole it would seem to suggest that there are many inverse relationships of global gene expression with chromosomal dosage in both yeast disomies and monosomies.

In a similar vein, the authors cite Muenzner et al 2024, Nature 630 149-157 that there is an attenuation of protein levels from aneuploid chromosomes while the mRNA levels correlate with gene dosage. This interpretation also seems to have been made in a vacuum of the evolutionary genomics data noted above and there was no consideration of transcriptome size change in the aneuploids. Also, Muenzner et al make the remarkable suggestion that there is degradation of overproduced proteins from hyperploidy, but for monosomies there is greater degradation of the proteins from the remainder of the genome.

To clarify the claims of this study, it would be informative to produce distributions of the various ratios of individual gene expression in monosomy versus diploid as performed by Hou et al. 2018. This will better express the trends of up and down regulation across the genome and whether there are any genes on the varied chromosome that are dosage compensated. The authors claim in the Abstract that "There is no evidence of increased (compensatory) gene expression on the monosomic chromosomes", but then note after describing the increased cell volume of monosomies that this observation likely signals an increased transcriptome size: "Indeed, one explanation for the observed epistasis for viability could be an ample overproduction of all transcripts, so that even those halved by monosomy are sufficiently abundant". It is not clear to this reviewer what conclusions can be made from this work other than the empirical observation that monosomy does not reflect the cumulative effect of multiple haplo-insufficiencies of individual heterozygous deletions and that there are some RELATIVE changes in gene expression, but it is unclear what the ABSOLUTE PER CELL expression is across the whole genome. Clarifying this issue would be important for understanding the nature of any epistasis and fitness consequences.

Reviewer #3 (Public review):

The current study examined 13 monosomic yeast strains that lost different individual chromosomes. By comparing the fitness of monosomic strains and several heterozygous deletion strains, the authors observed strong positive epistasis for fitness. The transcriptomes of monosomic strains indicated that general gene-dose compensation is not the reason for fitness gains. On the other hand, gene expression of ribosomal proteins was up-regulated and proteasome subunit expression was down-regulated in all tested monosomic strains. The authors speculated that overexpression in combination with decreased degradation of the insufficient proteins might explain the positive epistasis observed in monosomic strains. This study investigates an important biological question and has some interesting results. However, I have some reservations about the data interpretations listed below.

(1) In Figure 3b (and line 179), the authors stated that those haploinsufficient genes were not transcribed at elevated rates, but almost half of them are in reddish colors (indicating that the expression is higher than 1-fold). Obviously, many haploinsufficient genes are up-regulated in monosomic strains. What the data really show is that the level of overexpression is not correlated with the fitness effect of the deletion (since all the p values are not significant). The authors need to correct their conclusions.<br /> (2) Why are some monosomic strains removed from the transcriptomics analysis, especially when the chromosome IV and XV strains show very strong positive epistasis? The authors need to provide an explanation here.<br /> (3) The authors stated that diploidy observed in chromosome VII and XIII strains were due to endoreplication after losing the marked chromosomes (lines 97 and 117). Isn't chromosome missegregation an equally possible explanation? Since monosomic cells are generated by chromosome missegregation during mitosis, another chromosome missegregation event may occur to rescue the fitness (or viability) of monosomic cells in these strains.

Comments for the revised version:

The authors have addressed all my previous concerns and I have no further questions.

Reviewer #2 (Public review):

Summary:

The authors set out to study whether the cooling agent binding site in TRPM8, which is located between the S1-S4 and the TRP domain, is conserved within the TRPM family of ion channels. They specifically chose the TRPM4 channel as the model system, which is directly activated by intracellular Ca2+. Using electrophysiology, the authors characterized and compared the Ca2+ sensitivity and the voltage-dependence of TRPM4 channels in the absence and presence of synthetic cooling agonist icilin. They also analyzed the mutational effects of residues (A867G and R901H; equivalent mutations in TRPM8 were shown involved in icilin sensitivity) on Ca2+ sensitivity and voltage-dependence of TRPM4 in the absence and presence of Ca2+. Based on the results as well as structure/sequence alignment, the authors concluded that icilin likely binds to the same pocket in TRPM4 and suggested that this cooling agonist binding pocket is conserved in TRPM channels.

Strengths:

The authors gave a very thorough introduction of the TRPM channels. They have nicely characterized the Ca2+ sensitivity and the voltage-dependence of TRPM4 channels and demonstrated icilin potentiates the Ca2+ sensitivity and diminishes the outward rectification of TRPM4. These results indicate icilin modulates TRPM4 activation by Ca2+.

The authors have incorporated additional data analysis and control experiments in the revised manuscript to strengthen their findings. They have well addressed the concerns raised by reviewers in the responses.

Weaknesses:

The study is conducted based on an assumption that TRPM4 activation is controlled by Ca2+ binding to a single site in the S1-S4 pocket in each subunit, and the second Ca2+ site in the cytoplasmic MHRs is simplified.

Despite the technical reasons presented by the authors in the rebuttal, the conclusion of this study can be strengthened if more cooling compounds- the most well-studied natural cooling agonist menthol, and/or other cooling agonists such as WS-12 and/or C3-are tested for their effects on TRPM4 and several other TRPM channels.

Reviewer #1 (Public review):

In this manuscript, Yang et al report a novel regulatory role of SIRT4 in the progression of kidney fibrosis. The authors showed that in the fibrotic kidney, SIRT4 exhibited an increased nuclear localization. Deletion of Sirt4 in renal tubule epithelium attenuated the extent of kidney fibrosis following injury, while overexpression of SIRT4 aggravates kidney fibrosis. Employing a battery of in vitro and in vivo experiments, the authors demonstrated that SIRT4 interacts with U2AF2 in the nucleus upon TGF-β1 stimulation or kidney injury and deacetylates U2AF2 at K413, resulting in elevated CCN2 expression through alternative splicing of Ccn2 gene to promote kidney fibrosis. The authors further showed that the translocation of SIRT4 is through the BAX/BAK pore complex and is dependent on the ERK1/2-mediated phosphorylation of SIRT4 at S36, and consequently the binding of SIRT4 to importin α1. This fundamental work substantially advances our understanding of the progression of kidney fibrosis and uncovers a novel SIRT4-U2AF2-CCN2 axis as a potential therapeutic target for kidney fibrosis.

Comment on revised version:

In the new version of the manuscript, the authors have addressed most of my concerns . Overall, the authors have done an extensive, well-performed study. The results are convincing, and the conclusions are mostly well supported by the data. The message is interesting to a wider community working on kidney fibrosis, protein acetylation and SIRT4 biology. This work substantially advances our understanding of the mechanism of kidney fibrosis and uncovers a novel SIRT4-U2AF2-CCN2 axis as a potential therapeutic target for kidney fibrosis.

Reviewer #2 (Public review):

Summary:

The manuscript by Yang et al. presents a novel and significant investigation into the role of SIRT4 For CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing and its impact on the development of kidney fibrosis.

Strengths:

The authors' main conclusion is that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via pre-mRNA splicing. Additionally, the study reveals that SIRT4 translocates from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, further promoting its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Overall, the findings are fully convincing. The current study, to some extent, shows potential importance in this field.

Reviewer #3 (Public review):

Summary:

Yang et al reported in this paper that TGF-beta induces SIRT4 activation, TGF-beta activated SIRT4 then modulates U2AF2 alternative splicing, U2AF2 in turn causes CCN2 for expression. The mechanism is described as this: mitochondrial SIRT4 transport into the cytoplasm in response to TGF-β stimulation, phosphorylated by ERK in the cytoplasm, and pathway and then undergo nuclear translocation by forming the complex with importin α1. In the nucleus, SIRT4 can then deacetylate U2AF2 at K413 to facilitate the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Moreover, they used exosomes to deliver Sirt4 antibodies to mitigate renal fibrosis in a mouse model. TGF-beta has been widely reported for its role in fibrosis induction.

Strengths:

TGF-beta induction of SIRT4 translocation from mitochondria to nuclei for epigenetics or gene regulation remains largely unknown. The findings presented here that SIRT4 is involved in U2AF2 deacetylation and CCN2 expression are interesting.

Comments on revised version:

I went through the revised manuscript and the letter from the authors. I have no further concerns.

Reviewer #1 (Public review):

Summary:

The authors want to determine the role of the sperm hook of the house mouse sperm in movement through the uterus. They use transgenic lines with fluorescent labels to sperm proteins, and they cross these males to C57BL/6 females in pathogen-free conditions. They use 2-photon microscopy on ex vivo uteri within 3 hours of mating and the appearance of a copulation plug. There are a total of 10 post-mating uteri that were imaged with 3 different males. They provide 10 supplementary movies that form that basis for some of the quantitative analysis in the main body figures. Their data suggest that the role of the sperm hook is to facilitate movement along the uterine wall.

Strengths:

Ex vivo live imaging of fluorescently labeled sperm with 2-photon microscopy is a powerful tool for studying the behavior of sperm.

Weaknesses:

The paper is descriptive and the data are correlations.

The authors cannot directly test their proposed function of the sperm hook in sliding and preventing backward slipping.

Reviewer #1 (Public Review):

Summary:

This is a well-written and detailed manuscript showing important results on the molecular profile of 4 different cohorts of female patients with lung cancer.

Strengths:

The authors used several different methods to identify potential novel targets for therapeutic interventions.

Weaknesses:

Statistical test results need to be provided in comparisons between cohorts. This was addressed by the authors in the revisions.

Reviewer #2 (Public Review):

New comments are added after authors responses to my initial comments.

Summary:

Zhang et al. performed a proteogenomic analysis of lung adenocarcinoma (LUAD) in 169 female never-smokers from the Xuanwei area (XWLC) in China. These analyses reveal that XWLC is a distinct subtype of LUAD and that BaP is a major risk factor associated with EGFR G719X mutations found in the XWLC cohort. Four subtypes of XWLC were classified with unique features based on multi-omics data clustering.

Strengths:

The authors made great efforts in performing several large-scale proteogenomic analyses and characterizing molecular features of XWLCs. Datasets from this study will be a valuable resource to further explore the etiology and therapeutic strategies of air-pollution-associated lung cancers, particularly for XWLC.

Weaknesses:

[...]

(2) Importantly, while providing the large datasets, validating key findings is minimally performed, and surprisingly there is no interrogation of XWLC drug response/efficacy based on their findings, which makes this manuscript descriptive and incomplete rather than conclusive. For example, testing the efficacy of XWLC response to afatinib combined with other drugs targeting activated kinases in EGFR G719X mutated XWLC tumors would be one way to validate their datasets and new therapeutic options.

Response: We appreciate your suggestion. In reference to testing the efficacy of XWLC response to afatinib combined with drugs targeting kinases, we have planned to establish PDX and organoid models to validate the effectiveness of our therapeutic approach. Due to the extended timeframe required, we intend to present these results in a subsequent study.

Comments: All conclusions in the manuscript made by authors are based on interpretations of large-scale multi-omics data, which should be properly validated by other approaches and methods. Without validation, these are all speculations and any conclusions without supporting evidence are not acceptable. This reviewer suggested an example of validation experiment, and Reviewer #3 also pointed out several data that need to be validated. However, authors do not agree to perform any of these validation experiments without reasonable justification.

(3) The authors found MAD1 and TPRN are novel therapeutic targets in XWLC. Are these two genes more frequently mutated in one subtype than the other 3 XWLC subtypes? How these mutations could be targeted in patients?

Response: Thank you for your question. We have investigated the TPRN and MAD1 mutations in our dataset, identifying five TPRN mutations and eight MAD1 mutations. Among the TPRN mutations, XWLC_0046 and XWLC_0017 belong to the MCII subtype, XWLC_0012 belongs to the MCI subtype, and the subtype of the other three samples is undetermined, resulting in mutation frequencies of 1/16, 2/24, 0/15, and 0/13, respectively. Similarly, for the MAD1 mutations, XWLC_0115, XWLC_0021, and XWLC_0047 belong to the MCII subtype, XWLC_0055 containing two mutations belongs to the MCI subtype, and the subtype of the other three samples is undetermined, resulting in mutation frequencies of 1/16, 3/24, 0/15, and 0/13 across subtypes, respectively. Fisher's test did not reveal significant differences between the subtypes. For targeting novel therapeutic targets such as MAD1 and TPRN, we propose a multi-step approach. Firstly, we advocate for conducting functional in vivo and in vitro experiments to verify their roles during cancer progression. Secondly, we suggest conducting small molecule drug screening based on the pharmacophore of these proteins, which may lead to the identification of potential therapeutic drugs. Lastly, we recommend testing the efficacy of these drugs to further validate their potential as effective treatments.

Comments: Please properly incorporate the above explanation into the main text.

(4) In Figures 2a and b: while Figure 2a shows distinct genomic mutations among each LC cohort, Figure 2b shows similarity in affected oncogenic pathways (cell cycle, Hippo, NOTCH, PI3K, RTK-RAS, and WNT) between XWLC and TNLC/CNLC. Considering that different genomic mutations could converge into common pathways and biological processes, wouldn't these results indicate commonalities among XWLC, TNLC, and CNLC? How about other oncogenic pathways not shown in Figure 2b?

Response: Thank you for your question. Based on the data presented in Fig. 2a, which encompasses all genomic mutations, it appears that the mutation landscape of XWLC bears the closest resemblance to TSLC (Fig. 2a). However, when considering oncogenic pathways (Fig. 2b) and genes (Fig. 2c), there is a notable disparity between the two cohorts. These findings suggest that while XWLC and TSLC exhibit similarities in terms of genomic mutations, they possess distinct characteristics in terms of oncogenic pathways and genes.<br /> Regarding the oncogenic signaling pathways, we referred to ten well-established pathways identified from TCGA cohorts. These members of oncogenic pathways are likely to serve as cancer drivers (functional contributors) or therapeutic targets, as highlighted by Sanchez-Vega et al. in 2018(Sanchez-Vega et al., 2018).

Comments: It is unclear to this reviewer how authors defined "distinct characteristics" in terms of oncogenic pathways and genes. Would 10-20% differences in "Fraction of samples affected" in Fig2b be sufficient to claim significance? How could authors be sure whether mutations in genes involved in each oncogenic pathway are activating or inactivating mutations (rather than benign, thus non-affecting mutations)?

[...]

(6) Supplementary Table 11 shows a number of mutations at the interface and length of interface between a given protein-protein interaction pair. Such that, it does not provide what mutation(s) in a given PPI interface is found in each LC cohort. For example, it fails to provide whether MAD1 R558H and TPRN H550Q mutations are found significantly in each LC cohort.

Response: We appreciate your careful review. In Supplementary Table 11, we have provided significant onco_PPI data for each LC cohort, focusing on enriched mutations at the interface of two proteins. Our emphasis lies on onco_PPI rather than individual mutations, as any mutation occurring at the interface could potentially influence the function of the protein complex. Thus, our Supplementary Table 11 exclusively displays the onco_PPI rather than mutations. MAD1 R558H and TPRN H550Q were identified through onco_PPI analysis, and subsequent extensive literature research led us to focus specifically on these mutations.

Comments: Are authors referring to Table S9 (Onco_PPIs identified in four cohorts) instead of Supplementary Table 11? There is no Table 11 among submitted files. In Table S9, the Column N (length of protein product of gene1) does not make sense: MYO1C (8152), TP53 (3924), EGFR (12961). These should not be the number of amino acids residues of each protein. Then, what do these numbers mean?

(7) Figure 7c and d are simulation data not from an actual binding assay. The authors should perform a biochemical binding assay with proteins or show that the mutation significantly alters the interaction to support the conclusion.

Response: We appreciate your suggestion. The relevant experiments are currently in progress, and we anticipate presenting the corresponding data in a subsequent study.

Comments: The suggested experiment is to support the simulated data. Again, without supporting experimental results, authors could not make a conclusion simply based on simulated data. Where else could the supporting experimental results be presented?

Reviewer #1 (Public review):

Summary:

Arman Angaji and his team delved into the intricate world of tumor growth and evolution, utilizing a blend of computer simulations and real patient data from liver cancer.

Strengths:

Their analysis of how mutations and clones are distributed within tumors revealed an interesting finding: tumors don't just spread from their edges as previously believed. Instead, they expand both from within and the edges simultaneously, suggesting a unique growth mode. This mode naturally indicates that external forces may play a role in cancer cells dispersion within the tumor. Moreover, their research hints at an intriguing phenomenon - the high death rate of progenitor cells and extremely slow pace in growth in the initial phase of tumor expansion. Understanding this dynamic could significantly impact our comprehension of cancer development.

Weaknesses:

It's important to note, however, that this study relies on specific computer models, metrics derived from inferred clones, and a limited number of patient data. While the insights gained are promising, further investigation is essential to validate these findings. Nonetheless, this work opens up exciting avenues for comprehending the evolution of cancers.

Comments on revised submission:

The authors have effectively addressed my concerns. This revision is excellent.

Reviewer #2 (Public review):

Summary:

The article uses a cell-based model to investigate how mutations and cells spread throughout a tumour. The paper uses published data and the proposed model to understand how growth and death mechanisms lead to the observed data. This work provides an insight into the early stages of tumour development. From the work provided here, the results are solid, showing a thorough analysis. The article is well written and presents a very suitable and rigorous analysis to describe the data. The authors did a particularly nice job of the discussion and decision of their "metrics of interest", though this is not the main aim of this work.

Strengths:

Due to the particularly nice and tractable cell-based model, the authors are able to perform a thorough analysis to compare the published data to that simulated with their model. They then used their computational model to investigate different growth mechanisms of volume growth and surface growth. With this approach, the authors are able to compare the metric of interest (here, the direction angle of a new mutant clone, the dispersion of mutants throughout the tumour) to quantify how the different growth models compare to the observed data. The authors have also used inference methods to identify model parameters based on the data observed. The authors performed a rigorous analysis and have chosen the metrics in an appropriate manner to compare the different growth mechanisms.

Context:

Improved mechanistic understanding into the early developmental stages of tumours will further assist in disease treatment and quantification. Understanding how readily and quickly a tumour is evolving is key to understanding how it will develop and progress. This work provides a solid example as to how this can be achieved with data alongside simulated models.

Reviewer #1 (Public Review):

Summary:

This manuscript provides a solid advance to the scavenger receptor field by reporting the crystal structures of the domains of SCARF1 that bind modified LDL such as oxidized LDL and acylated LDL. The crystal packing reveals a new interface for homodimerization of SCARF1. The authors characterize SCARF1 binding to modified LDL using flow cytometry, ELISA, and fluorescent microscopy. They identify a positively-charged surface on the structure that they predict will bind the LDLs, and they support this hypothesis with several mutant constructs in binding experiments.

Strengths:

The authors have crystallized domains of an understudied scavenger receptor and used the structure to identify a putative binding site for modified LDL particles. An especially strong set of experiments are binding studies with chimeras of SCARF1 and SCARF2, where they show gain-of-function results (binding of modified LDLs) by SCARF2, a related protein that does not normally bind modified LDLs. The paper is a straightforward set of experiments that identify the likely binding site of modified LDL on SCARF1

Weaknesses:

In the current revision, the authors addressed my technical concerns.<br /> Two remaining considerations that may limit the broader impact of this paper are 1) that it does not explain the structural basis for specificity of the binding of SCARF1 to various lipoproteins (i.e. why SCARF1 binds oxLDL and AcLDL but not LDL or HDL) and 2) a lack of a biological assay to interpret the functional consequences of the SCARF1 mutants. These may be addressed in future work.

Reviewer #1 (Public review):

The authors have tried to identify the plausible Na+ entry pathway in an important SLC6 member GAT1, using computational approaches to assess residence times of the ions as they enter the vestibule of GAT1. The authors identify a patch of negative residues in TM6a and implicate them for being important to attract the Na+ ions during their movement towards the binding sites Na1 and Na2. Besides this they also suggest that sodium binding at site 1 is flexible and at times can occupy the primary binding site when the substrate is not available. Na2 site as other literature also suggests is demonstrated to be vital for the stability of the outward-open state.

Studies of ion permeation are challenging given that the states are challenging to trap through structural studies and computational methods are vital for understanding these steps. The authors suggest that two negatively charged residues are vital to attract Na+ ions to the vestibule. Using a combination of simulations and PCA analysis the authors identify the importance of Na+ binding at site 2 that stabilises the outward-open state and the flexibility observed in Na1 site for ion binding which happens alongside substrate in the GABA bound state. The study reconfirms earlier observations in the SLC6 family that Na2 site is critical for conformational transitions and Na1 site is substrate dependent in amino acid transporters.<br /> One of the challenges in such studies is to conclusively establish the presence of additional Na+ sites or regions of ion-binding with experimental structures as they are nearly impossible to trap. Such studies using simulations therefore become the only resort to understand such phenomena.

The work is likely to further provide insights into the transport mechanism of GAT1 and lends credence to some structural studies where the sodium at site1 is displaced but the ion remains proximal to the bound substrate.

Reviewer #1 (Public review):

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

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

The authors responded to all my comments and I have nothing to add. The evidence provided for durotaxis of non adherent (or low-adhering) cells is strong. I am particularly impressed by the fact that amoeboid cells can durotax even when not confined. I wish to congratulate the authors for the excellent work, which will fuel discussion in the field of cell adhesion and migration.

Reviewer #2 (Public review):

Summary:

The authors developed an imaging-based device, that provides both spatial confinement and stiffness gradient, to investigate if and how amoeboid cells, including T cells, neutrophils and Dictyostelium can durotax. Furthermore, the authors showed that the mechanism for the directional migration of T cells and neutrophils depends on non-muscle myosin IIA (NMIIA) polarized towards the soft-matrix-side. Finally, they developed a mathematical model of an active gel that captures the behavior of the cells described in vitro.

Strengths:

The topic is intriguing as durotaxis is essentially thought to be a direct consequence of mechanosensing at focal adhesions. To the best of my knowledge, this is the first report on amoeboid cells that are not dependent on FAs to exert durotaxis. The authors developed an imaging-based durotaxis device that provides both spatial confinement and stiffness gradient and they also utilized several techniques such as quantitative fluorescent speckle microscopy and expansion microscopy. The results of this study have well-designed control experiments and are therefore convincing.

Weaknesses:

Overall this study is well performed but there are still some minor issues I recommend the authors address:<br /> (1) When using NMIIA/NMIIB knockdown cell lines to distinguish the role of NMIIA and NMIIB in amoeboid durotaxis, it would be better if the authors take compensatory effects into account.<br /> (2) The expansion microscopy assay is not clearly described and some details are missed such as how the assay is performed on cells under confinement.<br /> (3) In this study, an active gel model was employed to capture experimental observations. Previously, some active nematic models were also considered to describe cell migration, which is controlled by filament contraction. I suggest the authors provide a short discussion on the comparison between the present theory and those prior models.<br /> (4) In the present model, actin flow contributes to cell migration while myosin distribution determines cell polarity. How does this model couple actin and myosin together?

Reviewer #1 (Public review):

Summary:

In the manuscript "FABP4-mediated lipid accumulation and lipolysis in tumor-associated macrophages promote breast cancer metastasis", Yorek, et al. provide a novel mechanism explaining how unsaturated fatty acids induce macrophages to accumulate lipid droplets, which when contained in tumor-associated macrophages (TAMs) are associated with increased metastasis in breast cancer. The authors conclude that unsaturated fatty acids are transported into macrophages by the chaperone FABP4 where they induce C/EBPalpha expression and transcriptional activity resulting in upregulation of enzymes involved in triacylglycerol and lipid droplet biosynthesis. The resulting accumulation of lipid droplets in macrophages creates a store of fatty acids that can subsequently be released through FABP4-dependent lipolysis and thereby stimulate the migration of nearby breast cancer cells. While generally well-written and developed, there are a few concerns about the rigor of experimental evidence that supports some conclusions, including the existence of a FABP4-C/EBPalpha pathway. Overall, the mechanism identified is a valuable contribution to our understanding of how tumor-associated macrophages may influenced by available metabolites to promote the aggressiveness of certain cancers. FABP4 has the potential to be used as a novel biomarker of macrophage-induced cancer aggressiveness and/or a therapeutic target to prevent metastasis.

Strengths:

(1) The study is logically organized and provides extensive evidence in support of the overall model proposed.

(2) Multiple complementary techniques are used to identify and quantify lipid droplets.

(3) Primary macrophages and macrophage cell lines are used and provide consistent data.

(4) Knock-down and knock-out cells are used to assess the contributions of FABP4 and C/EBPalpha to gene expression.

(5) Public gene expression data (GEO, TCGA) is used effectively throughout.

Weaknesses:

(1) After Figure 1, a single saturated (palmitic acid; PA) and a single unsaturated (linoleic acid; LA) fatty acid are used for the remaining studies, bringing into question whether effects are in fact the result of a difference in saturation vs. other potential differences.

(2) While primary macrophages are used in several mechanistic studies, tumor-associated macrophages (TAMs) are not used. Rather, correlative evidence is provided to connect mechanistic studies in macrophage cell lines and primary macrophages to TAMs.

(3) C/EBPalpha and FABP4 clearly regulate LA-induced changes in gene expression. However, whether these two key proteins act in parallel or as a pathway is not resolved by presented data.

(4) It is very interesting that FABP4 regulates both lipid droplet formation and lipolysis, yet is unclear if the regulation of lipolysis is direct or if the accumulation of lipid droplets - likely plus some other signal(s) - induces upregulation of lipolysis genes.

(5) In several places increased expression of genes coding for enzymes with known functions in lipid biology is conflated with an increase in the lipid biology process the enzymes mediate. Additional evidence would be needed to show these processes are in fact increased in a manner dependent on increased enzyme expression.

Reviewer #2 (Public review):

The manuscript by Yorek et al explores the role of fatty acids, particularly unsaturated fatty acids, in lipid droplet accumulation and lipolysis in tumor-associated macrophages (TAMs). Using flow cytometry, immunofluorescent imaging, and TEM, the authors observed that unsaturated fatty acids, such as linoleic acids (LA), tend to induce lipid droplet accumulation in the ER of macrophages, but not in the lysosomes. This phenomenon led them to examine the key enzymes involved in lipid droplet/TAG biosynthesis, where they found incubation of LA upregulates GPAT/DGAT and C/EBPα. In vitro studies, data from public databases, single-cell RNA sequencing of splenic macrophages, and more show that FABP4 emerges as an important mediator for C/EBPα activation. This is further confirmed by FABP4-knockout macrophages, where lipid accumulation and utilization of unsaturated fatty acids were compromised in macrophages through inhibition of LA-induced lipolysis. Using the co-culture system and immunohistochemical analysis, they found that the high FABP4 expression in TAMs, which are observed in metastatic breast cancer tissue, promotes breast cancer cell migration in vitro.

This study is important since the impact of tumor microenvironment is crucial for the development of breast cancer. The individual experiments are well-designed and structured. However, the logic connecting to the next step is a bit difficult to follow, especially when combined with incomplete statistical analysis in some figures, making the conclusion less convincing. For instance, the comparison of macrophage FABP4 expression between breast cancer patients with or without metastasis illustrates the importance of FABP4 expression in metastasis, yet there is no examination of the expression of other key enzymes in the lipolysis or lipid biosynthesis pathway nor there is any correlation with parameters that would reflect patients' consumption of fatty acids. Similarly, an in vivo study comparing FABP4 knockout mice with or without unsaturated fatty acids would yield more compelling evidence. The statistical analysis was largely focused on the sets of unsaturated fatty acids when data from both types of fatty acids were present. In some cases, significant changes are observed in the sets of saturated fat, but there is no explanation of why only the data from unsaturated fats are important for investigation.

Overall, there is solid evidence for the importance of FABP4 expression in TAMs on metastatic breast cancer as well as lipid accumulation by LA in the ER of macrophages. A stronger rationale for the exclusive contribution of unsaturated fatty acids to the utilization of TAMs in breast cancer and a more detailed description and statistical analysis of data will strengthen the findings and resulting claims.

Reviewer #3 (Public review):

Summary:

Regulated metabolism has only recently been recognized as a key component of cancer biology, and even more recently recognized as a significant modulator of the tumor microenvironment (TME). TAMs in the TME play a major role in supporting cancer cell survival and growth/spread, as well as generating an immunosuppressive ME to suppress anti-tumor immunity. Specific regulation of lipid metabolism in this context, in particular how lipids are stored and subsequently mobilized for metabolism, is largely unexplored - especially in the immunological components of the TME.

In this manuscript, the authors build on their previous observations that the fatty acid-binding protein FABP4 plays an important role in macrophage function and that FABP4 expression in tumor associated macrophages (TAM) promotes breast cancer progression. They demonstrate:

(1) Unlike saturated fatty acids (FA), unsaturated FA promotes lipid droplet (LD) accumulation in murine macrophages. LD is the primary intracellular storage depot for FA.

(2) Unsaturated FA activates the FABP4-C/EBPalpha axis to upregulate transcription of the enzymes involved in the synthesis of neutral triacylglycerol (TAG) is an essential step in the formation of the neutral lipid core of LD. It should be noted that the authors speculate that UFA-activated FABP4 translocates to the nucleus to activate PPARgamma, which is known to induce C/EBPalpha expression, but do not directly test the involvement of PPARgamma in this axis.

(3) FABP4 deficiency compromises unsaturated FA-mediated lipid accumulation and utilization in murine macrophages.

(4) FABP4-mediated lipid metabolism in macrophages (TAMS) contributes to breast cancer metastasis, in in-vitro of tumor migration induced by murine macrophages and in correlative studies from human patient breast cancer biopsies.

From these studies, the manuscript concludes that FABP4 plays a pivotal role in mediating lipid droplet formation and lipolysis in TAM, which provides lipids to breast cancer cells that contribute to their growth and metastasis.

These are significant findings, as they provide new insight into the mechanistic regulation of TAM biology via regulation of lipid metabolism, as well as define new biomarkers and potential novel therapeutic targets.

The findings are strong in the studies that mechanistically define the role of FAB4 in lipid accumulation and utilization in murine macrophages. However, evidence is less compelling regarding TAM biology and human breast cancer in 3 main areas:

First, while there is clear in vitro evidence that co-cultured murine macrophages genetically deficient in FABP4 (or their conditioned media) do not enhance breast cancer cell motility and invasion, these macrophages are not bonafide TAM - which may have different biology. The use of actual TAM in these experiments would be more compelling. Perhaps more importantly, there is no in vivo data in tumor-bearing mice that macrophage deficiency of FABP4 affects tumor growth or metastasis - which are doable experiments given the availability of the FABP4 KO mice.

Second, no data is presented that the mechanisms/biology that are elegantly demonstrated in the murine macrophages also occur in human macrophages - which would be foundational to translating these findings into human breast cancer. It seems like straightforward in vitro studies in human monocytes/macrophages could be done to recapitulate the main characteristics seen in the murine macrophages.

Third, while the data from the human breast cancer specimens is very intriguing, it is difficult to ascertain how accurate IHC is in determining that the CD163+ cells (TAM) are in fact the same cells expressing FABP4 - which is the central premise of these studies. Demonstrating that IHC has the resolution to do this would be important. Additionally, the in vitro characterization of FABP4 expression in human macrophages would also add strength to these findings.

In summary, the strengths of this manuscript are the significance of metabolic regulation of the immune tumor microenvironment (TME), and the careful mechanistic delineation of FABP4 involvement in mediating lipid droplet formation and lipolysis in murine macrophages. The weaknesses of the work are the lack of direct experimental evidence that human macrophages behave in the same way as murine macrophages, the incomplete characterization of the role of FABP4 expression in TAM in modulating tumor growth in vivo (in murine models), and whether it can be definitively determined that FABP4 is being primarily expressed in the CD163+ macrophages in human breast cancer samples.

Strengths:

(1) Regulated metabolism has only recently been recognized as a key component of cancer biology, and even more recently recognized as a significant modulator of the tumor microenvironment (TME). TAMs in the TME play a major role in supporting cancer cell survival and growth/spread, as well as generating an immunosuppressive ME to suppress anti-tumor immunity.

(2) Regulation of lipid metabolism in this context is largely unexplored, especially in the immunological components of the TME.

(3) The work builds on the authors' previous work on the role of FABP4 plays an important role in macrophage function including FABP4 expression in TAM promotes breast cancer progression (Hao et al, Cancer Res 2018). This paper identified FABP4-expressing macrophages as being pro-tumorigenic via upregulation of IL-6STAT3 signaling.

(4) The careful and thorough mechanistic delineation of FABP4 involvement in mediating lipid droplet formation and lipolysis in murine macrophages.

(5) The intriguing observations that FABP4-mediated lipid metabolism in macrophages contributes to breast cancer metastasis, in in vitro of tumor migration induced by murine macrophages and in correlative studies from human patient breast cancer biopsies that CD163+ cell numbers (putatively TAM) and FABP4 expression was associated with increased metastatic disease and poor overall survival.

(6) Identification of FABP4 both a prognostic biomarker and a potential therapeutic target to modulate the pro-tumor immune TME.

Weaknesses:

(1) While the authors speculate that UFA-activated FABP4 translocates to the nucleus to activate PPARgamma, which is known to induce C/EBPalpha expression, they do not directly test involvement of PPARgamma in this axis.

(2) While there is clear in vitro evidence that co-cultured murine macrophages genetically deficient in FABP4 (or their conditioned media) do not enhance breast cancer cell motility and invasion, these macrophages are not bonafide TAM - which may have different biology. Use of actual TAM in these experiments would be more compelling. Perhaps more importantly, there is no in vivo data in tumor bearing mice that macrophage-deficiency of FABP4 affects tumor growth or metastasis.

(3) Related to this, the authors find FABP4 in the media and propose that macrophage secreted FABP4 is mediating the tumor migration - but don't do antibody neutralizing experiments to directly demonstrate this.

(4) No data is presented that the mechanisms/biology that are elegantly demonstrated in the murine macrophages also occurs in human macrophages - which would be foundational to translating these findings into human breast cancer.

(5) While the data from the human breast cancer specimens is very intriguing, it is difficult to ascertain how accurate IHC is in determining that the CD163+ cells (TAM) are in fact the same cells expressing FABP4 - which is central premise of these studies. Demonstration that IHC has the resolution to do this would be important. Additionally, the in vitro characterization of FABP4 expression in human macrophages would also add strength to these findings.

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Weaknesses:

(1) Important methodological details regarding the treatment of mAC membrane preps with fatty acids are missing.

(2) It is not evident that fatty acid regulators can be considered as "signaling molecules" since it is not clear (at least to this reviewer) how concentrations of free fatty acids in plasma or endocytic membranes are hormonally or otherwise regulated.

Reviewer #2 (Public review):

Summary:

The authors extend their earlier findings with bacterial adenylyl cyclases to mammalian enzymes. They show that certain aliphatic lipids activate adenylyl cyclases in the absence of stimulatory G proteins and that lipids can modulate activation by G proteins. Adding lipids to cells expressing specific isoforms of adenylyl cyclases could regulate cAMP production, suggesting that adenylyl cyclases could serve as 'receptors'.

Strengths:

This is the first report of lipids regulating mammalian adenylyl cyclases directly. The evidence is based on biochemical assays with purified proteins, or in cells expressing specific isoforms of adenylyl cyclases.

Weaknesses:

It is not clear if the concentrations of lipids used in assays are physiologically relevant. Nor is there evidence to show that the specific lipids that activate or inhibit adenylyl cyclases are present at the concentrations required in cell membranes. Nor is there any evidence to indicate that this method of regulation is seen in cells under relevant stimuli.

Reviewer #3 (Public review):

Summary:

Landau et al. have submitted a manuscript describing for the first time that mammalian adenylyl cyclases can serve as membrane receptors. They have also identified the respective endogenouse ligands which act via AC membrane linkers to modify and control Gs-stimulated AC activity either towards enhancement or inhibition of ACs which is family and ligand-specific. Overall, they have used classical assays such as adenylyl cyclase and cAMP accumulation assays combined with molecular cloning and mutagenesis to provide exceptionally strong biochemical evidence for the mechanism of the involved pathway regulation.

Strengths:

The authors have gone the whole long classical way from having a hypothesis that ACs could be receptors to a series of MS studies aimed at ligand indentification, to functional studies of how these candidate substances affect the activity of various AC families in intact cells. They have used a large array of techniques with a paper having clear conceptual story and several strong lines of evidence.

Weaknesses:

(1) At the beginning of the results section, the authors say "We have expected lipids as ligands". It is not quite clear why these could not have been other substances. It is because they were expected to bind in the lipophilic membrane anchors? Various lipophilic and hydrophilic ligands are known for GPCR which also have transmembrane domains. Maybe 1-2 additional sentences could be helpful here.

(2) In stably transfected HEK cells expressing mAC3 or mAC5, they have used only one dose of isoproterenol (2.5 uM) for submaximal AC activation. The reference 28 provided here (PMID: 33208818) did not specifically look at Iso and endogenous beta2 adrenergic receptors expressed in HEK cells. As far as I remember from the old pharmacological literature, this concentration is indeed submaximal in receptor binding assays but regarding AC activity and cAMP generation (which happen after signal amplification with a so-called receptor reserve), lower Iso amounts would be submaximal. When we measure cAMP, these are rather 10 to 100 nM but no more than 1 uM at which concentration response dependencies usually saturate. Have the authors tried lower Iso concentrations to prestimulate intracellular cAMP formation? I am asking this because, with lower Iso prestimulation, the subsequent stimulatory effects of AC ligands could be even greater.

(3) The authors refer to HEK cell models as "in vivo". I agree that these are intact cells and an important model to start with. It would be very nice to see the effects of the new ligands in other physiologically relevant types of cells, and how they modulate cAMP production under even more physiological conditions. Probably, this is a topic for follow-up studies.

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

The authors have achieved their aims to a very high degree, their results do nicely support their conclusions. There is only one point (various classical GPCR concentrations, please see above) that would be beneficial to address.

Without any doubt, this is a groundbreaking study that will have profound implications in the field for the next years/decades. Since it is now clear that mammalian adenylyl cyclases are receptors for aliphatic fatty acids and anandamide, this will change our view on the whole signaling pathway and initiate many new studies looking at the biological function and pathophysiological implications of this mechanism. The manuscript is outstanding.

Reviewer #1 (Public review):

Summary:

The paper demonstrated through a comprehensive multi-omics study of the oviduct that the transcriptomic and proteomic landscape of the oviduct at 4 different preimplantation periods was dynamic during natural fertilization, pseudopregnancy, and superovulation using three independent cell/tissue isolation and analytical techniques. This work is very important for understanding oviductal biology and physiology. In addition, the authors have made all the results available in a web search format, which will maximize the public's access and foster and accelerate research in the field.

Strengths:

(1) The manuscript addresses an important and interesting question in the field of reproduction: how does the oviduct at different regions adapt to the sperm and embryos for facilitating fertilization and preimplantation embryo development and transport?

(2) Authors used cutting-edge techniques: Integrated multi-modal datasets followed by in vivo confirmation and machine learning prediction.

(3) RNA-seq, scRNA-seq, and proteomic results are immediately available to the scientific community in a web search format.

(4) Substantiated results indicate the source of inflammatory responses was the secretory cell population in the IU region when compared to other cell types; sperm modulate inflammatory responses in the oviduct; the oviduct displays immuno-dynamism.

Weaknesses:

(1) The rationale for using the superovulation model is not clear. The oviductal response to sperm and embryos can be studied by comparing mating with normal and vasectomized mice and comparing pregnancy vs pseudopregnancy (induced by mating with vasectomized males). Superovulation causes supraphysiological hormone levels and other confounding conditions.

(2) This study involves a very complex dataset with three different models at four time points. If possible, it would be very informative to generate a graphic abstract/summary of their major findings in oviductal responses in different models and time points

(3) The resolution of Figures 3A-3C in the submitted file was not high enough to assess the authors' conclusion.

(4) The authors need to double-check influential transcription factors identified by machine learning. Apparently, some of them (such as Anxa2, Ift88, Ccdc40) are not transcription factors at all.

Reviewer #2 (Public review):

The manuscript investigates oviductal responses to the presence of gametes and embryos using a multi-omics and machine learning-based approach. By applying RNA sequencing (RNA-seq), single-cell RNA sequencing (sc-RNA-seq), and proteomics, the authors identified distinct molecular signatures in different regions of the oviduct, proximal versus distal. The study revealed that sperm presence triggers an inflammatory response in the proximal oviduct, while embryo presence activates metabolic genes essential for providing nutrients to the developing embryos. Overall, this study offers valuable insights and is likely to be of great interest to reproductive biologists and researchers in the field of oviduct biology. However, further investigation into the impact of sperm on the immune cell population in the oviduct is necessary to strengthen the overall findings.

Reviewer #1 (Public review):

Summary:

This work seeks to provide genetic evidence for a role for beta-adrenergic receptors that regulate heart rate and blood flow on cavernous malformation development using a zebrafish model, and to extend information regarding beta-adrenergic drug blockade in cavernous malformation development, with the idea that these drugs may be useful therapeutically.

Strengths:

The work shows that genetic loss of a specific beta-adrenergic receptor in zebrafish, adrb1, prevents embryonic venous malformations and CCM in adult zebrafish brains. Two drugs, propranolol and metoprolol, also blunt CCM in the adult fish brain. These findings are predicted to potentially impact the treatment of human CCM, and they increase understanding of the factors leading to CCM.

Weaknesses:

There are minor weaknesses that detract slightly from enthusiasm, including poor annotation of the Figure panels and lack of a baseline control for the study of Klf2 expression (Figure 4).

Reviewer #2 (Public review):

Summary:

Previously, the authors developed a zebrafish model for cerebral cavernous malformations (CCMs) via CRISPR/Cas9-based mosaic inactivation of the ccm2 gene. This model yields CCM-like lesions in the caudal venous plexus of 2 days post-fertilization embryos and classical CNS cavernomas in 8-week fish that depend, like the mouse model, on the upregulation of the KLF2 transcription factor. Remarkably, the morpholino-based knockdown of the gene encoding the Beta1 adrenergic receptor or B1AR (adrb1; a hemodynamic regulator) in fish and treatment with the anti-adrenergic S enantiomer of propranolol in both fish and mice reduce the frequency and size of CMM lesions.

In the present study, the authors aim to test the model that adrb1 is required for CCM lesion development using adrb1 mutant fish (rather than morpholino-mediated knockdown and pharmacological treatments with the anti-adrenergic S enantiomer of propranolol or a racemic mix of metoprolol (a selective B1AR antagonist).

Strengths:

The goal of the work is important, and the findings are potentially highly relevant to cardiovascular medicine.

Weaknesses:

(1) The following figures do not report sample sizes, making it difficult to assess the validity of the findings: Figures 1B and D (the number of scored embryos is missing), Figures 2G and 3B (should report both the number of fish and lesions scored, with color-coding to label the lesions corresponding to individual fish in which they where found).

(2) Figure 4 has a few caveats. First, the use of adrb1 morphants (rather than morphants) is at odds with the authors' goal of using genetic validation to test the involvement of adrb1 in CCM2-induced lesion development.

Second, the authors should clarify if they have validated that the tnnt (tnnt2a) morpholino phenocopies tnnt2a mutants in the context in which they are using it (this reviewer found that the tnnt2a morpholino blocks the heartbeat just like the mutant, but induces additional phenotypes not observed in the mutants).

Third, the data in Figure 4E is from just two embryos per treatment, a tiny sample size. Furthermore, judging from the number of points in the graph, only a few endothelial PCV cells appear to have been sampled per embryo. Also, judging from the photos and white arrowheads and arrows (Figure 4A-D), only the cells at the ventral side of the vessel were scored (if so, the rationale behind this choice requires clarification).

Fourth, it is unclear whether and how the Tg(kdrl:mcherry)is5 endothelial reporter was used to mask the signals from the klf2a reporter. The reviewer knows by experience that accuracy suffers if a cytosolic or cell membrane signal is used to mask a nuclear green signal.

Finally, the text and legend related to Figure 4 could be more explicit. What do the authors mean by a mosaic pattern of endothelial nuclear EGFP intensity, and how is that observation reflected in graph 4E? When I look at the graph, I understand that klf2a is decreased in C-D compared to A-B. Are some controls missing? Suppose the point is to show mosaicism of Klf2a levels upon ccm2 CRISPR. Don't you need embryos without ccm2 CRISPR to show that Klf2a levels in those backgrounds have average levels that vary within a defined range and that in the presence of ccm2 mosaicism, some cells have values significantly outside that range? Also, in 4A-D, what are the white arrowheads and arrows? The legend does not mention them.

Given the practical relevance of the findings to cardiovascular medicine, increasing the strength of the evidence would greatly enhance the value of this work.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

No critical weaknesses were noted.

Reviewer #2 (Public review):

Summary:

The manuscript by Ma et. al. utilizes a zebrafish melanoma model, single-cell RNA sequencing (scRNA-seq), a mammalian in vitro co-culture system, and quantitative PCR (Q-PCR) gene expression analysis to investigate the role keratinocytes might play within the melanoma microenvironment. Convincing evidence is presented from scRNA-seq analysis showing that a small cluster of melanoma-associated keratinocytes upregulates the master EMT regulator, transcription factor, Twist1a. To investigate how Twist-expressing keratinocytes might influence melanoma development, the authors use an in vivo zebrafish model to induce melanoma initiation while overexpressing Twist in keratinocytes through somatic transgene expression. This approach reveals that Twist overexpression in keratinocytes suppresses invasive melanoma growth. Using a complementary in vitro human cell line co-culture model, the authors demonstrate reduced migration of melanoma cells into the keratinocyte monolayer when keratinocytes overexpress Twist. Further scRNA-seq analysis of zebrafish melanoma tissues reveals that in the presence of Twist-expressing keratinocytes, subpopulations of melanoma cells show altered gene expression, with one unique melanoma cell cluster appearing more terminally differentiated. Finally, the authors use computational methods to predict putative receptor-ligand pairs that might mediate the interaction between Twist-expressing keratinocytes and melanoma cells.

Strengths:

The scRNA-seq approach reveals a small proportion of keratinocytes undergoing EMT within melanoma tissue. The use of a zebrafish somatic transgenic model to study melanoma initiation and progression provides an opportunity to manipulate host cells within the melanoma microenvironment and evaluate their impact on tumour progression. Solid data demonstrate that Twist-expressing keratinocytes can constrain melanoma invasive development in vivo and reduce melanoma cell migration in vitro, establishing that Twist-overexpressing keratinocytes can suppress at least one aspect of tumour progression.

Weaknesses:

While the scRNA-seq analysis of melanoma tissue and RT-PCR analysis of EMT gene expression in isolated keratinocytes provide evidence that a subpopulation of host keratinocytes upregulates Twist and other EMT marker genes and potentially undergoes EMT, the in vivo evidence for keratinocyte EMT within the melanoma microenvironment is based on cell morphology in a single image without detailed characterization and quantification. No EMT marker gene expression was examined in melanoma tissue sections to determine the proportion and localization of Twist+ve keratinocytes within the melanoma microenvironment.

The scRNA-seq UMAP suggests the proportion of EMT keratinocytes within the melanoma microenvironment is very small, raising questions about their precise location and significance within the tumour microenvironment. Although both in vivo and in vitro evidence demonstrates that Twist-expressing keratinocytes can suppress melanoma progression, the conditions modelled by the authors involve over-expression of Twist in all keratinocytes, which do not naturally occur within the melanoma microenvironment and, therefore, might not be relevant to naturally occurring melanoma progression. The author did not test whether blocking EMT through down-regulation of Twist in keratinocytes may influence melanoma development, which would establish the role of Twist expression keratinocytes in the melanoma microenvironment.

To address the potential mechanism by which Twist-expressing keratinocytes suppress melanoma progression, a second scRNA-seq analysis was conducted. However, this analysis is not adequately presented to provide strong evidence for proposed mechanisms for how Twist-expressing keratinocytes suppress melanoma cell invasion. CellChat analysis was used to attempt to identify receptor-ligand pairs that might mediate keratinocyte-melanoma cell interaction, but the interactions between tumour-associated keratinocytes (TAK) and melanoma cells were not included in the analysis. Furthermore, although genetic reporters were used to label both keratinocytes and melanoma cells, no images showing the detailed distribution and positional information of these cells within melanoma tissue are presented in the report. None of the gene expression changes detected through Q-PCR or scRNA-seq were validated using immunostaining or in situ hybridization.

Overall, the data presented in this report draw attention to a less-studied host cell type within the tumour microenvironment, the keratinocytes, which, similar to well-studied immune cells and fibroblasts, could play important roles in either promoting or constraining melanoma development.

Counterintuitively, the authors show that Twist-expressing EMT keratinocytes can constrain melanoma progression. While the detailed mechanisms remain to be uncovered, this is an interesting observation.

Reviewer #3 (Public review):

Summary:

In this study the authors use the zebrafish model and in vitro co-cultures with human cell lines, to study how keratinocytes modulate the early stages of melanoma development/migration. The authors demonstrate that keratinocytes undergo an EMT-like transformation in the presence of melanoma cells which leads to a reduction in melanoma cell migration. This EMT transformation occurs via Twist; and resulted in an improvement in OS in zebrafish melanoma models. Authors suggest that the limitation of melanoma cell migration by Twist-overexpressing keratinocytes was through altered cell-cell interactions (Jam3b) that caused a physical blockage of melanoma cell migration.

Strengths:

The authors describe a new cross-talk between melanoma and its major initial microenvironment: the keratinocytes and how instructed by melanoma cells keratinocytes undergo an EMT transformation, which then controls melanoma migration.

Overall, the paper is very well written, and the results are clearly organized and presented.

Weaknesses:

(1) To really show their last point it would be important to CRISPR KO Jam3b in melanoma with twist OE keratinocytes, in vivo or in vitro.

(2) The use of patient biopsies from early-stage melanomas vs healthy tissue to assess if there is a similar alteration of morphology of adjacent keratinocytes and an increase in vimentin in human samples would strengthen the author's findings.

(3) The cell-cell junctions and borders between cells (melanoma/ keratinocytes) should be characterized better, with cellular and sub-cellular resolution. Since melanocytes can "touch" with their dendrites ~40 keratinocytes - can authors expand and explain better their model? Can this explain that in some images we cannot observe a direct interface between the cells?

Reviewer #1 (Public review):

In this study, Hama et al. explored the molecular regulatory mechanisms underlying the formation of the ULK1 complex. By employing the AlphaFold structural prediction tool, they showed notable differences in the complex formation mechanisms between ULK1 in mammalian cells and Atg1 in yeast cells. Their findings revealed that in mammalian cells, ULK1, ATG13, and FIP200 form a complex with a stoichiometry of 1:1:2. These predicted interaction regions were validated through both in vivo and in vitro assays, enhancing our understanding of the molecular mechanisms governing ULK1 complex formation in mammalian cells. Importantly, they identified a direct interaction between ULK1 and FIP200, which is crucial for autophagy. However, some aspects of this manuscript require further clarification, validation, and correction by the authors.

Reviewer #2 (Public review):

Summary:

This is important work that helps to uncover how the process of autophagy is initiated - via structural analyses of the initiating ULK1 complex. High-resolution structural details and a mechanistic insight of this complex have been lacking and understanding how it assembles and functions is a major goal of a field that impacts many aspects of cell and disease biology. While we know components of the ULK1 complex are essential for autophagy, how they physically interact is far from clear. The work presented makes use of AlphaFold2 to structurally predict interaction sites between the different subunits of the ULK1 complex (namely ULK1, ATG13, and FIP200). Importantly, the authors go on to experimentally validate that these predicted sites are critical for complex formation by using site-directed mutagenesis and then go on to show that the three-way interaction between these components is necessary to induce autophagy in cells.

Strengths:

The data are very clear. Each binding interface of ATG13 (ATG13 with FIP300/ATG13 with ULK1) is confirmed biochemically with ITC and IP experiments from cells. Likewise, IP experiments with ULK1 and FIP200 also validate interaction domains. A real strength of the work in in their analyses of the consequences of disrupting ATG13's interactions in cells. The authors make CRISPR KI mutations of the binding interface point mutants. This is not a trivial task and is the best approach as everything is monitored under endogenous conditions. Using these cells the authors show that ATG13's ability to interact with both ULK1 and FIP200 is essential for a full autophagy response.

Weaknesses:

I think a main weakness here is the failure to acknowledge and compare results with an earlier preprint that shows essentially the same thing (https://doi.org/10.1101/2023.06.01.543278). Arguably this earlier work is much stronger from a structural point of view as it relies not only on AlphaFold2 but also actual experimental structural determinations (and takes the mechanisms of autophagy activation further by providing evidence for a super complex between the ULK1 and VPS34 complexes). That is not to say that this work is not important, as in the least it independently helps to build a consensus for ULK1 complex structure. Another weakness is that the downstream "functional" consequences of disrupting the ULK1 complex are only minimally addressed. The authors perform a Halotag-LC3 autophagy assay, which essentially monitors the endpoint of the process. There are a lot of steps in between, knowledge of which could help with mechanistic understanding. Not in the least is the kinase activity of ULK1 - how is this altered by disrupting its interactions with ATG13 and/or FIP200?

Reviewer #3 (Public review):

In this study, the authors employed the protein complex structure prediction tool AlphaFold-Multimer to obtain a predicted structure of the protein complex composed of ULK1-ATG13-FIP200 and validated the structure using mutational analysis. This complex plays a central role in the initiation of autophagy in mammals. Previous attempts at resolving its structure have failed to obtain high-resolution structures that can reveal atomic details of the interactions within the complex. The results obtained in this study reveal extensive binary interactions between ULK1 and ATG13, between ULK1 and FIP200, and between ATG13 and FIP200, and pinpoint the critical residues at each interaction interface. Mutating these critical residues led to the loss of binary interactions. Interestingly, the authors showed that the ATG13-ULK1 interaction and the ATG13-FIP200 interaction are partially redundant for maintaining the complex.

The experimental data presented by the authors are of high quality and convincing. However, given the core importance of the AlphaFold-Multimer prediction for this study, I recommend the authors improve the presentation and documentation related to the prediction, including the following:

(1) I suggest the authors consider depositing the predicted structure to a database (e.g. ModelArchive) so that it can be accessed by the readers.

(2) I suggest the authors provide more details on the prediction, including explaining why they chose to use the 1:1:2 stoichiometry for ULK1-ATG13-FIP200 and whether they have tried other stoichiometries, and explaining why they chose to use the specific fragments of the three proteins and whether they have used other fragments.

(3) I suggest the authors present the PAE plot generated by AlphaFold-Multimer in Figure S1. The PAE plot provides valuable information on the prediction.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteasomal function in an ATP-independent manner. Although there are multiple works that have highlighted the role of this protein in tumours, this study specifically tried to develop a correlation with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.

Strengths:

The observations of the authors hint that beyond PA28y's association with the proteasome, it might also stabilize certain proteins such as C1QBP which influences energy metabolism.

Weaknesses:

The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.<br /> In most of the experiments, the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex that is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in a more convincing manner.

In all of the assays, C1QBP has been detected as doublet. However, the expression pattern of the two bands varies depending on the experiment. In some cases, the upper band is intensely stained and in some the lower bands. Do C1QBP isoforms exist and are they differentially regulated depending on experiment conditions/tissue types?

Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of works of literature that have dealt with the metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond the estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, the significance of this study and how it will extend our understanding of OSCC needs to be elaborated.

Reviewer #2 (Public review):

Summary:

The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.

Strengths:

They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria, the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.

Weaknesses:

Many data sets are shown in figures that cannot be understood without more descriptions, either in the text or the legend, e.g., Figure 1A. Similarly, many abbreviations are not defined.

Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Figure 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Figure 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Figure 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.

The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation and be analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.

To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and also show whether a mutation that disrupts the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Figure 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Figure 1G is a quantification of Western blot data that should be shown.

The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Figure 1I, more Flag-C1QBP 1-167 was pulled down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?

The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect

Reviewer #1 (Public review):

Summary:

The authors investigate the role of HSPA2 during mouse preimplantation development. Knocking down HSPA2 in zygotes, the authors describe lower chances of developing into blastocysts, which show a reduced number of inner cell mass cells. They find that HSPA2 mRNA and protein levels show some heterogeneity among blastomeres at the 4-cell stage and propose that HSPA2 could contribute to skewing their relative contribution to embryonic lineages. To test this, the authors try to reduce HSPA2 expression in one of the 2-cell stage blastomere and propose that it biases their contribution to towards extra-embryonic lineages. To explain this, the authors propose that HSPA2 would interact with CARM1, which controls chromatin accessibility around genes regulating differentiation into embryonic lineage.

Strengths:

(1) The study offers simple and straightforward experiments with large sample sizes.

(2) Unlike most studies in the field, this research often relies on both mRNA and protein levels to analyse gene expression and differentiation.

Weaknesses:

(1) Image and statistical analyses are not well described.

(2) The functionality of the overexpression construct is not validated.

(3) Tracking of KD cells in embryos injected at the 2-cell stage with GFP is unclear.

(4) A key rationale of the study relies on measuring small differences in the levels of mRNA and proteins using semi-quantitative methods to compare blastomeres. As such, it is not possible to know whether those subtle differences are biologically meaningful. For example, the lowest HSPA2 level of the embryo with the highest level is much higher than the top cell from the embryo with the lowest level. What does this level mean then? Does this mean that some blastomeres grafted from strong embryos would systematically outcompete all other blastomeres from weaker embryos? That would be very surprising. I think the authors should be more careful and consider the lack of quantitative power of their approach before reaching firm conclusions. Although to be fair, the authors only follow a long trend of studies with the same intrinsic flaw of this approach.

(5) Some of the analyses on immunostaining do not take into account that this technique only allows for semi-quantitative measurements and comparisons.<br /> a) Some of the microscopy images are shown with an incorrect look-up table.<br /> b) Some of the schematics are incorrect and misleading.

Reviewer #2 (Public review):

Summary:

In this study, Gao et al. use RNA-seq to identify Hspa2 as one of the earliest transcripts heterogeneously distributed between blastomeres. Functional studies are performed using siRNA knockdown showing Hspa2 may bias cells toward the ICM lineage via interaction with the known methyltransferase CARM1.

Strengths:

This study tackles an important question regarding the origins of the first cell fate decision in the preimplantation embryo. It provides novelty in its identification of Hspa2 as a heterogeneous transcript in the early embryo and proposes a plausible mechanism showing interactions with Carm1. Multiple approaches are used to validate their functional studies (FISH, WB, development rates, proteomics). Given only 4 other transcripts/RNA have been identified at or before the 4-cell stage (LincGET, CARM1, PRDM14, HMGA1), this would be an important addition to our understanding of how TE vs ICM fate is established.

Weaknesses:

The RNA-seq results leading the authors to focus on Hspa2 are not included in the manuscript. This dataset would serve as an important resource but is neither included nor discussed. Nor is it mentioned whether Hspa2 was identified in prior RNA-seq embryos studies (for example Deng Science 2014).

In addition, the functional studies are centered on Hspa2 knockdown at the zygote (1-cell) stage, which would largely target maternal transcript. Given the proposed mechanism relies on Hspa2 heterogeneity post-ZGA (late 2-cell stage), the knockdown studies don't necessarily test this and thus don't provide direct support to the authors' conclusions. The relevance of the study would be improved if the authors could show that zygotic knockdown leads to symmetric Hspa2 levels at the late 2-cell and/or 4-cell stage. It may be possible that zygotic knockdown leads to lower global Hspa2 levels, but that asymmetry is still generated at the 4-cell stage.

Furthermore, the authors show that Hspa2 knockdown at the 1-cell stage lowers total Carm1 levels at the 4-cell stage. However, it is unclear how total abundance within the embryo alters lineage specification within blastomeres. The authors go on to propose a plausible mechanism involving Hspa2 and Carm1 interaction, but do not discuss how expression levels may be involved.

Reviewer #1 (Public review):

Summary:

This is a comprehensive study that clearly and deeply investigates the function of GATA6 in human early cardiac development.

Strengths:

This study combines hESC engineering, differentiation, detailed gene expression, genome occupancy, and pathway modulation to elucidate the role of GATA6 in early cardiac differentiation. The work is carefully executed and the results support the conclusions. The use of publicly available data is well integrated throughout the manuscript. The RIME experiments are excellent.

Weaknesses:

Much has been known about GATA6 in mesendoderm development, and this is acknowledged by the authors.

Reviewer #2 (Public review):

Summary:

This manuscript by Bisson et al describes the role of GATA6 to regulate cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation using human embryonic stem cells (hESCs). The authors found that GATA6 loss-of-function hESC exhibits early defects in mesendoderm and lateral mesoderm patterning stages. Using RNA-seq and CUT&RUN assays the genes of the Wnt and BMP programs were found to be affected by the loss of GATA6 expression. Modulating Wnt and BMP during early cardiac differentiation can partially rescue CPC and CM defects in GATA6 hetero- and homozygous mutant hESCs.

Strengths:

The studies performed were rigorous and the rationale for the experimental design was logical. The results obtained were clear and supported the conclusions that the authors made regarding the role of GATA6 on Wnt and BMP pathway gene expression.

Weaknesses:

Given the wealth of studies that have been performed in this research area previously, the amount of new information provided in this study is relatively modest. Nevertheless, the results and quite clear and should make a strong contribution to the field.

Reviewer #3 (Public review):

In this study, Bison et al. analyzed the role of the GATA6 transcription factor in patterning the early mesoderm and generating cardiomyocytes, using human embryonic stem cell differentiation assays and patient-derived hiPSCs with heart defects associated with mutations in the GATA6 gene. They identified a novel role for GATA6 in regulating genes involved in the WNT and BMP pathways -findings not previously noted in earlier analyses of GATA6 mutant hiPSCs during early cardiac mesoderm specification (Sharma et al., 2020). Modulation of the WNT and BMP pathways may partially rescue early cardiac mesoderm defects in GATA6 mutant hESCs. These results provide significant insights into how GATA6 loss-of-function and heterozygous mutations contribute to heart defects.

I have the following comments:

(1) Throughout the manuscript, Bison et al. alternate between different protocols to generate cardiomyocytes, which creates some confusion (e.g., Figure 1 vs. Supplemental Figure 2A). The authors should provide a clear justification for using alternative protocols.

(2) The authors should characterise the mesodermal identity and cardiomyocyte subtypes generated with the activin/BMP-induction protocol thoroughly and clarify whether defects in the expression of BMP and WNT-related gene affect the formation of specific cardiomyocyte subtypes in a chamber-specific manner. This analysis is important, as Sharma et al. suggested a role for GATA6 in orchestrating outflow tract formation, and Bison et al. similarly identified decreased expression of NRP1, a gene involved in outflow tract septation, in their GATA6 mutant cells.

(3) The authors developed an iPSC line derived from a congenital heart disease (CHD) patient with an atrial septal defect and observed that these cells generate cTnnT+ cells less efficiently. However, it remains unclear whether atrial cardiomyocytes (or those localised specifically at the septum) are being generated using the activin/BMP-induction protocol and the patient-derived iPSC line.

(4) The authors should also justify the necessity of using the patient-derived line to further analyse GATA6 function.

(5) Figure 3 suggests an enrichment of paraxial mesoderm genes in the context of GATA6 loss-of-function, which is intriguing given the well-established role of GATA6 in specifying cardiac versus pharyngeal mesoderm lineages in model organisms. Could the authors expand their analysis beyond GO term enrichment to explore which alternative fates GATA6 mutant cells may acquire? Additionally, how does the potential enrichment of paraxial mesoderm, rather than pharyngeal mesoderm, relate to the initial mesodermal induction from their differentiation protocol? Could the authors also rule out the possibility of increased neuronal cell fates?

Reviewer #1 (Public Review):

In the current manuscript, the authors use theoretical and analytical tools to examine the possibility of neural projections to engage ensembles of synaptic clusters in active dendrites. The analysis is divided into multiple models that differ in the connectivity parameters, speed of interactions, and identity of the signal (electric vs. second messenger). They first show that random connectivity almost ensures the representation of presynaptic ensembles. As expected, this convergence is much more likely for small group sizes and slow processes, such as calcium dynamics. Conversely, fast signals (spikes and postsynaptic potentials) and large groups are much less likely to recruit spatially clustered inputs. Dendritic nonlinearity in the postsynaptic cells was found to play a highly important role in distinguishing these clustered activation patterns, both when activated simultaneously and in sequence. The authors tackled the difficult issue of noise, showing a beneficiary effect when noise 'happens' to fill in gaps in a sequential pattern but degraded performance at higher background activity levels. Last, the authors simulated selectivity to chemical and electrical signals. While they find that longer sequences are less perturbed by noise, in more realistic activation conditions, the signals are not well resolved in the soma.

While I think the premise of the manuscript is worth exploring, I have a number of reservations regarding the results.

(1) In the analysis, the authors made a simplifying assumption that the chemical and electrical processes are independent. However, this is not the case; excitatory inputs to spines often trigger depolarization combined with pronounced calcium influx; this mixed signaling could have dramatic implications on the analysis, particularly if the dendrites are nonlinear (see below)

(2) Sequence detection in active dendrites is often simplified to investigating activation in a part of or the entirety of individual branches. However, the authors did not do that for most of their analysis. Instead, they treat the entire dendritic tree as one long branch and count how many inputs form clusters. I fail to see why simplification is required and suspect it can lead to wrong results. For example, two inputs that are mapped to different dendrites in the 'original' morphology but then happen to fall next to each other when the branches are staggered to form the long dendrites would be counted as neighbors.

(3) The simulations were poorly executed. Figures 5 and 6 show examples but no summary statistics. The authors emphasize the importance of nonlinear dendritic interactions, but they do not include them in their analysis of the ectopic signals! I find it to be wholly expected that the effects of dendritic ensembles are not pronounced when the dendrites are linear.

To provide a comprehensive analysis of dendritic integration, the authors could simulate more realistic synaptic conductances and voltage-gated channels. They would find much more complicated interactions between inputs on a single site, a sliding temporal and spatial window of nonlinear integration that depends on dendritic morphology, active and passive parameters, and synaptic properties. At different activation levels, the rules of synaptic integration shift to cooperativity between different dendrites and cellular compartments, further complicated by nonlinear interactions between somatic spikes and dendritic events.

While it is tempting to extend back-of-the-napkin calculations of how many inputs can recruit nonlinear integration in active dendrites, the biological implementation is very different from this hypothetical. It is important to consider these questions, but I am not convinced that this manuscript adequately addressed the questions it set out to probe, nor does it provide information that was unknown beforehand.

Reviewer #2 (Public Review):

Summary:

If synaptic input is functionally clustered on dendrites, nonlinear integration could increase the computational power of neural networks. But this requires the right synapses to be located in the right places. This paper aims to address the question of whether such synaptic arrangements could arise by chance (i.e. without special rules for axon guidance or structural plasticity), and could therefore be exploited even in randomly connected networks. This is important, particularly for the dendrites and biological computation communities, where there is a pressing need to integrate decades of work at the single-neuron level with contemporary ideas about network function.

Using an abstract model where ensembles of neurons project randomly to a postsynaptic population, back-of-envelope calculations are presented that predict the probability of finding clustered synapses and spatiotemporal sequences. Using data-constrained parameters, the authors conclude that clustering and sequences are indeed likely to occur by chance (for large enough ensembles), but require strong dendritic nonlinearities and low background noise to be useful.

Strengths:

(1) The back-of-envelope reasoning presented can provide fast and valuable intuition. The authors have also made the effort to connect the model parameters with measured values. Even an approximate understanding of cluster probability can direct theory and experiments towards promising directions, or away from lost causes.

(2) I found the general approach to be refreshingly transparent and objective. Assumptions are stated clearly about the model and statistics of different circuits. Along with some positive results, many of the computed cluster probabilities are vanishingly small, and noise is found to be quite detrimental in several cases. This is important to know, and I was happy to see the authors take a balanced look at conditions that help/hinder clustering, rather than to just focus on a particular regime that works.

(3) This paper is also a timely reminder that synaptic clusters and sequences can exist on multiple spatial and temporal scales. The authors present results pertaining to the standard `electrical' regime (~50-100 µm, <50 ms), as well as two modes of chemical signaling (~10 µm, 100-1000 ms). The senior author is indeed an authority on the latter, and the simulations in Figure 5, extending those from Bhalla (2017), are unique in this area. In my view, the role of chemical signaling in neural computation is understudied theoretically, but research will be increasingly important as experimental technologies continue to develop.

Weaknesses:

(1) The paper is mostly let down by the presentation. In the current form, some patience is needed to grasp the main questions and results, and it is hard to keep track of the many abbreviations and definitions. A paper like this can be impactful, but the writing needs to be crisp, and the logic of the derivation accessible to non-experts. See, for instance, Stepanyants, Hof & Chklovskii (2002) for a relevant example.

It would be good to see a restructure that communicates the main points clearly and concisely, perhaps leaving other observations to an optional appendix. For the interested but time-pressed reader, I recommend starting with the last paragraph of the introduction, working through the main derivation on page 7, and writing out the full expression with key parameters exposed. Next, look at Table 1 and Figure 2J to see where different circuits and mechanisms fit in this scheme. Beyond this, the sequence derivation on page 15 and biophysical simulations in Figures 5 and 6 are also highlights.

(2) I wonder if the authors are being overly conservative at times. The result highlighted in the abstract is that 10/100000 postsynaptic neurons are expected to exhibit synaptic clustering. This seems like a very small number, especially if circuits are to rely on such a mechanism. However, this figure assumes the convergence of 3-5 distinct ensembles. Convergence of inputs from just 2 ensembles would be much more prevalent, but still advantageous computationally. There has been excitement in the field about experiments showing the clustering of synapses encoding even a single feature.

(3) The analysis supporting the claim that strong nonlinearities are needed for cluster/sequence detection is unconvincing. In the analysis, different synapse distributions on a single long dendrite are convolved with a sigmoid function and then the sum is taken to reflect the somatic response. In reality, dendritic nonlinearities influence the soma in a complex and dynamic manner. It may be that the abstract approach the authors use captures some of this, but it needs to be validated with simulations to be trusted (in line with previous work, e.g. Poirazi, Brannon & Mel, (2003)).

(4) It is unclear whether some of the conclusions would hold in the presence of learning. In the signal-to-noise analysis, all synaptic strengths are assumed equal. But if synapses involved in salient clusters or sequences were potentiated, presumably detection would become easier? Similarly, if presynaptic tuning and/or timing were reorganized through learning, the conditions for synaptic arrangements to be useful could be relaxed. Answering these questions is beyond the scope of the study, but there is a caveat there nonetheless.

Reviewer #1 (Public review):

Summary

In this manuscript, Day et al. present a high-throughput version of expansion microscopy to increase the throughput of this well-established super-resolution imaging technique. Through technical innovations in liquid handling with custom-fabricated tools and modifications to how the expandable hydrogels are polymerized, the authors show robust ~4-fold expansion of cultured cells in 96-well plates. They go on to show that HiExM can be used for applications such as drug screens by testing the effect of doxorubicin on human cardiomyocytes. Interestingly, the effects of this drug on changing DNA organization were only detectable by ExM, demonstrating the utility of HiExM for such studies.

Overall, this is a very well-written manuscript presenting an important technical advance that overcomes a major limitation of ExM - throughput. As a method, HiExM appears extremely useful and the data generally support the conclusions.

Strengths

Hi-ExM overcomes a major limitation of ExM by increasing the throughput and reducing the need for manual handling of gels. The authors do an excellent job of explaining each variation introduced to HiExM to make this work and thoroughly characterize the impressive expansion isotropy. The dox experiments are generally well-controlled and the comparison to an alternative stressor (H2O2) significantly strengthens the conclusions.

Weaknesses

(1) It is still unclear to me whether or not cells that do not expand remain in the well given the response to point 1. The authors say the cells are digested and washed away but then say that there is a remaining signal from the unexpanded DNA in some cases. I believe this is still a concern that potential users of the protocol should be aware of.

(2) Regarding the response to point 9, I think this information should be included in the manuscript, possibly in the methods. It is important for others to have a sense of how long imaging may take if they were to adopt this method.

Reviewer #2 (Public review):

Summary:

In the present work, the authors present an engineering solution to sample preparation in 96-well plates for high-throughput super resolution microscopy via Expansion Microscopy. This is not a trivial problem, as the well cannot be filled with the gel, which would prohibit expansion of the gel. They thus engineered a device that can spot a small droplet of hydrogel solution and keep it in place as it polymerises. It occupies only a small portion space at the center of each well, the gel can expand into all directions and imaging and staining can proceed by liquid handling robots and an automated microscope.

Strengths:

In contrast to Reference 8, the authors system is compatible with standard 96 well imaging plates for high-throughput automated microscopy and automated liquid handling for most parts of the protocol. They thus provide a clear path towards high throughput exM and high throughout super resolution microscopy, which is a timely and important goal.

Addition upon revision:

The authors addressed this reviewer's suggestions.

Reviewer #3 (Public review):

Summary:

Day et al. introduced high-throughput expansion microscopy (HiExM), a method facilitating the simultaneous adaptation of expansion microscopy for cells cultured in a 96-well plate format. The distinctive features of this method include: 1) the use of a specialized device for delivering a minimal amount (~230 nL) of gel solution to each well of a conventional 96-well plate, and 2) the application of the photochemical initiator, Irgacure 2959, to successfully form and expand toroidal gel within each well.

Addition upon revision:

Overall, the authors have adequately addressed most of the concerns raised. There are a few minor issues that require attention.

Minor comments:

Figure S10: There appears to be a discrepancy in the panel labeling. The current labels are E-H, but it is unclear whether panels A-D exist. Also, this reviewer thought that panels G and H would benefit from statistical testing to strengthen the conclusions. As a general rule for scientific graph presentation, the y-axis of all graphs should start at zero unless there is a compelling reason not to do so.

Joint Public Review:

TRPML1 functions as a lysosomal ion channel whose variants are associated with lysosomal storage disorder mucolipidosis type IV. Understanding the structure and function of sites involved in the allosteric control TRPML1 may provide new molecular moieties to target with prototypic drugs.

Gan et al provide the first high resolution cryo-EM structure of a mutant (Y404W) TRPML1 channel in the open state without any activating ligands. This new structure demonstrates how a mutation at a site some distance away from the pore can influence channel gating. The authors provide compelling electrophysiology evidence which supports the proposed Y404W gain of function effect.

The authors propose an allosteric mechanism whereby the engineered W404 sidechain provides extra van der Waals contacts within a pocket surrounded by helices of the voltage sensor-like domain (VSLD) and causes S4 bending which in turn opens the pore through the S4-S5 linker. Conversely, the authors functionally demonstrate that an alanine mutation at this site causes a loss of function. Although the authors do not provide a structure of the Y404A mutant, they propose that the alanine substitution disrupts the sidechain packing and likely destabilizes the open conformation.

TRPML1 channels are regulated by PIP2 species in the cell. In the lysosomal membrane, PI(3,5)P2 activates the channel, whereas in the plasma membrane PI(4,5)P2 inhibits it. Towards understanding its lipid regulation, the authors solve a cryo-EM structure of TRPML1 bound to PI(4,5)P2 in the closed state and provide functional evidence that PI(4,5)P2 occupancy inhibits TRPML1 currents.

Within this same structure, the authors observe a density which may be attributed to sphingomyelin (or possibly phosphocholine). Using electrophysiology on WT and Y404W channels, the authors report an antagonist effect of sphingomyelin on TRPML1 currents.

Taken together, the study provides convincing evidence for a gating (opening/closing) mechanism of the TRPML1 pore which can be allosterically regulated by altered side-chain packing and by lipid interactions within the VSLD.

Reviewer #1 (Public Review):

Summary:

This manuscript by Meissner and colleagues described a novel take on a classic social cognition paradigm developed for marmosets. The classic pull task is a powerful paradigm that has been used for many years across numerous species, but its analog approach has several key limitations. As such, it has not been feasible to adopt the task for neuroscience experiments. Here the authors capture the spirit of the classic task but provide several fundamental innovations that modernize the paradigm - technically and conceptually. By developing the paradigm for marmosets, the authors leverage the many advantages of this primate model for studies of social brain functions and their particular amenability to freely-moving naturalistic approaches.

Strengths:

The current manuscript describes one of the most exciting paradigms in primate social cognition to be developed in many years. By allowing for freely-moving marmosets to engage in high numbers of trials, while precisely quantifying their visual behavior (e.g. gaze) and recording neural activity this paradigm has the potential to usher in a new wave of research on the cognitive and neural mechanisms underlying primate social cognition and decision-making. This paradigm is an elegant illustration of how naturalistic questions can be adapted to more rigorous experimental paradigms. Overall, I thought the manuscript was well written and provided sufficient details for others to adopt this paradigm.

Reviewer #2 (Public Review):

Summary:

This important work by Meisner et al., developed an automated apparatus (MarmoAPP) to collect a wide array of behavioral data (lever pulling, gaze direction, vocalizations) in marmoset monkeys, with the goal of modernizing collection of behavioral data to coincide with the investigation of neurological mechanisms governing behavioral decision making in an important primate neuroscience model. The authors show a variety of "proof-of-principle" concepts that this apparatus can collect a wide range of behavioral data, with higher behavioral resolution than traditional methods. For example, the authors highlight that typical behavioral experiments on primate cooperation provide around 10 trials per session, while using their approach the authors were able to collect over 100 trials per 20-minute session with the MarmoAAP.

Overall the authors argue that this approach has a few notable advantages:

(1) It enhances behavioral output which is important for measuring small or nuanced effects/changes in behavior;

(2) Allows for more advanced analyses given the higher number of trials per session;

(3) Significantly reduces the human labor of manually coding behavioral outcomes and experimenter interventions such as reloading apparatuses for food or position;

(4) Allows for more flexibility and experimental rigor in measuring behavior and neural activity simultaneously.

Strengths:

The paper is well-written and the MarmoAPP appears to be highly successful at integrating behavioral data across many important contexts (cooperation, gaze, vocalizations), with the ability to measure significantly many more behavioral contexts (many of which the authors make suggestions for).

The authors provide substantive information about the design of the apparatus, how the apparatus can be obtained via a long list of information Apparatus parts and information, and provide data outcomes from a wide number of behavioral and neurological outcomes. The significance of the findings is important for the field of social neuroscience and the strength of evidence is solid in terms of the ability of the apparatus to perform as described, at least in marmoset monkeys. The advantage of collecting neural and freely-behaving behavioral data concurrently is a significant advantage.

Reviewer #3 (Public Review):

Summary:

The authors set out to devise a system for the neural and behavioral study of socially cooperative behaviors in nonhuman primates (common marmosets). They describe instrumentation to allow for a "cooperative pulling" paradigm, the training process, and how both behavioral and neural data can be collected and analyzed. This is a valuable approach to an important topic, as the marmoset stands as a great platform to study primate social cognition. Given that the goals of such a methods paper are to (a) describe the approach and instrumentation, (b) show the feasibility of use, and (c) quantitatively compare to related approaches, the work is easily able to meet those criteria. My specific feedback on both strengths and weaknesses is therefore relatively limited in scope and depth.

Strengths:

The device is well-described, and the authors should be commended for their efforts in both designing this system but also in "writing it up" so that others can benefit from their R&D.

The device appears to generate more repetitions of key behavior than other approaches used in prior work (with other species).

The device allows for quantitative control and adjustment to control behaviour.

The approach also supports the integration of markerless behavioral analysis as well as neurophysiological data.

Reviewer #1 (Public review):

Summary:

Understanding large-scale neural activity remains a formidable challenge in neuroscience. While several methods have been proposed to discover the assemblies from such large-scale recordings, most of previous studies do not explicit modeling the temporal dynamics. This study is an attempt to uncover the temporal dynamics of assemblies using a tool that have been establish in other domains.

The authors previously introduced the compositional Restricted Boltzmann Machine (cRBM) to identify neuron assemblies in zebrafish brain activity. Building upon this, they now employ the Recurrent Temporal Restricted Boltzmann Machine (RTRBM) to elucidate the temporal dynamics within these assemblies. By introducing recurrent connections between hidden units, RTRBM could retrieve neural assemblies and their temporal dynamics from simulated and zebrafish brain data.

Strengths:

The RTRBM has been previously used in other domains. Training the model has been already established. This study is an application of such model to neuroscience. Overall, the paper is well-structured and the methodology is robust, the analysis is solid to support the authors claim.

Weaknesses:

The overall degree of advance is very limited. The performance improvement by RTRBM compared to their cRBM is marginal, and insights into assembly dynamics are limited.

(1) The biological insights from this method are constrained. Though the aim is to unravel neural ensemble dynamics, the paper lacks in-depth discussion on how this method enhances our understanding of zebrafish neural dynamics. For example, the dynamics of assemblies can be analyzed using various tools such as dimensionality reduction methods once we have identified them using cRBM. What information can we gain by knowing the effective recurrent connection between them? It would be more convincing to show this in real data.

(2) Including predicted and measured neural activity traces could aid readers in evaluating model efficacy. The current version only contains comparison of the statistics, such as mean and covariance.

Reviewer #2 (Public review):

Summary:

In this work, the authors propose an extension to some of the last author's previous work, where a compositional restricted Boltzmann machine was considered as a generative model of neuron-assembly interaction. They augment this model by recurrent connections between the Boltzmann machine's hidden units, which allow them to explicitly account for temporal dynamics of the assembly activity. Since their model formulation does not allow the training towards a compositional phase (as in the previous model), they employ a transfer learning approach according to which they initialise their model with a weight matrix that was pre-trained using the earlier model so as to essentially start the actually training in a compositional phase. Finally, they test this model on synthetic and actual data of whole-brain light-sheet-microscopy recordings of spontaneous activity from the brain of larval zebrafish.

Strengths:

This work introduces a new model for neural assembly activity. Importantly, being able to capture temporal assembly dynamics is an interesting feature that goes beyond many existing models. While this work clearly focuses on the method (or the model) itself, it opens up an avenue for experimental research where it will be interesting to see if one can obtain any biologically meaningful insights considering these temporal dynamics when one is able to, for instance, relate them to development or behaviour.

Weaknesses:

For most of the work, the authors present their RTRBM model as an improvement over the earlier cRBM model. Yet, when considering synthetic data, they actually seem to compare with a "standard" RBM model. This seems odd considering the overall narrative and that when considering whole-brain zebrafish data, the comparisons were made between RTRBM and cRBM models. For that, the RTRBM model was initialised with the cRBM weight matrix to overcome the fact that RTRBM alone does not seem to converge to a compositional phase, so to cite the latter as reason does not really make sense.

Furthermore, whether the clusters shown in Figure 3E can indeed be described as "spatially localized" is debatable. Especially in view of clusters 3 and 4, this seems a stretch. If receptive fields are described as "spatially localized", arguably, one would expect that they are contained in some small (compared to the overall size of the brain) or specific anatomical brain region. However, this is clearly not the case here.

In addition, the performance comparison for the temporal dynamics of the hidden units actually suggests that the RTRBM (significantly) underperforms where the text says (Line 235f) it outperforms the cRBM model.

Reviewer #1 (Public Review):

Summary:

This study provides valuable insights into the therapeutic effects of two parathyroid hormone (PTH) analogs on bone regeneration and osseointegration. The research is methodologically sound, employing a robust animal model and a comprehensive array of analytical techniques, including micro-CT, histological/histomorphometric analyses, and serum biochemical analysis.

Strengths:

The use of a large animal model, which closely mimics postmenopausal osteoporosis in humans, enhances the study's relevance to clinical applications. The study is well-structured, with clear objectives, detailed methods, and a logical flow from introduction to conclusion. The findings are significant, demonstrating the potential of rhPTH(1-34) and dimeric R25CPTH(1-34) in enhancing bone regeneration, particularly in the context of osteoporosis.

Weaknesses:

There are no major weaknesses.

Reviewer #2 (Public Review):

Summary:

This article explores the regenerative effects of recombinant PTH analogues on osteogenesis.

Strengths:

Although PTH has known to induce the activity of osteoclasts, accelerating bone resorption, paradoxically its intermittent use has become a common treat for osteoporosis. Previous studies successfully demonstrated this phenomenon in vivo, but most of them used rodent animal models, inevitably having a limitation. In this article, the authors tried to address this, using a beagle model, and assessed the osseointegrative effect of recombinant PTH analogues. As a result, the authors clearly observed the regenerative effects of PTH analogues, and compared the efficacy, using histologic, biochemical, and radiologic measurement for surgical-endocrinal combined large animal models. The data seem to be solid, and has potential clinical implications.

Weaknesses:

All the issues that I raised have been resolved in the revision process.

Overall, this paper is well-written and has clarity and consistency for a broader readership.

Reviewer #3 (Public Review):

Summary:

The work submitted by Dr. Jeong-Oh Shin and co-workers aims to investigate the therapeutic efficacy of rhPTH(1-34) and R25CPTH(1-34) on bone regeneration and osseointegration of titanium implants using a postmenopausal osteoporosis animal model.

In my opinion the findings presented are not strongly supported by the provided data since the methods utilized do not allow to significantly support the primary claims.

Strengths:

Strengths include certain good technologies utilized to perform histological sections (i.e. the EXAKT system).

Weaknesses:

Certain weaknesses continue to significantly lower the enthusiasm for this work. Most important: the limited number of samples/group. In fact, as presented, the work has an n=4 for each treatment group. This limited number of samples/group significantly impairs the statistical power of the study. In addition, the implants were surgically inserted following a "conventional implant surgery", implying that no precise/guided insertion was utilized. This weakness is, in my opinion, particularly significant since the amount of bone osteointegration may greatly depend on the bucco-lingual positioning of each implant at the time of the surgical insertion (which should, therefore, be precisely standardized across all animals and for all surgical procedures).

Reviewer #1 (Public review):

Summary:

This paper presents a mechanistic study of rDNA origin regulation in yeast by SIR2. Each of the ~180 tandemly repeated rDNA gene copies contains a potential replication origin. Early-efficient initiation of these origins is suppressed by Sir2, reducing competition with origins distributed throughout the genome for rate-limiting initiation factors. Previous studies by these authors showed that SIR2 deletion advances replication timing of rDNA origins by a complex mechanism of transcriptional de-repression of a local PolII promoter causing licensed origin proteins (MCMcomplexes) to re-localize (slide along the DNA) to a different (and altered) chromatin environment. In this study, they identify a chromatin remodeler, FUN30, that suppresses the sir2∆ effect, and remarkably, results in a contraction of the rDNA to about one-quarter it's normal length/number of repeats, implicating replication defects of the rDNA. Through examination of replication timing, MCM occupancy and nucleosome occupancy on the chromatin in sir2, fun30, and double mutants, they propose a model where nucleosome position relative to the licensed origin (MCM complexes) intrinsically determines origin timing/efficiency. While their interpretations of the data are largely reasonable and can be interpreted to support their model, a key weakness is the connection between Mcm ChEC signal disappearance and origin firing. While the cyclical chromatin association-dissociation of MCM proteins with potential origin sequences may be generally interpreted as licensing followed by firing, dissociation may also result from passive replication and as shown here, displacement by transcription and/or chromatin remodeling. Moreover, linking its disappearance from chromatin in the ChEC method with such precise resolution needs to be validated against an independent method to determine the initiation site(s). Differences in rDNA copy number and relative transcription levels also are not directly accounted for, obscuring a clearer interpretation of the results. Nevertheless, this paper makes a valuable advance with the finding of Fun30 involvement, which substantially reduces rDNA repeat number in sir2∆ background. The model they develop is compelling and I am inclined to agree, but I think the evidence on this specific point is purely correlative and a better method is needed to address the initiation site question. The authors deserve credit for their efforts to elucidate our obscure understanding of the intricacies of chromatin regulation. At a minimum, I suggest their conclusions on these points of concern should be softened and caveats discussed. Statistical analysis is lacking for some claims.

Strengths are the identification of FUN30 as suppressor, examination of specific mutants of FUN30 to distinguish likely functional involvement. Use of multiple methods to analyze replication and protein occupancies on chromatin. Development of a coherent model.

Weaknesses are failure to address copy number as a variable; insufficient validation of ChEC method relationship to exact initiation locus; lack of statistical analysis in some cases.

Review of revised version and response letter:

In the response, the authors make some improvements by better quantifying 2D gels, adding some missing statistical analyses, analyzing the effect of fun30 on rDNA replication in strains with reduced rDNA copy number, and using ChIP-seq of MCMs to support the ChEC-seq data. However, these additions do not address the main issue that is at the heart of their model: where initiation precisely occurs and whether the location is altered in the mutant(s). Thus, mechanistic insight is limited.

Under the section "Addressing Alternative Explanations", the authors claim that processes like transcription and passive replication cannot affect the displaced complex specifically. Why? They are not on same DNA (as mentioned in the Fig 1 legend).

The model in Fig 7 implies that initiation sites are different in WT versus the mutants and this determines their timing/efficiency. But they also suggest that the same site might be used with different efficiencies in this response. I agree that both are possibilities and are not resolved.

Supporting their model requires better resolution to determine the actual replication initiation site. While this may be challenging, it should be feasible with methods to map nascent strands like DNAscent, or Okazaki fragment mapping.

The 2D gel analysis of strains with reduced rDNA copy numbers adequately addresses the copy number variable with regard to the replication effect.

Overall, the paper is improved by providing additional data and improved analysis. The paper nicely characterizes the effect of Fun30. The model is reasonable but remains lacking in precise details of mechanism.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors follow up on their previous work showing that in the absence of the Sir2 deacetylase the MCM replicative helicase at the rDNA spacer region is repositioned to a region of low nucleosome occupancy. Here they show that the repositioned displaced MCMs have increased firing propensity relative to non-displaced MCMs. In addition, they show that activation of the repositioned MCMs and low nucleosome occupancy in the adjacent region depend on the chromatin remodeling activity of Fun30.

Strengths:

The paper provides new information on the role of a conserved chromatin remodeling protein in regulation of origin firing and in addition provides evidence that not all loaded MCMs fire and that origin firing is regulated at a step downstream of MCM loading.

Weaknesses:

The relationship between the authors results and prior work on the role of Sir2 (and Fob1) in regulation of rDNA recombination and copy number maintenance is not explored, making it difficult to place the results in a broader context. Sir2 has previously been shown to be recruited by Fob1, which is also required for DSB formation and recombination-mediated changes in rDNA copy number. Are the changes that the authors observe specifically in fun30 sir2 cells related to this pathway? Is Fob1 required for the reduced rDNA copy number in fun30 sir2 double mutant cells?

Reviewer #3 (Public review):

Summary:

Heterochromatin is characterized by low transcription activity and late replication timing, both dependent on the NAD-dependent protein deacetylase Sir2, the founding member of the sirtuins. This manuscript addresses the mechanism by which Sir2 delays replication timing at the rDNA in budding yeast. Previous work from the same laboratory (Foss et al. PLoS Genetics 15, e1008138) showed that Sir2 represses transcription-dependent displacement of the Mcm helicase in the rDNA. In this manuscript, the authors show convincingly that the repositioned Mcms fire earlier and that this early firing partly depends on the ATPase activity of the nucleosome remodeler Fun30. Using read-depth analysis of sorted G1/S cells, fun30 was the only chromatin remodeler mutant that somewhat delayed replication timing in sir2 mutants, while nhp10, chd1, isw1, htl1, swr1, isw2, and irc5 had no effect. The conclusion was corroborated with orthogonal assays including two-dimensional gel electrophoresis and analysis of EdU incorporation at early origins. Using an insightful analysis with an Mcm-MNase fusion (Mcm-ChEC), the authors show that the repositioned Mcms in sir2 mutants fire earlier than the Mcm at the normal position in wild type. This early firing at the repositioned Mcms is partially suppressed by Fun30. In addition, the authors show Fun30 affects nucleosome occupancy at the sites of the repositioned Mcm, providing a plausible mechanism for the effect of Fun30 on Mcm firing at that position. However, the results from the MNAse-seq and ChEC-seq assays are not fully congruent for the fun30 single mutant. Overall, the results support the conclusions providing a much better mechanistic understanding how Sir2 affects replication timing at rDNA,

Strengths

(1) The data clearly show that the repositioned Mcm helicase fires earlier than the Mcm in the wild type position.<br /> (2) The study identifies a specific role for Fun30 in replication timing and an effect on nucleosome occupancy around the newly positioned Mcm helicase in sir2 cells.

Weaknesses

(1) It is unclear which strains were used in each experiment.<br /> (2) The relevance of the fun30 phospho-site mutant (S20AS28A) is unclear.<br /> (3) For some experiments (Figs. 3, 4, 6) it is unclear whether the data are reproducible and the differences significant. Information about the number of independent experiments and quantitation is lacking. This affects the interpretation, as fun30 seems to affect the +3 nucleosome much more than let on in the description.

Reviewer #1 (Public review):

Summary:

In this work, Qiu and colleagues examined the effects of preovulatory (i.e., proestrous or late follicular phase) levels of circulating estradiol on multiple calcium and potassium channel conductances in arcuate nucleus kisspeptin neurons. Although these cells are strongly linked to a role as the "GnRH pulse generator," the goal here was to examine the physiological properties of these cells in a hormonal milieu mimicking late proestrus, the time of the preovulatory GnRH-LH surge. Computational modeling is used to manipulate multiple conductances simultaneously and support a role for certain calcium channels in facilitating a switch in firing mode from tonic to bursting. CRISPR knockdown of the TRPC5 channel reduced overall excitability, but this was only examined in cells from ovariectomized mice without estradiol treatment.

Comments to address most recent author response:

The concern regarding the CRISPR experiments being confined to OVX mice is that the results can only suggest that CRISPR-mediated knockdown of TRPC5 can, at best, phenocopy the OVX+E condition. A reciprocal experiment in the opposite direction (for example, that returning TRPC5 to OVX levels in OVX+E mice prevents the changes in firing activity and pattern typical of the OVX+E2 condition) would strengthen the indication that E2-sensitive changes in TRPC5 expression and function are critically important to surge function. Acknowledging this as a limitation of the studies would help to better contextualize the value of the CRISPR experiments to an understanding of surge mechanisms when done only in OVX conditions.

The nature of the confusion regarding the consideration of OVX+E2 conditions in the computational model primarily arises from the methods description in the supplemental file: "The effect of E2 on ionic currents is modelled as a change in the maximum conductance parameter. For currents IM,IT, ICa and ITRPC5 this change is inferred from the qPCR data assuming that the conductance is directly proportional to the mRNA expression." If these were instead based on the whole-cell recordings as the authors now indicate in their response, then this description needs to be edited and clarified accordingly. Furthermore, the section states, "For ISK, IBK, Ileak, the OVX and OVX+E2 conductances are obtained from current-voltage relationships recorded from Kiss1ARH neurons in the absence/presence of iberiotoxin (BK blocker) and apamin (SK blocker). All other currents were assumed to be unaffected by E2." This section thus does not directly indicate that the recordings in the stated figures were used in the model, and moreover suggests that currents besides ISK, IBK, and Ileak were not different in OVX+E2 conditions.

The prior evidence stated for correlation of mRNA and channel conductance is not explicitly cited in the manuscript. It is well known that post-translational modifications, physiological modulation of individual channel biophysical properties, and many other factors can influence the end output of a membrane conductance. Therefore, the authors should, at minimum, provide a literature citation supporting the assumption used here.

Reviewer #2 (Public review):

Summary:

Kisspeptin neurons of the arcuate nucleus (ARC) are thought to be responsible for the pulsatile GnRH secretory pattern and to mediate feedback regulation of GnRH secretion by estradiol (E2). Evidence in the literature, including the work of the authors, indicates that ARC kisspeptin coordinate their activity through reciprocal synaptic interactions and the release of glutamate and of neuropeptide neurokinin B (NKB), which they co-express. The authors show here that E2 regulates the expression of genes encoding different voltage-dependent calcium channels, calcium-dependent potassium channels and canonical transient receptor potential (TRPC5) channels and of the corresponding ionic currents in ARC kisspeptin neurons. Using computer simulations of the electrical activity of ARC kisspeptin neurons, the authors also provide evidence of what these changes translate into in terms of these cells' firing patterns. The experiments reveal that E2 upregulates various voltage-gated calcium currents as well as 2 subtypes of calcium-dependent potassium currents, while decreasing TRPC5 expression (an ion channel downstream of NKB receptor activation), the slow excitatory synaptic potentials (slow EPSP) elicited in ARC kisspeptin neurons by NKB release and expression of the G protein-associated inward-rectifying potassium channel (GIRK). Based on these results, and on those of computer simulations, the authors propose that E2 promotes a functional transition of ARC kisspeptin neurons from neuropeptide-mediated sustained firing that supports coordinated activity for pulsatile GnRH secretion to a less intense burst-like firing pattern that could favor glutamate release from ARC kisspeptin. The authors suggest that the latter might be important for the generation of the preovulatory surge in females.

Strengths:

The authors combined multiple approaches in vitro and in silico to gain insights into the impact of E2 on the electrical activity of ARC kisspeptin neurons. These include patch-clamp electrophysiology combined with selective optogenetic stimulation of ARC kisspeptin neurons, reverse transcriptase quantitative PCR, pharmacology and CRISPR-Cas9-mediated knockdown of the Trpc5 gene. The addition of computer simulations for understanding the impact of E2 on the electrical activity of ARC kisspeptin cells is also a strength.

The authors add interesting information on the complement of ionic currents in ARC kisspeptin neurons and on their regulation by E2 to what was already known in the literature. Pharmacological and electrophysiological experiments appear of the highest standards and robust statistical analyses are provided throughout. The impact of E2 replacement on calcium and potassium currents is compelling. Likewise, the results of Trpc5 gene knockdown do provide good evidence that the TRPC5 channel plays a key role in mediating the NKB-mediated slow EPSP. Surprisingly, this also revealed an unsuspected role for this channel in regulating the membrane potential and excitability of ARC kisspeptin neurons.

Weaknesses:

The manuscript also has weaknesses that obscure some of the conclusions drawn by the authors.

One is that the authors compare here two conditions, OVX versus OVX replaced with high E2, that may not reflect the physiological conditions under which the proposed transition between neuropeptide-dependent sustained firing and less intense burst firing might take place (i.e. the diestrous [low E2] and proestrous [high E2] stages of the estrous cycle). This is an important caveat to keep in mind when interpreting the authors' findings. Indeed, that E2 alters certain ionic currents when added back to OVX females, does not mean that the magnitude of all of these ionic currents will vary during the estrous cycle.<br /> In addition, although the computational modeling indicates a role of the various E2-modulated conductances in causing a transition in ARC kisspeptin neuron firing pattern, their role is not directly tested in physiological recordings, weakening the link between these changes and the shift in firing patterns.

Overall, the manuscript provides interesting information about the effects of E2 on specific ionic currents in ARC kisspeptin neurons and some insights into the functional impact of these changes. However, some of the conclusions of the work, with regard, in particular, to the role of these changes in ion channels and to their implications for the LH surge, are not fully supported by the findings.

Reviewer #1 (Public review):

This manuscript describes soluble Uric Acid (sUA) as an endogenous inhibitor of CD38, affecting CD38 activity and NAD+ levels both in vitro and in vivo. Importantly, the inhibition constants calculated supports the claim that sUA inhibits CD38 under physiological conditions. These findings are of extreme importance to understand the regulation of an enzyme that has been shown to be the main NAD+/NMN-degrading enzyme in mammals, which impacts several metabolic processes and has major implications to understanding aging diseases. The manuscript is well written, the figures are self explanatory, and in the experiments presented, the data is very solid. The authors discuss the main limitations of the study, especially in regard to the in vivo results. As a whole, I believe that this is a very interesting manuscript that will be appreciated by the scientific community and that opens a lot of new questions in the field of metabolism and aging.

During the revision process, the authors have performed new experiments to respond to relevant questions raised by the reviewers. In other cases, they have made changes in the text to improve the manuscript.

I believe that this manuscript in its current form is a mature and relevant set of findings that deserve attention and future developments.

Reviewer #2 (Public review):

Summary:

This is an interesting work where Wen et al. aimed to shed light on the mechanisms driving the protective role of soluble uric acid (sUA) toward avoiding excessive inflammation. They present biochemical data to support that sUA inhibits the enzymatic activity of CD38 (Figures 1 and 2). In a mouse model of acute response to sUA and using mice deficient in CD38, they find evidence that sUA increases the plasma levels of nicotinamide nucleotides (NAD+ and NMN) (Figure 3) and that sUA reduces the plasma levels of inflammasome-driven cytokines IL-1b and IL-18 in response to endotoxin, both dependent on CD38 (Figure 4). Their work is an important advance in the understanding of the physiological role of sUA, with mechanistic insight that can have important clinical implications.

Strengths:

The authors present evidence from different approaches to support that sUA inhibits CD38, impacts NAD+ levels, and regulates inflammatory responses through CD38.

Weaknesses:

The authors investigate macrophages as the cells affected by sUA in promoting immunoregulation, proposing that sUA's inhibition of CD38 and the resulting increase in NAD+ promotes inflammasome inhibition through a previously established mechanism of NLRP3 regulation by NAD+-dependent sirtuins. However, they were unable to validate their in vivo findings using murine bone marrow-derived macrophages, a standard model for assessing inflammasome activation, due to the low uptake of sUA in these cells. Pharmacological blockage in THP-1 cells provides mechanistic evidence that sUA inhibits NLRP3-mediated secretion of IL-1β through CD38, but genetic evidence and direct assessment of the activation of inflammasome components would be necessary to fully validate the model.

Reviewer #3 (Public review):

Summary:

In the present manuscript, the authors propose that soluble Uric acid (sUA) is an enzymatic inhibitor of the NADase CD38 and that it controls levels of NAD modulating inflammatory response. Although interesting the studies are at this stage preliminary and validation is needed.

Strengths:

The study characterizes the potential relevance of sUA in NAD metabolism.

Comment on revised version:

The authors have responded the majority of my criticism.

Reviewer #1 (Public review):

Summary:

In previous work, the authors described necrosis-induced apoptosis (NiA) as a consequence of induced necrosis. Specifically, experimentally induced necrosis in the distal pouch of larval wing imaginal discs triggers NiA in the lateral pouch. In this manuscript, the authors confirmed this observation and found that while necrosis can kill all areas of the disc, NiA is limited to the pouch and to some extent to the notum, but is excluded from the hinge region. Interestingly and unexpectedly, signaling by the Jak/Stat and Wg pathways inhibits NiA. Further characterization of NiA by the authors reveals that NiA also triggers regenerative proliferation which can last up to 64 hours following necrosis induction. This regenerative response to necrosis is significantly stronger compared to discs ablated by apoptosis. Furthermore, the regenerative proliferation induced by necrosis is dependent on the apoptotic pathway because RNAi targeting the RHG genes is sufficient to block proliferation. However, NiA does not promote proliferation through the previously described apoptosis-induced proliferation (AiP) pathway, although cells at the wound edge undergo AiP. Further examination of the caspase levels in NiA cells allowed the authors to group these cells into two clusters: some cells (NiA) undergo apoptosis and are removed, while others referred to as Necrosis-induced Caspase Positive (NiCP) cells survive despite caspase activity. It is the NiCP cells that repair cellular damage including DNA damage and that promote regenerative proliferation. Caspase sensors demonstrate that both groups of cells have initiator caspase activity, while only the NiA cells contain effector caspase activity. Under certain conditions, the authors were also able to visualize effector caspase activity in NiCP cells, but the level was low, likely below the threshold for apoptosis. Finally, the authors found that loss of the initiator caspase Dronc blocks regenerative proliferation, while inhibiting effector caspases by expression of p35 does not, suggesting that Dronc can induce regenerative proliferation following necrosis in a non-apoptotic manner. This last finding is very interesting as it implies that Dronc can induce proliferation in at least two ways in addition to its requirement in AiP.

Strengths:

This is a very interesting manuscript. The authors demonstrate that epithelial tissue that contains a significant number of necrotic cells is able to regenerate. This regenerative response is dependent on the apoptotic pathway which is induced at a distance from the necrotic cells. Although regenerative proliferation following necrosis requires the initiator caspase Dronc, Dronc does not induce a classical AiP response for this type of regenerative response. In future work, it will be very interesting to dissect this regenerative response pathway genetically.

Weaknesses:<br /> No weaknesses were identified.

Reviewer #2 (Public review):

Summary / Strengths:

In this manuscript, Klemm et al., build on past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors aim to investigate how tissues regenerate following necrotic cell death. They find that:<br /> (1) the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),<br /> (2) two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may explain the region specificity,<br /> (3) the presence of NiA cells promotes regenerative proliferation in late stages of regeneration,<br /> (4) not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells,<br /> (5) the caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration.

Weaknesses:

However, here are some of my current concerns for the manuscript in its current version:

(1) The presence of cells with activated caspase that don't die (NiCP cells) is an interesting biological phenomenon but is not described until Figure 5. How does the existence of NiCP cells impact the earlier findings presented? Is late proliferation due to NiA, NiCP, or both? Does Wg and JAK/STAT signaling act to prevent the formation of both NiA and NiCP cells or only NiA cells? Moreover, the authors are able to specifically manipulate the wound edge (WE) and lateral pouch cells (LP), but don't show how these manipulations within these distinct populations impact regeneration. The authors provide evidence that driving UAS-mir(RHG) throughout the pouch, in the LP or the WE all decrease the amount of NiA/NiCP in Figure 3G-O, but no data on final regenerative outcomes for these manipulations is presented (such as those presented for Dronc-/+ in Fig 7M). The manuscript would be greatly enhanced by quantification of more of the findings, especially in describing if the specific manipulations that impacted NiA /NiCP cells disrupt end-point regeneration phenotypes.

(2) How fast does apoptosis take within the wing disc epithelium? How many of the caspase(+) cells are present for the whole 48 hours of regeneration? Are new cells also induced to activate caspase during this time window? The author presented a number of interesting experiments characterizing the NiCP cells. For the caspase sensor GC3Ai experiments in Figure 5, is there a way to differentiate between cells that have maintained fluorescent CG3Ai from cells that have newly activated caspase? What is the timeline for when NiA and NiCP are specified? In addition, what fraction of NiCP cells contribute to the regenerated epithelium? Additional information about the temporal dynamics of NiA and NiCP specification/commitment would be greatly appreciated.

(3) The notum also does not express developmental JAK/STAT, yet little NiA was observed within the notum. Do the authors have any additional insights into the differential response between the pouch and notum? What makes the pouch unique? Are NiA/NiCP cells created within other imaginal discs and other tissues? Are they similarly important for regenerative responses in other contexts?

Reviewer #3 (Public review):

The manuscript "Regeneration following tissue necrosis is mediated by non-apoptotic caspase activity" by Klemm et al. is an exploration of what happens to a group of cells that experience caspase activation after necrosis occurs some distance away from the cells of interest. These experiments have been conducted in the Drosophila wing imaginal disc, which has been used extensively to study the response of a developing epithelium to damage and stress. The authors revise and refine their earlier discovery of apoptosis initiated by necrosis, here showing that many of those presumed apoptotic cells do not complete apoptosis. Thus, the most interesting aspect of the paper is the characterization of a group of cells that experience mild caspase activation in response to an unknown signal, followed by some effector caspase activation and DNA damage, but that then recover from the DNA damage, avoid apoptosis, and proliferate instead. Many questions remain unanswered, including the signal that stimulates the mild caspase activation, and the mechanism through which this activation stimulates enhanced proliferation.

The authors should consider answering additional questions, clarifying some points, and making some minor corrections:

Major concerns affecting the interpretation of experimental results:

Expression of STAT92E RNAi had no apparent effect on the ability of hinge cells to undergo NiA, leading the authors to conclude that other protective signals must exist. However, the authors have not shown that this STAT92E RNAi is capable of eliminating JAK/STAT signaling in the hinge under these experimental conditions. Using a reporter for JAK/STAT signaling, such as the STAT-GFP, as a readout would confirm the reduction or elimination of signaling. This confirmation would be necessary to support the negative result as presented.

Similarly, the authors should confirm that the Zfh2 RNAi is reducing or eliminating Zfh2 levels in the hinge under these experimental conditions, before concluding that Zfh2 does not play a role in stopping hinge cells from undergoing NiA.

EdU incorporation was quantified by measuring the fluorescence intensity of the pouch and normalizing it to the fluorescence intensity of the whole disc. However, the images show that EdU fluorescence intensity of other regions of the disc, especially the notum, varied substantially when comparing the different genetic backgrounds (for example, note the substantially reduced EdU in the notum of Figure 3 B' and B'). Indeed, it has been shown that tissue damage can lead to suppression of proliferation in the notum and elsewhere in the disc, unless the signaling that induces the suppression is altered. Therefore, the normalization may be skewing the results because the notum EdU is not consistent across samples, possibly because the damage-induced suppression of proliferation in the notum is different across the different genetic backgrounds.

The authors expressed p35 to attempt to generate "undead cells". They take an absence of mitogen secretion or increased proliferation as evidence that undead cells were not generated. However, there could be undead cells that do not stimulate proliferation non-autonomously, which could be detected by the persistence of caspase activity in cells that do not complete apoptosis. Indeed, expressing p35 and observing sustained effector caspase activation could help answer the later question of what percentage of this cell population would otherwise complete apoptosis (NiA, rescued by p35) vs reverse course and proliferate (NiCP, unaffected by p35).

It is unclear if the authors' model is that the NiCP cells lead to autonomous or non-autonomous cell proliferation, or both. Could the lineage-tracing experiments and/or the experiments marking mitosis relative to caspase activity answer this question?

Many of the conclusions rely on single images. Quantification of many samples should be included wherever possible.

Why does the reduction of Dronc appear to affect regenerative growth in females but not males?

Reviewer #1 (Public review):

Summary:

This work develops a simple, rapid, low-cost methodology for assembling combinatorially complete microbial consortia using basic laboratory equipment. The motivation behind this work is to make the study of microbial community interactions more accessible to laboratories that lack specialized equipment such as robotic liquid handlers or microfluidic devices. The method was tested on a library of Pseudomonas aeruginosa strains to demonstrate its practicality and effectiveness. It provided a means to explore the complex functional interactions within microbial communities and identify optimal consortia for specific functions, such as biomass production.

Strengths:

The primary strength of this manuscript lies in its accessibility and practicality. The method proposed by the authors allows any laboratory with standard equipment, such as multichannel pipettes and 96-well plates, to readily construct all possible combinations of microbial consortia from a given set of species. This greatly enhances access to full factorial designs, which were previously limited to labs with advanced technology.

Another strength of the manuscript is the measurement and analysis of the biomass of all possible combinations of 8 strains of P. aeruginosa. This analysis provides a concrete example of how the authors' new methodology can be used to identify the best-performing communities and map pairwise and higher-order functional interactions.

Notably, the authors do exceptionally well in providing a thorough description of the methodology, including detailed protocols and an R script for customizing the method to different experimental needs. This enhances the reproducibility and adaptability of the methodology, making it a valuable resource for researchers wishing to adopt this methodology.

Weaknesses:

While the methodology is robust and well-presented, there are some limitations that should be acknowledged more thoroughly. First, the method's scalability is an important factor. The authors indicate that it should be effective for up to 10-12 species, but there is no discussion of what sets this scale: time, amount of labor, consumables, the likelihood of error, sample volume, etc. Second, this methodology is tailored to construct communities where the abundance of each strain is identical in each combination. Therefore, combinations with a different number of strains also differ in the total initial amount of microbial cells. Second, variations in the initial proportions of the same set of strains cannot be readily explored. Third, the manuscript only discusses how to construct the combinations, and not how to assay them afterward (e.g. for community function, interspecific interactions, etc'). While details on how to achieve these goals are clearly outside the scope of this work, the use of biomass as an example function may obfuscate this caveat, which should be stated more explicitly.

Reviewer #2 (Public review):

Summary:

A simple and effective method for combinatorial assembly of microbes in synthetic communities of <12 species.

Strengths:

Overall this manuscript is a useful contribution. The efficiency of the method and clarity of the presentation is a strength. It is well-written and easy to follow. The figures are great, the pedagogical narrative is crisp. I can imagine the method being used in lots of other contexts too.

Weaknesses:

The authors could better clarify what HOIs mean. They could address challenges with assaying community function. However, neither of these "weaknesses" affects the primary goal of the paper which is methodological.

Reviewer #3 (Public review):

The authors developed a useful methodology for generating all combinations of multiple reagents using standard lab equipment. This methodology has clear uses for studying microbial ecology as they demonstrated. The methodology will likely be useful for other types of experiments that require exhaustive testing of all possible combinations of a given set of reagents (e.g., drug-drug antagonism and synergy).

The authors provided a useful R script that generates a detailed experimental protocol for building the desired combination from any number of reagents. The produced document is useful and has clear instructions. The output of the computer script will be strengthened if graphical output is also provided (similar to the one provided in Figure 1C).

The authors show that the error rate of the method doesn't go up with the number of combinations using dyes (Figure 2).

The authors demonstrate the value of their methodology for studying interactions within microbial consortia by assembling all possible combinations of eight strains of Pseudomonas aeruginosa. The value of their methodology for this application is well-founded. However, it is also unclear why specific experimental choices were made for this application. It is unclear why authors continue to show the absorbance measurements of strain assemblies over the entire wavelength spectrum and not just for ABS 600 nm (Figures 3 and 4). It is also unclear why the authors provided information on the "sum of the three spectra" as this reference line is meaningless and not a reasonable null model for estimating how well specific strain combinations will grow together.

Figure 5 illustrates the various analysis types that can be performed on the data collected from growing combinations of eight Pseudomonas aeruginosa strains. It is a very informative figure since it provides a "roadmap" on the various ways in which the dataset produced can be explored. The information in Figures 5 and S6 will likely be very useful for a wide audience.

Reviewer #1 (Public review):

Summary:

Odor- and taste-sensing are mediated by two different systems, the olfactory and gustatory systems, and have different behavioral roles. In this study, Wei et al. challenge this dichotomy by showing that odors can activate gustatory receptor neurons (GRNs) in Drosophila to promote feeding responses, including the proboscis extension response (PER) that was previously thought to be driven only by taste. While previous studies suggested that odors can promote PER to appetitive tastants, Wei et al. go further to show that odors alone cause PER, this effect is mediated through sweet-sensing GRNs, and sugar receptors are required. The study also shows that odor detection by bitter-sensing GRNs suppresses PER. The authors' conclusions are supported by behavioral assays, calcium imaging, electrophysiological recordings, and genetic manipulations. The observation that both attractive and aversive odors promote PER leaves an open question as to why this effect is adaptive. Overall, the study sheds new light on chemosensation and multimodal integration by showing that odor and taste detection converge at the level of sensory neurons, a finding that is interesting and surprising while also being supported by another recent study (Dweck & Carlson, Sci Advances 2023).

Strengths:

(1) The main finding that odors alone can promote PER by activating sweet-sensing GRNs is interesting and novel.

(2) The study uses video tracking of the proboscis to quantify PER rather than manual scoring, which is typically used in the field. The tracking method is less subjective and provides a higher-resolution readout of the behavior.

(3) The study uses calcium imaging and electrophysiology to show that odors activate GRNs. These represent complementary techniques that measure activity at different parts of the GRN (axons versus dendrites, respectively) and strengthen the evidence for this conclusion.

(4) Genetic manipulations show that odor-evoked PER is primarily driven by sugar GRNs and sugar receptors rather than olfactory neurons. This is a major finding that distinguishes this work from previous studies of odor effects on PER and feeding (e.g., Reisenman & Scott, 2019; Shiraiwa, 2008) that assumed or demonstrated that odors were acting through olfactory neurons.

Weaknesses/Limitations:

1) The authors may want to discuss why PER to odors alone has not been previously reported, especially as they argue that this is a broad effect evoked by many different odors. Previous studies testing the effect of odors on PER only observed odor enhancement of PER to sugar (Oh et al., 2021; Reisenman & Scott, 2019; Shiraiwa, 2008) and some of these studies explicitly show no effect of odor alone or odor with low sugar concentration; regardless, the authors likely would have noticed if PER to odor alone had occurred. Readers of this paper may also be aware of unpublished studies failing to observe an effect of PER on odor alone (including studies performed by this reviewer and unrelated work by other colleagues in the field), which of course the authors are not expected to directly address but may further motivate the authors to provide possible explanations.

(2) Many of the odor effects on behavior or neuronal responses were only observed at very high concentrations. Most effects seemed to require concentrations of at least 10-2 (0.01 v/v), which is at the high end of the concentration range used in olfactory studies (e.g., Hallem et al., 2004), and most experiments in the paper used a far higher concentration of 0.5 v/v. It is unclear whether these are concentrations that would be naturally encountered by flies.

(3) The calcium imaging data showing that sugar GRNs respond to a broad set of odors contrasts with results from Dweck & Carlson (Sci Adv, 2023) who recorded sugar neurons with electrophysiology and observed responses to organic acids, but not other odors. This discrepancy is not discussed.

(4) Related to point #1, it would be useful to see a quantification of the percent of flies or trials showing PER for the key experiments in the paper, as this is the standard metric used in most studies and would help readers compare PER in this study to other studies. This is especially important for cases where the authors are claiming that odor-evoked PER is modulated in the same way as previously shown for sugar (e.g., the effect of starvation in Figure S4).

(5) Given the novelty of the finding that odors activate sugar GRNs, it would be useful to show more examples of GCaMP traces (or overlaid traces for all flies/trials) in Figure 3. Only one example trace is shown, and the boxplots do not give us a sense of the reliability or time course of the response. A related issue is that the GRNs appear to be persistently activated long after the odor is removed, which does not occur with tastes. Why should that occur? Does the time course of GRN activation align with the time course of PER, and do different odors show differences in the latency of GRN activation that correspond with differences in the latency of PER (Figure S1A)?

(6) Several controls are missing, and in some cases, experimental and control groups are not directly compared. In general, Gal4/UAS experiments should include comparisons to both the Gal4/+ and UAS/+ controls, at least in cases where control responses vary substantially, which appears to be the case for this study. These controls are often missing, e.g. the Gal4/+ controls are not shown in Figure 2C-G and the UAS/+ controls are not shown in Figure 2J-L (also, the legend for the latter panels should be revised to clarify what the "control" flies are). For the experiments in Figure S5, the data are not directly compared to any control group. For several other experiments, the control and experimental groups are plotted in separate graphs (e.g., Figure 2C-G), and they would be easier to visually compare if they were together. In addition, for each experiment, the authors should denote which comparisons are statistically significant rather than just reporting an overall p-value in the legend (e.g., Figure 2H-L).

(7) Additional controls would be useful in supporting the conclusions. For the Kir experiments, how do we know that Kir is effective, especially in cases where odor-evoked PER was not impaired (e.g., Orco/Kir)? The authors could perform controls testing odor aversion, for example. For the Gr5a mutant, few details are provided on the nature of the control line used and whether it is in the same genetic background as the mutant. Regardless, it would be important to verify that the Gr5a mutant retains a normal sense of smell and shows normal levels of PER to stimuli other than sugar, ruling out more general deficits. Finally, as the method of using DeepLabCut tracking to quantify PER was newly developed, it is important to show the accuracy and specificity of detecting PER events compared to manual scoring.

(8) The authors' explanation of why both attractive and aversive odors promote PER (lines 249-259) did not seem convincing. The explanation discusses the different roles of smell and taste but does not address the core question of why it would be adaptive for an aversive odor, which flies naturally avoid, to promote feeding behavior.

Reviewer #2 (Public review):

Summary:

A gustatory receptor and neuron enhances an olfactory behavioral response, proboscis extension.

This manuscript clearly establishes a novel mechanism by which a gustatory receptor and neuron evokes an olfactory-driven behavioral response. The study expands recent observations by Dweck and Carlson (2023) that suggest new and remarkable properties among GRNs in Drosophila. Here, the authors articulate a clear instance of a novel neural and behavioral mechanism for gustatory receptors in an olfactory response.

Strengths:

The systematic and logical use of genetic manipulation, imaging and physiology, and behavioral analysis makes a clear case that gustatory neurons are bona fide olfactory neurons with respect to proboscis extension behavior.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #3 (Public review):

Summary:

Using flies, Kazama et al. combined behavioral analysis, electrophysiological recordings, and calcium imaging experiments to elucidate how odors activate gustatory receptor neurons (GRNs) and elicit a proboscis extension response, which is interpreted as a feeding response.

The authors used DeepLabCut v2.0 to estimate the extension of the proboscis, which represents an unbiased and more precise method for describing this behavior compared to manual scoring.

They demonstrated that the probability of eliciting a proboscis extension increases with higher odor concentrations. The most robust response occurs at a 0.5 v/v concentration, which, despite being diluted in the air stream, remains a relatively high concentration. Although the probability of response is not particularly high it is higher than control stimuli. Notably, flies respond with a proboscis extension to both odors that are considered positive and those regarded as negative.

The authors used various transgenic lines to show that the response is mediated by GRNs. Specifically, inhibiting Gr5a reduces the response, while inhibiting Gr66a increases it in fed flies. Additionally, they find that odors induce a strong positive response in both types of GRNs, which is abolished when the labella of the proboscis are covered. This response was also confirmed through electrophysiological tip recordings.

Finally, the authors demonstrated that the response increases when two stimuli of different modalities, such as sucrose and odors, are presented together, suggesting clear multimodal integration.

Strengths:

The integration of various techniques, that collectively support the robustness of the results.

The assessment of electrophysiological recordings in intact animals, preserving natural physiological conditions.

Weaknesses:

The behavioral response is observed in only a small proportion of animals.

Reviewer #1 (Public review):

Summary:

Here the authors address how reinforcement-based sensorimotor adaptation changes throughout development. To address this question, they collected many participants in ages that ranged from small children (3 years old) to adulthood (18+ years old). The authors used four experiments to manipulate whether binary and positive reinforcement was provided probabilistically (e.g., 30 or 50%) versus deterministically (e.g.,100%), and continuous (infinite possible locations) versus discrete (binned possible locations) when the probability of reinforcement varied along the span of a large redundant target. The authors found that both movement variability and the extent of adaptation changed with age.

Strengths:

The major strength of the paper is the number of participants collected (n = 385). The authors also answer their primary question, that reinforcement-based sensorimotor adaptation changes throughout development, which was shown by utilizing established experimental designs and computational modelling.

Weaknesses:

Potential concerns involve inconsistent findings with secondary analyses, current assumptions that impact both interpretation and computational modelling, and a lack of clearly stated hypotheses.

(1) Multiple regression and Mediation Analyses.

The challenge with these secondary analyses is that:<br /> (a) The results are inconsistent between Experiments 1 and 2, and the analysis was not performed for Experiments 3 and 4,<br /> (b) The authors used a two-stage procedure of using multiple regression to determine what variables to use for the mediation analysis, and<br /> (c) The authors already have a trial-by-trial model that is arguably more insightful.

Given this, some suggested changes are to:<br /> (a) Perform the mediation analysis with all the possible variables (i.e., not informed by multiple regression) to see if the results are consistent.<br /> (b) Move the regression/mediation analysis to Supplementary, since it is slightly distracting given current inconsistencies and that the trial-by-trial model is arguably more insightful.

(2) Variability for different phases and model assumptions:

A nice feature of the experimental design is the use of success and failure clamps. These clamped phases, along with baseline, are useful because they can provide insights into the partitioning of motor and exploratory noise. Based on the assumptions of the model, the success clamp would only reflect variability due to motor noise (excludes variability due to exploratory noise and any variability due to updates in reach aim). Thus, it is reasonable to expect that the success clamps would have lower variability than the failure clamps (which it obviously does in Figure 6), and presumably baseline (which provides success and failure feedback, thus would contain motor noise and likely some exploratory noise).

However, in Figure 6, one visually observes greater variability during the success clamp (where it is assumed variability only comes from motor noise) compared to baseline (where variability would come from:<br /> (a) Motor noise.<br /> (b) Likely some exploratory noise since there were some failures.<br /> (c) Updates in reach aim.

Given the comment above, can the authors please:<br /> (a) Statistically compare movement variability between the baseline, success clamp, and failure clamp phases.<br /> (b) The authors have examined how their model predicts variability during success clamps and failure clamps, but can they also please show predictions for baseline (similar to that of Cashaback et al., 2019; Supplementary B, which alternatively used a no feedback baseline)?<br /> (c) Can the authors show whether participants updated their aim towards their last successful reach during the success clamp? This would be a particularly insightful analysis of model assumptions.<br /> (d) Different sources of movement variability have been proposed in the literature, as have different related models. One possibility is that the nervous system has knowledge of 'planned (noise)' movement variability that is always present, irrespective of success (van Beers, R. J. (2009). Motor learning is optimally tuned to the properties of motor noise. Neuron, 63(3), 406-417). The authors have used slightly different variations of their model in the past. Roth et al (2023) directly compared several different plausible models with various combinations of motor, planned, and exploratory noise (Roth A, 2023, "Reinforcement-based processes actively regulate motor exploration along redundant solution manifolds." Proceedings of the Royal Society B 290: 20231475: see Supplemental). Their best-fit model seems similar to the one the authors propose here, but the current paper has the added benefit of the success and failure clamps to tease the different potential models apart. In light of the results of a), b), and c), the authors are encouraged to provide a paragraph on how their model relates to the various sources of movement variability and other models proposed in the literature.<br /> (e) line 155. Why would the success clamp be composed of both motor and exploratory noise? Please clarify in the text

(3) Hypotheses:

The introduction did not have any hypotheses of development and reinforcement, despite the discussion above setting up potential hypotheses. Did the authors have any hypotheses related to why they might expect age to change motor noise, exploratory noise, and learning rates? If so, what would the experimental behaviour look like to confirm these hypotheses? Currently, the manuscript reads more as an exploratory study, which is certainly fine if true, it should just be explicitly stated in the introduction. Note: on line 144, this is a prediction, not a hypothesis. Line 225: this idea could be sharpened. I believe the authors are speaking to the idea of having more explicit knowledge of action-target pairings changing behaviour.

Reviewer #2 (Public review):

Summary:

In this study, Hill and colleagues use a novel reinforcement-based motor learning task ("RML"), asking how aspects of RML change over the course of development from toddler years through adolescence. Multiple versions of the RML task were used in different samples, which varied on two dimensions: whether the reward probability of a given hand movement direction was deterministic or probabilistic, and whether the solution space had continuous reach targets or discrete reach targets. Using analyses of both raw behavioral data and model fits, the authors report four main results: First, developmental improvements reflected 3 clear changes, including increases in exploration, an increase in the RL learning rate, and a reduction of intrinsic motor noise. Second, changes to the task that made it discrete and/or deterministic both rescued performance in the youngest age groups, suggesting that observed deficits could be linked to continuous/probabilistic learning settings. Overall, the results shed light on how RML changes throughout human development, and the modeling characterizes the specific learning deficits seen in the youngest ages.

Strengths:

(1) This impressive work addresses an understudied subfield of motor control/psychology - the developmental trajectory of motor learning. It is thus timely and will interest many researchers.

(2) The task, analysis, and modeling methods are very strong. The empirical findings are rather clear and compelling, and the analysis approaches are convincing. Thus, at the empirical level, this study has very few weaknesses.

(3) The large sample sizes and in-lab replications further reflect the laudable rigor of the study.

(4) The main and supplemental figures are clear and concise.

Weaknesses:

(1) Framing.<br /> One weakness of the current paper is the framing, namely w/r/t what can be considered "cognitive" versus "non-cognitive" ("procedural?") here. In the Intro, for example, it is stated that there are specific features of RML tasks that deviate from cognitive tasks. This is of course true in terms of having a continuous choice space and motor noise, but spatially correlated reward functions are not a unique feature of motor learning (see e.g. Giron et al., 2023, NHB). Given the result here that simplifying the spatial memory demands of the task greatly improved learning for the youngest cohort, it is hard to say whether the task is truly getting at a motor learning process or more generic cognitive capacities for spatial learning, working memory, and hypothesis testing. This is not a logical problem with the design, as spatial reasoning and working memory are intrinsically tied to motor learning. However, I think the framing of the study could be revised to focus in on what the authors truly think is motor about the task versus more general psychological mechanisms. Indeed, it may be the case that deficits in motor learning in young children are mostly about cognitive factors, which is still an interesting result!

(2) Links to other scholarship.<br /> If I'm not mistaken a common observation in studies of the development of reinforcement learning is a decrease in exploration over-development (e.g., Nussenbaum and Hartley, 2019; Giron et al., 2023; Schulz et al., 2019); this contrasts with the current results which instead show an increase. It would be nice to see a more direct discussion of previous findings showing decreases in exploration over development, and why the current study deviates from that. It could also be useful for the authors to bring in concepts of different types of exploration (e.g. "directed" vs "random"), in their interpretations and potentially in their modeling.

(3) Modeling.<br /> First, I may have missed something, but it is unclear to me if the model is actually accounting for the gradient of rewards (e.g., if I get a probabilistic reward moving at 45˚, but then don't get one at 40˚, I should be more likely to try 50˚ next then 35˚). I couldn't tell from the current equations if this was the case, or if exploration was essentially "unsigned," nor if the multiple-trials-back regression analysis would truly capture signed behavior. If the model is sensitive to the gradient, it would be nice if this was more clear in the Methods. If not, it would be interesting to have a model that does "function approximation" of the task space, and see if that improves the fit or explains developmental changes. Second, I am curious if the current modeling approach could incorporate a kind of "action hysteresis" (aka perseveration), such that regardless of previous outcomes, the same action is biased to be repeated (or, based on parameter settings, avoided).

(4) Psychological mechanisms.<br /> There is a line of work that shows that when children and adults perform RL tasks they use a combination of working memory and trial-by-trial incremental learning processes (e.g., Master et al., 2020; Collins and Frank 2012). Thus, the observed increase in the learning rate over development could in theory reflect improvements in instrumental learning, working memory, or both. Could it be that older participants are better at remembering their recent movements in short-term memory (Hadjiosif et al., 2023; Hillman et al., 2024)?

Reviewer #3 (Public review):

Summary:

The study investigates reinforcement learning across the lifespan with a large sample of participants recruited for an online game. It finds that children gradually develop their abilities to learn reward probability, possibly hindered by their immature spatial processing and probabilistic reasoning abilities. Motor noise, reinforcement learning rate, and exploration after a failure all contribute to children's subpar performance.

Strengths:

(1) The paradigm is novel because it requires continuous movement to indicate people's choices, as opposed to discrete actions in previous studies.

(2) A large sample of participants were recruited.

(3) The model-based analysis provides further insights into the development of reinforcement learning ability.

Weaknesses:

(1) The adequacy of model-based analysis is questionable, given the current presentation and some inconsistency in the results.

(2) The task should not be labeled as reinforcement motor learning, as it is not about learning a motor skill or adapting to sensorimotor perturbations. It is a classical reinforcement learning paradigm.

Reviewer #1 (Public review):

Summary:

Sidarta-Oliveira et al. present TopOMetry, a novel dimensionality reduction method based on the eigendecomposition of approximated Laplace-Beltrami Operator. Shortly, TopOMetry is an iterative version of the existing spectral methods (e.g., Laplacian Eigenmap or Diffusion map). It approximates the Laplacian operators twice, once in a "phenotypic space" and then once again in the eigenbases space. By doing this the approximated operator will contain more information of the manifold, which allows for more robust and accurate downstream analyses.

Strengths:

(1) The approach was rigorously tested based on synthetic and real single-cell RNA-seq datasets.

(2) The package is well-made and easily scalable to millions of cells.

(3) The comprehensive documentation helps the end-users to run desired analyses.

Weaknesses:

(1) The method is an extension of the current state-of-art methods, not a fundamentally new one.

(2) Considering the target readers, the paper contains a lot of jargon.

Reviewer #2 (Public review):

Summary:

This work introduces a novel framework to systematically learn the latent dimensions of single-cell data, grounded in the theory of the Riemannian manifold. The authors demonstrate how this framework can be applied to various important tasks, such as estimating intrinsic dimensionalities, annotating cell types, etc. They did a great job of tackling an important but not yet established problem in the field and approaching it with a theoretically sound and novel approach. I think after a more rigorous and comprehensive validation, this work could be impactful.

Strengths:

(1) Dimensionality reduction is a routine step in analyzing many high-dimensional data, such as molecular data. While the downstream analysis results depend heavily on this step, existing methods rely on strong assumptions and are sometimes heuristic. The authors present a novel, theoretically grounded approach to address this important problem.

(2) The authors demonstrated its usability in downstream analysis in a comprehensive manner. In particular, they show evidence suggesting novel T-cell subpopulations.

(3) I commend the authors for releasing and maintaining their software well with comprehensive documentation. This significantly increases the usability and accessibility of the method.

Weaknesses:

(1) To encourage the single-cell community to adopt this method, the authors should more clearly demonstrate its advantages over existing methods. There are many single cell analysis algorithms that are proposed in each task and some of them are widely used by biologists. However, the comparison in this work is somewhat limited. For example, Even methods mentioned in the relevant work paragraph (2nd paragraph) on page 2 are not all compared, or the reason why they are not included is not discussed. Also, I am curious how PC dimensions are determined. The choice of 300 PCs on page 11 seems arbitrary. Furthermore, the usefulness of dimension-reduced data also depends a lot on the preceding processing steps, such as highly variable gene selection. I understand it is hard to control all those factors, but I think there is room for improvement.

(2) The paper lacks experiments that validate the results. It would be beneficial to see additional evaluation settings with better-established ground truths to more strongly demonstrate the method's effectiveness.

(3) The effect of various parameters, such as those involved in k-nearest neighbors (KNN) or choosing the appropriate Laplacian operator, is not comprehensively explored. How can we ensure the analysis is not overly sensitive to these parameters?

(4) Batch effects are prevalent in single-cell data. The paper does not adequately address how the proposed method handles this issue.

Reviewer #1 (Public review):

Summary:

The signaling pathways regulating the immune response to bacteria and fungi have been well characterized in Drosophila. Using the recently identified anti-parasitoid effector Lectin24A as a read-out, this article describes the signaling pathways regulating the humoral response against parasites.

Strengths:

This study reveals a role of JAK-STAT, Toll, and GATA in the fat body in the regulation of Lectin24A. They also observe an enrichment of binding sites for NF-kB, STAT, and GATA factors upstream of ORFs of genes induced upon encapsulation. Based on this observation, they generalize their findings on the involvement of JAK-STAT, Toll, and Gata in the humoral response to encapsulation. Although roles for the Toll and JAK-STAT pathways in capsule formation have previously been identified, the merit of this article is in analyzing the roles of these pathways in the humoral response using a new gene readout that will be a precious tool in the community.

Weaknesses:

The data are mostly convincing, but not always analyzed with sufficient detail; their conclusions should be reinforced by monitoring Lectin24A gene expression by RT-qPCR, by adding additional time points and by using alternative genetic tools. Using read-outs of the Toll and Imd pathways as comparisons is also important. Thus, this paper is interesting and important in advancing our understanding of Drosophila immunity but not yet enough solid to reach definitive conclusions on the proposed claims.

Reviewer #2 (Public review):

Summary:

In a previous study, the investigators had identified through genetic analysis of lines derived from natural populations that lectin-24A was an important gene required for protection against the parasitoid wasp Leptopilina boulardii, albeit only in a specific genetic context depending on an unidentified locus on the third chromosome (Arunkumar, et al., PNAS, 2023). They had documented that the gene is induced upon wasp infection and that the corresponding Lectin-24A binds to the wasp egg prior to hemocyte, mediating a faster encapsulating cellular response. They had identified a polymorphism in susceptible lines that correlated with a 21 nt deficiency in the lectin-24A promoter that removed a proximal NF-kappaB binding site. Here, they follow up this work by first performing a transgenic dissection of this promoter, including the mutations of putative transcription factor binding sites (TFBS) of the JAK-STAT, the Toll pathway, and the GATA family transcription factors. Secondly, they directly affect the expression of genes of the JAK-STAT pathway, of the DIF or Dorsal NF-kappaB transcription factors (and also Relish), and of pannier, the one induced gene of five GATA family members. Of note, the lectin is preferentially expressed in the posterior part of the fat body.

Strengths:

The combination of the analysis of the expression of the lectin-24A gene in cis through mutations in putative TFBS for three families of transcription factors and the analysis in trans of either the genetic pathway (JAK-STAT) or the STAT/DIF/Dorsal/Pannier transcription factors provides a fine-grained description of the regulation of the expression of a humoral effector gene that is induced by parasitoid wasp infestation. Thus, this work goes much beyond the bioinformatics analysis by using a rather thorough experimental approach. The finding of an induction of lectin-24A in the posterior rather than the anterior fat body is interesting yet puzzling. Is it known whether this species of parasitoid wasps deposits its eggs preferentially in the posterior part of the larva?

Weaknesses:

There are some discrepancies between the "cis" and "trans" approaches as regards their effects on basal or induced expression of lectin-24A:

JAK-STAT:<br /> Figure 4D shows that mutating three of six predicted STAT TFBS in the 314 bp promoter leads to a reduction of both basal and induced lectin-24A expression levels, with the gene still being inducible. In contrast, knocking down or out the Drosophila JAK and STAT genes abolished the inducibility of the lectin-24A reporter down or close to basal levels. Conversely, the overactivation of the JAK-STAT pathway led to basal levels that increased to those of induced ones.

Toll pathway:<br /> Figure 4D shows that mutating the proximal Dif-Dorsal TFBS reduces both basal and induced levels of the reporter gene to a common level that is below that of the wild-type basal activity. These data suggest that NF-kappaB signaling is required for both basal and induced expression of Lectin-24A. Affecting either Dif or dorsal gene expression led to opposite changes essentially in the basal expression level of the lectin-24A reporter. Conversely, dorsal overexpression in the fat body and other tissues (hemocytes) led to an enhanced basal expression of the lectin gene.

GATA:<br /> The mutation of the single GATA TFBS in the promoter led to a reduced expression phenotype very similar to that of JAK-STAT TFBS mutations. In contrast, ubiquitous somatic KO mutations of pannier did not affect the basal or induced lectin-24A expression levels. The overactivation of pannier using an allele that cannot be negatively regulated leads to a higher induction of Lectin-24A gene expression, strikingly with basal expression going up to induced levels.

Reviewer #3 (Public review):

Summary:

In this very thorough manuscript, the authors provide further evidence that the lectin-24A gene in Drosophila melanogaster is directly involved in the anti-parasitoid wasp humoral immune reaction.

Strengths:

In this study in particular they use a fluorescent reporter and promoter-bashing to determine how this gene is regulated. They find that JAK/STAT, Pannier, and NF-κB signaling are integral to the regulation of lectin-24A and to the humoral anti-parasitoid immune response. These claims are well supported by the experimental design, results, and analysis.

Weaknesses:

A bit of clarity is needed regarding Figure 4a as well as on the rationale for the lengths of the promoter intervals used.

Reviewer #1 (Public review):

Summary:

Tian et al. investigated the effects of emotional signals in biological motion on pupil responses. In this study, subjects were presented with point-light biological motion stimuli with happy, neutral, and sad emotions. Their pupil responses were recorded with an eye tracker. Throughout the study, emotion type (i.e., happy/sad/neutral) and BM stimulus type (intact/inverted/non-BM/local) were systematically manipulated. For intact BM stimuli, happy BM induced a larger pupil diameter than neutral BM, and neutral BM also induced a larger pupil diameter than sad BM. Importantly, the diameter difference between happy and sad BM correlated with the autistic trait of individuals. These effects disappeared for the inverted BM and non-BM stimuli. Interestingly, both happy and sad emotions show superiority in pupil diameter.

Strengths:

(1) The experimental conditions and results are very easy to understand.<br /> (2) The writing and data presentation are clear.<br /> (3) The methods are sound. I have no problems with the experimental design and results.

Reviewer #2 (Public review):

Summary:

Through a serial of four experiments, Yuan, Wang and Jiang examined pupil size responses to emotion signals in point-light motion stimuli. Experiment 1 examined upright happy, sad and neutral point-light biological motion (BM) walkers. The happy BM induced a significantly larger pupil response than the neutral, whereas the sad BM evoked a significantly smaller pupil size than the neutral BM. Experiment 2 examined inverted BM walkers. Experiment 3 examined BM stimuli with acceleration removed. No significant effects of emotion were found in neither Experiment 2 nor Experiment 3. Experiment 4 examined scrambled BM stimuli, in which local motion features were preserved while the global configuration was disrupted. Interestingly, the scrambled happy and sad BM led to significant greater pupil size than the scrambled neutral BM at a relatively early time, while no significant difference between the scrambled happy and sad BM was found. Thus, the authors argue that these results suggest multi-level processing of emotions in life motion signals.

Strengths:

The experiments were carefully designed and well-executed, with point-light stimuli that eliminate many potential confounding effects of low-level visual features such as luminance, contrast, and spatial frequency.

Overall, I think this is a well-written paper with solid experimental results that support the claim of the authors, i.e., the human visual system may process emotional information in biological motion at multiple levels. Given the key role of emotion processing in normal social cognition, the results will be of interest not only to basic scientists who study visual perception, but also to clinical researchers who work with patients of social cognitive disorders. In addition, this paper suggests that examining pupil size responses could be a very useful methodological tool to study brain mechanisms underlying emotion processing.

Reviewer #3 (Public review):

Summary:<br /> The overarching goal of the authors was to understand whether emotional information conveyed through point-light biological motion can trigger automatic physiological responses, as reflected in pupil size.

Strengths:<br /> This manuscript has several noticeable strengths: it addresses an intriguing research question that fills that gap in existing literature, presents a clear and accurate presentation of the current literature, and conducts a series of experiments and control experiments with adequete sample size. Yet, it also entails several noticeable limitations - especially in the study design and statistical analyses.

Assessment of the revision:

The authors have done a thorough job revising the manuscript, effectively addressing all of my previous concerns.

Reviewer #1 (Public Review):

The authors use a previously established reporter comprising a slow- and a fast-folding fluorescent protein fused to a randomly-generated library of penta-peptides at its amino-terminus and a signal sequence for import into the endoplasmic reticulum (ER). They then determine the stability of these constructs in a high throughput FACS-sorting procedure and identify a set of peptides that route the construct to proteasomal degradation. Increasing the copy number of one of these peptides further decreases the stability of the construct. This polypeptide resembles a "degron" for ER proteins, because it also targets other ER proteins with different topological and folding properties for degradation. It only works when placed at the amino-terminus of a protein and utilizes components of the Hrd1 ubiquitin ligase complex, a well-established quality control ubiquitin ligase in the ER membrane. Importantly, the degron also targets ER-proteins in mammalian cells.

The authors convincingly show that fusion of their newly identified degron to the amino terminus of ER-resident proteins with different topology suffices to target them for proteasomal degradation. The data for this are well-founded and contain appropriate controls. While technically sound, the study does only give superficial information on general properties of the degron and its recognition by cellular factors. Further simple experiments would have addressed a number of important points. The authors only provide data about the composition of the identified amino acid sections from the high-throughput approach and the statistical preference for certain amino acids at individual positions. They do not study degron composition experimentally by substituting individual amino acids with other residues and analyzing protein stability. Increasing the numbers of the initially identified degron pentamer increases substrate turnover, but the basis for this remains unclear. Each copy may be actively involved in better recognition, elongation of the degron may facilitate accessibility by recognition factors or multiplying the short amino acid stretch may generate new signatures at the amino-terminus that are more readily recognized by a quality control machinery. Consequently, this study does not allow conclusions to be drawn about general properties of degron composition and/or structure. The degron also functions with cytoplasmic proteins, suggesting that similar characteristics of a polypeptide attract the attention of quality control systems also in other cellular compartments. However, the authors did not pursue this finding further, e.g. by identifying factors for degron recognition in the cytoplasm. It would have been particularly interesting to test whether the degron would initiate degradation when placed at cytoplasmically-exposed amino termini of membrane-bound ER proteins. Information on degron properties is required to better understand principles of substrate recognition by protein quality control pathways and to design constructs for targeting endogenous proteins via proteolysis targeting chimeras (PROTACs).

Reviewer #2 (Public review):

Summary:

Sharninghausen et al use a generic screening platform to search for short (5 amino acid) degrons that function in the lumen of the endoplasmic reticulum (ER) of budding yeast. The screen did indeed identify a number of sequences which increased the rate of degradation of their test proteins. Although the effect of the single degron was rather modest the authors could show that by mutimerising the sequence (4x) they obtained degrons that functioned fairly efficiently. Further characterisation indicated that the degrons only functioned when placed at the N-terminus of the target protein and, were dependent on both the proteasome and the segregase Cdc48 (p97) for degradation. The authors also demonstrated that degradation was via the ERAD pathway.

Strengths:

In general, the data presented is supportive of the conclusions drawn and the authors have thus identified a sequence that can be appended onto other ER targeted proteins to mediate their degradation within the lumen of the ER. How useful this will be to the community remains to be seen.

Weaknesses:

While the observation that such mutimerised sequences can act as degrons is an interesting curiosity, it is not clear that such sequences function in vivo. In fact the DegV1 sequence used throughout the paper is not present in any yeast or fungal proteins and the fact that it has to be located at the N-terminus of the protein to induce degradation is at odds with the idea that proteins to be degraded need to be unfolded. Thus, the role of such sequences in vivo is questionable.

Comments on revised manuscript:

Although the role of such degron sequences remains to be determined in vivo, it is clear that the authors have developed a tool that could be useful to the scientific community. The specific points raised were appropriately addressed by the authors.

Reviewer 1 (Public Review):

Multiple sclerosis (MS) is a debilitating autoimmune disease that causes loss of myelin in neurons of the central nervous system. MS is characterized by the presence of inflammatory immune cells in several brain regions as well as the brain barriers (meninges). This study aims to understand the local immune hallmarks in regions of the brain parenchyma that are adjacent to the leptomeninges in a mouse model of MS. The leptomeninges are known to be a foci of inflammation in MS and perhaps "bleed" inflammatory cells and molecules to adjacent brain parenchyma regions. To do so, they use novel technology called spatial transcriptomics so that the spatial relationships between the two regions remain intact. The study identifies canonical inflammatory genes and gene sets such as complement and B cells enriched in the parenchyma in close proximity to the leptomeninges in the mouse model of MS but not control. The manuscript is very well written and easy to follow. The results will become a useful resource to others working in the field and can be followed by time series experiments where the same technology can be applied to the different stages of the disease.

Reviewer 2 (Public Review):

Accumulating data suggests that the presence of immune cell infiltrates in the meninges of the multiple sclerosis brain contributes to the tissue damage in the underlying cortical grey matter by the release of inflammatory and cytotoxic factors that diffuse into the brain parenchyma. However, little is known about the identity and direct and indirect effects of these mediators at a molecular level. This study addresses the vital link between an adaptive immune response in the CSF space and the molecular mechanisms of tissue damage that drive clinical progression. In this short report the authors use a spatial transcriptomics approach using Visium Gene Expression technology from 10x Genomics, to identify gene expression signatures in the meninges and the underlying brain parenchyma, and their interrelationship, in the PLP-induced EAE model of MS in the SJL mouse. MRI imaging using a high field strength (11.7T) scanner was used to identify areas of meningeal infiltration for further study. They report, as might be expected, the upregulation of genes associated with the complement cascade, immune cell infiltration, antigen presentation, and astrocyte activation. Pathway analysis revealed the presence of TNF, JAK-STAT and NFkB signaling, amongst others, close to sites of meningeal inflammation in the EAE animals, although the spatial resolution is insufficient to indicate whether this is in the meninges, grey matter, or both.

UMAP clustering illuminated a major distinct cluster of upregulated genes in the meninges and smaller clusters associated with the grey matter parenchyma underlying the infiltrates. The meningeal cluster contained genes associated with immune cell functions and interactions, cytokine production, and action. The parenchymal clusters included genes and pathways related to glial activation, but also adaptive/B-cell mediated immunity and antigen presentation. This again suggests a technical inability to resolve fully between the compartments as immune cells do not penetrate the pial surface in this model or in MS. Finally, a trajectory analysis based on distance from the meningeal gene cluster successfully demonstrated descending and ascending gradients of gene expression, in particular a decline in pathway enrichment for immune processes with distance from the meninges.

Reviewer #1 (Public Review):

This is a technically sound paper focused on a useful resource around the DRGP phenotypes which the authors have curated, pooled, and provided a user-friendly website. This is aimed to be a crowd-sourced resource for this in the future. The authors should make sure they coordinate as well as possible with the NC datasets and community and broader fly community.

Reviewer #2 (Public Review):

In the present study, Gardeux et al provide a web-based tool for curated association mapping results from DRP studies. The tool lets users view association results for phenotypes and compare mean phenotype ~ phenotype correlations between studies. In the manuscript, the authors provide several example utilities associated with this new resource, including pan-study summary statistics for sex, traits, and loci. They highlight cross-trait correlations by comparing studies focused on longevity with phenotypes such as oxphos and activity. Strengths: -Considerable efforts were dedicated toward curating the many DRG studies provided. -Available tools to query large DRP studies are sparse and so new tools present appeal Weaknesses: The creation of a tool to query these studies for a more detailed understanding of physiologic outcomes seems underdeveloped. These could be improved by enabling usages such as more comprehensive queries of meta-analyses, molecular information to investigate given genes or pathways, and links to other information such as in mouse rat or human associations.

Reviewer #1 (Public Review):

Kainate receptors play various important roles in synaptic transmission. The receptors can be divided into low affinity kainate receptors (GluK1-3) and high affinity kainate receptos (GluK4-5). The receptors can assemble as homomers (GluK1-3) or low-high affinity heteromers (GluK4-5). The functional diversity is further increased by RNA splicing. Previous studies have investigated C-terminal splice variants of GluK1, but GluK1 N-terminal (exon 9) insertions have not been previously characterized. In this study Dhingra et al investigate the functional implications of a GluK1 splice variant that inserts a 15 amino acid segment into the extracellular N-terminal region of the protein using whole-cell and excised outside-out electrophysiology.

The authors convincingly show that the insertion profoundly impacts the function of GluK1-1a - the channels that have the insertion are slower to desensitize. The data also shows that the insertion changes the modulatory effects of Neto proteins, resulting in altered rates of desensitization and recovery from desensitization. To determine the mechanism by which the insertion exerts these functional effects, the authors perform pull-down assays of Neto proteins, and extensive mutagenesis on the insert.<br /> The electrophysiological part of the study is very rigorous and meticulous.

The biggest weakness of the manuscript is the structural work. Due to issues with preferred orientation (a common problem in cryo-EM), the 3D reconstructions are at a low resolution (in the 5-8 Å range) and cannot offer much mechanistic insight into the effects of the insertion. The authors have opted to keep this data unchanged in the revised manuscript.

Despite this, the study is a valuable contribution to the field because it characterizes a GluK1 variant that has not been studied before and highlights the functional diversity that exists within the kainate receptor family.

Reviewer #1 (Public Review):

This manuscript addresses the regulation of the osmosensing protein kinases, WNK1 and WNK3. Prior work by the authors has shown that these enzymes are activated by PEG400 or ethylene glycol and inhibited by chloride ion, and that activation is associated with a conformational transition from dimer to monomer. In X-ray structures of the WNK1/SA inactive dimer, a water-mediated hydrogen bond network was observed between the catalytic loop (CL) and the activation loop (AL), named CWN1. This led to the proposal that bound water may be part of the osmosensing mechanism.

The current study carries this work further, by applying PEG400 to Xtals of dimeric WNK1/SA. This results in a change in kinase conformation and space group, along with 4-9 fewer waters in CWN1 and the complete disappearance of another water cluster (CWN2) located at the dimer interface. Six conserved residues lining the CWN1 pocket in WNK3 are mutated to determine effects on activity and inhibition by chloride ion (measured by AL autophosphorylation) and monomer-dimer interconversion (light scattering).

The results show that two mutants (E314Q/A in WNK3) at a site central to the water cluster result in increased kinase activity (autophosphorylation), and increased SLS, interpreted as aggregation. Three sites (D279A, Y346F, M301A) inhibit kinase activity with varying effects on oligomerization - Y346A and M301A retain monomer-dimer ratios similar to WT while D279N promotes aggregation. K236A and K307A show activity and monomer:dimer ratios similar to WT. Selected mutants (E314Q, D279N, Y346F) and WT appear to retain osmosensitivity with comparable activation by PEG400.

The study concludes that osmolytes may activate the kinase by removing waters from the CWN1 and CWN2 clusters, suggesting that waters might be considered allosteric ligands that promote the inactive structure of WNKs. The differing effects of mutations may be ascribed to disruption of the water networks as well as inhibitory perturbations at the active site.

Comments on latest version:

The revised manuscript incorporated new experiments that satisfactorily addressed my concerns.

Reviewer #2 (Public Review):

This work tests the hypothesis that water coordination in WNK kinases is linked to allosteric control of activity. It is proposed that dimeric WNK is inactive and bound to some conserved water molecules, and that monomerization/activation involves departure of these waters. New data here include a crystal structure of monomeric WNK1 which shows missing waters compared to the dimeric structure, in support of the hypothesis. Mutant proteins of a different isozyme (WNK3) designed to disrupt water coordination were produced, and activity and quaternary structure were measured.

Comments on latest version:

The authors have largely addressed my concerns by making sure collection of mutants analyzed for autophosphorylation in Figure 6 are consistent with the measurement of osmotic sensitivity in Figure 7. The other changes in response to reviews have made a stronger manuscript in my opinion.

Reviewer #1 (Public review):

The authors construct a pair of E. coli populations that differ by a single gene duplication in a selectable fluorescent protein. They then evolve the two populations under differing selective regimes to assess whether the end result of the selective process is a "better" phenotype when starting with duplicated copies. Importantly, their starting duplicated population is structured to avoid the duplication-amplification process often seen in bacterial artificial evolution experiments. They find that while duplication increases robustness and speed of adaptation, it does not result in more highly adapted final states, in contrast to Ohno's hypothesis.

Comments on revised version:

The authors have addressed my prior concerns, and I have no further comments on the manuscript.

Reviewer #2 (Public review):

Summary:

Drawing from tools of synthetic biology, Mihajlovic et al. use a cleverly designed experimental system to dissect Ohno's hypothesis, which describes the evolution of functional novelty on the gene-level through the process of duplication & divergence.<br /> Ohno's original idea posits that the redundancy gained from having two copies of the same gene allows one of them to freely evolve a new function. To directly test this, the authors make use of a fluorescent protein with two emission maxima, which allows to apply different selection regimes (e.g. selection for green AND blue, or, for green NOT blue). To achieve this feat without being distracted by more complex evolutionary dynamics caused by the frequent recombination between duplicates, the authors employ a well-controlled synthetic system to prevent recombination: Duplicates are placed on a plasmid as indirect repeats in a recombination-deficient strain of E.coli. The authors implement their directed evolution approach through in vitro mutagenesis and selection using fluorescent-activated cell sorting. Their in-depth analysis of evolved mutants in single-copy versus double-copy genotypes provides clear evidence for Ohno's postulate that redundant copies experience relaxed purifying selection. In contrast to Ohno's original postulate, however, the authors go on to show that this does not in fact lead to more rapid phenotypic evolution, but rather, the rapid inactivation of one of the copies.

Strengths:

This paper contributes with great experimental detail to an area where the literature predominantly leans on genomics data. Through the use of a carefully-designed, well-controlled synthetic system the authors are able to directly determine the phenotype & genotype of all individuals in their evolving populations and compare differences between genotypes with a single or double copy of coGFP. With it they find clear evidence for what critics of Ohno's original model have termed "Ohno's dilemma", the rapid non-functionalization by predominantly deleterious mutations.

Including an expressed but non-functional coGFP in (phenotypically) single copy genotypes provides an especially thoughtful control that allows determining a baseline dN/dS ratio in the absence of selection. All in all the study is an exciting example of how the clever use of synthetic biology can lead to new insights.

Weaknesses:

In the revised version of the paper, the authors now discuss one potential weakness of their study, which is tied to its biggest strength (as often in experimental biology there is a trade-off between 'resolution' and 'realism').<br /> The experimental set-up leaves out an important component of the evolutionary process in order to disentangle dosage effects from other effects that carrying two copies might have on their evolution. Specifically, by employing a recombination-deficient strain and constructing their duplicates as inverted repeats their experimental design completely abolishes recombination between the two copies. This was pointed out in my first review to be problematic for two reasons:

(i) In nature, new duplicates do not arise as inverted, but rather as direct (tandem) repeats and - as the authors correctly point out - these are very unstable, due to the fact that repeated DNA is prone to recA-dependent homologous recombination (which arise orders of magnitude more frequently than point mutations).

(ii) This instability often leads to further amplification of the duplicates under dosage selection both in the lab and in the wild (e.g. Andersson & Hughes, Annu. Rev. Genet. 2009), and would presumably also be an outcome under the current experimental set-up if it was not prevented from happening?

In their revised version, the authors now address this point and with much clarity explain why their experimental system is so powerful to study the fate of a gene duplicate, not despite lacking recombination, but *because* it lacks recombination.

Reviewer #3 (Public review):

Summary:

In this manuscript the authors explore the contribution of metabolism to the response of two subpopulations of macrophages to bacterial pathogens commonly encountered in the human lung, as well as the influence of priming signals typically produced at a site of inflammation. The two subpopulations are resident airway macrophages (AM) isolated via bronchoalveolar lavage and monocyte-derived macrophages (MDM) isolated from human blood and differentiated using human serum. The two cell types were primed using IFNγ and Il-4, which are produced at sites of inflammation as part of initiation and resolution of inflammation respectively, followed by stimulation with either heat-killed tuberculosis (Mtb) or LPS to simulate interaction with a bacterial pathogen that is either gram-negative in the case of Mtb or gram-positive in the case of LPS. The authors use human cells for this work, which makes use of widely reported and thoroughly described priming signals, as well as model antigens. This makes the observations on the functional response of these two subpopulations relevant to human health and disease to a greater extent that the mouse models typically used to interrogate these interactions. To examine the relationship between metabolism and functional response, the authors measure rates of oxidative phosphorylation and glycolysis under baseline conditions, primed using IFNγ or IL-4, and primed and stimulated with Mtb or LPS.

Overall, this study reveals how inflammatory and anti-inflammatory cytokine priming contributes to the metabolic reprogramming of AM and MDM populations. Their conclusions regarding the relationship between cytokine secretion and inflammatory molecule expression in response to bacterial stimuli are supported by the data. The involvement of metabolism in innate immune cell function is relevant when devising treatment strategies that target the innate immune response during infection. The data presented in this paper further our understanding of that relationship and advance the field of innate immune cell biology.

Reviewer #1 (Public review):

Summary:

The authors were seeking to define the roles of the Drosophila caspar gene in embryonic development and primordial germ cell (PGC) formation. They demonstrate that PGC number, and the distribution of the germ cell determinant Oskar, change as a result of changes in caspar expression; reduction of caspar reduces PGC number and the domain of Oskar protein expression, while overexpression of caspar does the reverse. They also observe defects in syncytial nuclear divisions in embryos produced from caspar mutant mothers. Previous work from the same group demonstrated that Caspar protein interacts with two partners, TER94 and Vap33. In this paper, they show that maternal knockdown of TER94 results in embryonic lethality and some overlap of phenotypes with reduction of caspar, supporting the idea may work together in their developmental roles. The authors propose models for how Caspar might carry out its developmental functions. The most specific of these is that Caspar and its partners might regulate oskar mRNA stability by recruiting ubiquitin to the translational regulator Smaug.

Strengths:

The work identifies a new factor that is involved in PGC specification and points toward an additional pathway that may be involved in establishing and maintaining an appropriate distribution of Oskar at the posterior pole of the embryo. It also ties together earlier observations about the presence of TER94 in the pole plasm that have not heretofore been linked to a function.

Weaknesses:

(1) A PiggyBac insertion allele casp[c04227] is used throughout the paper and referred to as a loss-of-function allele (casp[lof]). While the authors avoid the terms 'null' or 'amorph' and on one occasion refer to the allele as a 'strong hypomorph', nevertheless terming it a 'loss-of-function' allele is misleading. This is because the phenotype of the allele when homozygous is different from the phenotype produced when heterozygous over a deficiency.

(2) The peptide counts in the mass spectrometry experiment aimed at finding protein partners for Casp are extremely low, except for Casp itself and TER94. Peptide counts of 1-2 seem to me to be of questionable significance.

(3) The pole bud phenotypes from TER94 knockdown and casp mutant shown in Fig 5 appear to be quite different. These differences are unexplained and seem inconsistent with the model proposed that the two proteins work in a common pathway. Whole embryos should also be shown, as the TER94 KD phenotype could result from a more general dysmorphism.

(4) Fig 6 is not quantitative, lacking even a second control staining to check for intensity variation artifacts. Therefore it shows that the distribution of Oskar protein changes in the various genotypes, but not convincingly that the level of Oskar changes as the paper claims.

(5) The error bars are huge in the graphs in Fig 7H, I, and J, and in fact these changes are not statistically significant. Therefore the conclusion that 'Reduction in Casp activity specifically affects Smaug degradation during the MZT' is not supported by the data in this figure.

Reviewer #2 (Public review):

Summary:

This study investigated the role of the Caspar (Casp) gene, a Drosophila homolog of human Fas-associated factor-1. It revealed that maternal loss of Casp led to centrosomal and cytoskeletal abnormalities during nuclear cycles in Drosophila early embryogenesis, resulting in defective gastrulation. Moreover, Casp regulates PGC numbers, likely by regulating the levels of Smaug and then Oskar. They demonstrate that Casp protein levels are linearly correlated to the PGC number. The partner protein TER94, an ER protein, shows similar but slightly distinct phenotypes. Based on the deletion mutant analysis, TER94 seems functionally relevant for the observed Casp phenotype. Additionally, it is likely involved in regulating protein degradation during PGC specification.

Strengths:

This paper uncovers a new function of the Casper (Casp) gene, previously known for its role in immune response regulation and NF-kB signaling inhibition. This new function includes nuclear division and PGC formation in early fly embryos. The findings provide crucial insights into how this pathway contributes to the proper establishment of both somatic cells and the germline, particularly in the context of early embryogenesis. This research is therefore of significant interest to cell and developmental biologists.

Future Research:

While this study has made significant strides in understanding the role of the Casp gene in early embryogenesis, the functional relationships among molecules shown here (Casp, TER94, Osk) and other genes previously known to regulate these processes remain unclear. This underscores the need for future studies to delve deeper into these relationships and their implications.

Reviewer #3 (Public review):

Summary:

Das et al. discovered a maternal role for Caspar (Casp), the Drosophila orthologue of human Fas-associated factor-1 (FAF1), in embryonic development and germ cell formation. They find that Casp interacts with Transitional endoplasmic reticulum 94 (TER94). Loss of Casp or TER94 leads to partial embryonic lethality, correlated with aberrant centrosome behavior and cytoskeletal abnormalities. This suggests that Casp, along with TER94, promotes embryonic development through a still unidentified mechanism. They also find that Casp regulates germ cell number by controlling a key determinant of germ cell formation, Oskar, through its negative regulator, Smaug.

Strengths:

Overall, the experiments are well-conducted, and the conclusions of this paper are mostly well-supported by data.

Weaknesses:

Some additional controls could be included, and the language could be clarified for accuracy.

Reviewer #1 (Public review):

Summary:

The manuscript studies nutrient intake rates for stationary and motile microorganisms to assess the effectiveness of swim vs. stay strategies. This work provides valuable insights on how the different strategies perform in the context of a simplified mathematical model that couples hydrodynamics to nutrient advection and diffusion. The swim and stay strategies are shown to yield similar nutrient flux under a range of conditions.

Strengths:

Strengths of the work include (i) the model prediction in Fig. 3 of nutrient flux applied to a range of microorganisms including an entire clade that are known to use different feeding strategies and (ii) a study of the interaction between cilia and absorption coverage showing the robustness of their predictions provided these regions have sufficient overlap.

Weaknesses:

In the revision, the authors have adequately addressed the weaknesses I raised in the first round of review.

Reviewer #2 (Public review):

Summary:

The authors have collected a significant amount of data from the literature on the flow regimes associated with microorganisms whose propulsion is achieved through the action of cilia or flagella, with particular interest in the competition between sessile and motile lifestyles. They then use several distinct hydrodynamic models for the cilia-driven flows to quantify the nutrient uptake and clearance rate, reported as a function of the Peclet number. Among the interesting conclusions the authors draw concerns the question of whether, for certain ciliates, there is a clear difference in nutrient uptake rates in the sessile versus motile forms. The authors show that this is not the case, thereby suggesting that the evolutionary pressure associated with such a difference is not present. The analysis also includes numerical calculations of the uptake rate for spherical swimmers in the regime of large Peclet numbers, where the authors note an enhancement due to advection-generated thinning of the solutal boundary layer around the organism.

Strengths:

In addressing the whole range of organism sizes and Peclet numbers the authors have achieved an important broad perspective on the problem of nutrient uptake of ciliates, with implications for understanding evolutionary driving forces toward particular lifestyles (e.g. sessile versus motile).

Reviewer #2 (Public review):

Summary:

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 in 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:

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. The authors have taken into account earlier review comments to revise their paper in ways that enhance its clarity.

Weaknesses:

One weakness could be that the proposed theory does rely on a fairly large number of assumptions. However, there is at least some biological support for these. Importantly, the authors do lay out and discuss their key assumptions in the Discussion section, so readers can assess their validity and implications for themselves.

Reviewer #4 (Public review):

Summary:

Wilmes and colleagues develop a model for the computation of uncertainty modulated prediction errors based on an experimentally inspired cortical circuit model for predictive processing. Predictive processing is a promising theory of cortical function. An essential aspect of the model is the idea of precision weighting of prediction errors. There is ample experimental evidence for prediction error responses in cortex. However, a central prediction of the theory is that these prediction error responses are regulated by the uncertainty of the input. Testing this idea experimentally has been difficult due to a lack of concrete models. This work provides one such model and makes experimentally testable predictions.

Strengths:

The model proposed is novel and well-implemented. It has sufficient biological accuracy to make useful and testable predictions.

Weaknesses:

One key idea the model hinges on is that stimulus uncertainty is encoded in the firing rate of parvalbumin positive interneurons. This assumption, however, is rather speculative and there is no direct evidence for this.

Joint Public Review:

Summary:

This study presents a strategy to efficiently isolate PcrV-specific BCRs from human donors with cystic fibrosis who have/had Pseudomonas aeruginosa (PA) infection. Isolation of mAbs that provide protection against PA may be a key to developing a new strategy to treat PA infection as the PA has intrinsic and acquired resistance to most antibiotic drug classes. Hale et al. developed fluorescently labeled antigen-hook and isolated mAbs with anti-PA activity. Overall, the authors' conclusion is supported by solid data analysis presented in the paper. Four of five recombinantly expressed PcrV-specific mAbs exhibited anti-PA activity in a murine pneumonia challenge model as potent as the V2L2MD mAb (equivalent to gremubamab). However, therapeutic potency for these isolated mAbs is uncertain as the gremubamab has failed in Phase 2 trials. Clarification of this point would greatly benefit this paper.

Strengths:

(1) High efficiency of isolating antigen-specific BCRs using an antigenic hook.

(2) The authors' conclusion is supported by data.

Weaknesses:

Although the authors state that the goal of this study was to generate novel protective mAbs for therapeutic use (P12; Para. 2), it is unclear whether PcrV-specific mAbs isolated in this study have therapeutic potential better than the gremubamab, which has failed in Phase 2 trials. Four of five PcrV-specific mAbs isolated in this study reduced bacterial burdens in mice as potent as, but not superior to, gremubamab-equivalent mAb. Clarification of this concern by revising the text or providing experimental results that show better potential than gremubamab would greatly benefit this paper.

Reviewer #1 (Public review):

Summary:

Animals in natural environments need to identify predator-associated cues and respond with the appropriate behavioral response to survive. In rodents, some chemical cues produced by predators (e.g., cat saliva) are detected by chemosensory neurons in the vomeronasal organ (VNO). The VNO transmits predator-associated information to the accessory olfactory bulb, which in turn projects to the medial amygdala and the bed nucleus of the stria terminalis, two regions implicated in the initiation of antipredator defensive behaviors. A downstream area to these two regions is the ventromedial hypothalamus (VMH), which has been shown to control both active (i.e., flight) and passive (i.e, freezing) antipredator defensive responses via distinct efferent projections to the anterior hypothalamic nucleus or the periaqueductal gray, respectively. However, whether differences in predator-associated sensory information initially processed in the VNO and further conveyed to the VMH can trigger different types of behavioral responses remained unexplored. To address this question, here the authors investigated the behavioral responses of mice exposed to either fresh or old cat saliva, and further compared the underlying neural circuits that are activated by cat saliva with different freshness.

The scientific question of the study is valid, the experiments were well-performed, and the statistical analyses are appropriate. However, there are some concerns that may directly affect the main interpretation of the results.

In this revised version of the manuscript, the authors have made important modifications in the text, inserted new experiments and performed additional data analyses, as recommended. These modifications have significantly improved the quality of the manuscript and addressed all the major concerns detected during the prior submission.

Reviewer #2 (Public review):

In this study, Nguyen et al. showed that cat saliva can robustly induce freezing behavior in mice. This effect is mediated through accessory olfactory system as it requires physical contact and is abolished in Trp2 KO mice. The authors further showed that V2R-A4 cluster is responsive to cat saliva. Lastly, they demonstrated c-Fos induction in AOB and VMHdm/c by the cat saliva. The c-Fos level in the VMHdm/c is correlated with freezing response.

Strength:

The study opens an interesting direction. It reveals the potential neural circuit for detecting cat saliva and driving defense behavior in mice. The behavior results and the critical role of accessory olfactory system in detecting cat saliva are clear and convincing.

Weakness:

The findings are relatively preliminary. The identities of the receptor and the ligand in the cat saliva that induces the behavior remain unclear. The identity of VMH cells that are activated by the cat saliva remains unclear. There is a lack of targeted functional manipulation to demonstrate the role of V2R-A4 or VMH cells in the behavioral response to the cat saliva.

Here are some specific comments:

(1) This result suggests that V2R-A4 may be the dominant VR for mice to detect cat saliva. Future studies should determine the identity of the receptor and the ligand in the cat saliva. Additionally, the functional importance of V2R-A4 remains unclear. It is important to knockout the receptor and test changes in cat saliva-induced freezing.

(2) AOB does not project to VMH directly. Other known important nodes for the predator defense circuit includes MeApv, BNST, PMd, AHN and PAG. It will be helpful to provide c-Fos data in those regions (especially MEA and BNST as they are between AOB and VMH) to provide a complete picture regarding how the brain process cat saliva to induce the behavior change.

(3) It is interesting that activation level difference in the VNO by old and fresh cat saliva does not transfer to AOB. It could be informative to examine correlation between VNO and AOB p6/c-Fos cell number and AOB and VMH c-Fos cell number across animals to understand whether the activation level across those regions are related. If they are not correlated, it could be helpful to add a discussion regarding potential reasons, e.g. neuromodulatory inputs to the AOB.

(4) Please indicate n in all figure plots and specify what individual dots means. In Figure 4h, there are 7 dots in old saliva group, presumably indicating 7 animals. In Figure 6b, there appear to be more than 7 dots for old cat saliva group. Are there more than 7 animals? If so, why are they not included in Figure 4h? If not, what does each dot mean? Note that each dot should represent independent sample. One animal should not contribute more than one dot.

(5) The identification of a cluster of VMHdm cells uniquely activated by fresh cat saliva urine is interesting. It will be important to identify the molecular handle of the cells to facilitate further investigation. This could be achieved using either activity dependent RNAseq or double in situ of saliva-induced c-Fos and candidate genes (candidate gene may be identified based on the known gene expression pattern).

Reviewer #3 (Public review):

Summary:

Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also suggest that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. The work also shows that different VMH populations are activated by fresh and old saliva, demonstrating that these stimuli create qualitatively different neural activity profiles. However, the exact components that differ between fresh and old saliva remain unknown and may be identified in future studies.

Strengths:

(1) Predator saliva is a stimulus of high ethological relevance<br /> (2) The authors performed a careful quantification of fos induction across the anterior-posterior axis<br /> (3) Authors show that different VMH populations are activated by fresh and old saliva

Weaknesses:

(1) There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing neural activity of relevant cell bodies and axonal projections

(2) Some of the findings are disconnected from the story. For example, the authors show V2R-A4-expressing cells are activated by predator odors, but the causal role of these cells in generating defensive actions is not shown