Some casual talk before you're investigated for money laundering and before the cartel kills you.

This is also why Michael is just keeping her on the phone for hours and hours. It's just painful to read. Like, there's no defense at this point, just hang up the phone for 10 minutes and this would come crashing down, but she can't.

Agreed. I don't mean that in a nasty way, just like... oof.

Two things:

A) our financial system is a mess and that actually contributes to scams, because people get confused about what's possible to do. It's true that identity theft can be a nightmare, and that makes people more scared of it than they should be, and the problem because reinforcing. Mostly because banks are terrible at security and credit unions don't care about protecting data or correcting mistakes.

B) Just because This American Life reported it was her boyfriend does not mean that the vast majority of ID theft isn't random. Most of it's random, this American Life just picked up on it because it was noteworthy and a more dramatic story, which is irresponsible on their part if they didn't try to clarify the reality for most people.

You don't check this information with them on the phone, not if they called you. Check it separately, then call Amazon customer service back using the number on the website.

This is something to do universally -- not when a call feels suspicious, just as a general policy always initiate the call yourself. What's bad is that not all businesses will tell you to do this, but if I tell a business "for security I need to hang up and call you back" I have never had a business be upset about that, they all understand. Should be universal practice even if you're 100% sure it's not a scam, it's about forming a habit.

Maybe explain this more intuitively? It's just because the u nodes cannot be part of a cycle or isolated so they must be part of a simple path and only one simple path

Tell that to the super-rich.

Instagram usually gives out recommendations on products. When I searched some products online, instagram will immediately recommend me the same products while I look through stories. It makes me feel bad because sometimes I just want to take a quick look on some products, but is not thinking about buying that. Further, it ignited my desire to buy products, which I would realize that it's a waste of money after I spent money on them.

More discussion about how sports gambling is taking over sports.

So if destiny is just one path, then we have many destinies? Seems rather agnostic in the fate/free will debate.

How do you think of destiny and prophesy then around your own greatness?

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors provide a method aiming to accurately reflect the individual deviation of longitudinal/temporal change compared to the normal temporal change characterized based on pre-trained population normative model (i.e., a Bayesian linear regression normative model), which was built based on cross-sectional data. This manuscript aims to solve a recently identified problem of using normative models based on cross-sectional data to make inferences about longitudinal change.

Although the proposed method was implemented with real data and shown to be more sensitive in capturing the differences confirmed by previous studies than conventional methods, there is still a lack of simulation studies to formally evaluate the performance of the proposed method in making accurate estimations and inferences about the longitudinal changes.

Strengths:

The efforts of this work make a good contribution to addressing an important question of normative modeling. With the greater availability of cross-sectional studies for normative modeling than longitudinal studies, and the inappropriateness of making inferences about longitudinal subject-specific changes using these cross-sectional data-based normative models, it's meaningful to try to address this gap from the perspective of methodological development.

Weaknesses:

• The organization and clarity of this manuscript need enhancement for better comprehension and flow. For example, in the first few paragraphs of the introduction, the wording is quite vague. A lot of information was scattered and repeated in the latter part of the introduction, and the actual challenges/motivation of this work were not introduced until the 5th paragraph.

• There are no simulation studies to evaluate whether the adjustment of the cross-sectional normative model to longitudinal data can make accurate estimations and inferences regarding the longitudinal changes. Also, there are some assumptions involved in the modeling procedure, for example, the deviation of a healthy control from the population over time is purely caused by noise and constant variability of error/noise across x_n, and these seem to be quite strong assumptions. The presentation of this work's method development would be strengthened if the authors can conduct a formal simulation study to evaluate the method's performance when such assumptions are violated, and, ideally, propose some methods to check these assumptions before performing the analyses.

• The proposed "z-diff score" still falls in the common form of z-score to describe the individual deviation from the population/reference level, but now is just specifically used to quantify the deviation of individual temporal change from the population level. The authors need to further highlight the difference between the "z-score" and "z-diff score", ideally at its first mention, in case readers get confused (I was confused at first until I reached the latter part of the manuscript). The z-score can also be called a measure of "standardized difference" which kind of collides with what "z-diff" implies by its name.

• Explaining that one component of the variance is related to the estimation of the model and the other is due to prediction would be helpful for non-statistical readers.

• It would be easier for the non-statistical reader if the authors consistently used precision or variance for all variance parameters. Probably variance would be more accessible.

• The functions psi were never explicitly described. This would be helpful to have in the supplement with a reference to that in the paper.

• What is the goal of equations (13) and (14)? The authors should clarify what the point of writing these equations is prior to showing the math. It seems like it is to obtain an estimate of \sigma_{\ksi}^2, which the reader only learns at the end.

• What is the definition of "adaption" as used to describe equation (15)? In this equation, I think norm on subsample was not defined.

• "(the sandwich part with A)" - maybe call this an inner product so that it is not confused with a sandwich variance estimator. This is a bit unclear. Equation (8) does have the inner product involving A and \beta^{-1} does include variability of \eta. It seems like you mean that equation (8) incorrectly includes variability of \eta and does not have the right term vector component of the inner product involving A, but this needs clarifying.

• One challenge with the z-diff score is that it does not account for whether a person sits above or below zero at the first time point. It might make it difficult to interpret the results, as the results for a particular pathology could change depending on what stage of the lifespan a person is in. I am not sure how the authors would address those challenges.

The world has been chaotic, but people were too preoccupied with survival to notice. As a result, chaos often went unnoticed. However, today is different. People have finally taken notice, but it's not because they've suddenly become aware of the world's problems. Rather, it's because their basic needs are finally being met, and they're no longer preoccupied with where their next meal is coming from.

Author response:

The following is the authors’ response to the current reviews.

We sincerely appreciate the reviewer’s dedication to evaluating our manuscript and raising essential considerations regarding the classification of the migration behavior we described. While the reviewer suggests that this behavior aligns with the concept of itinerancy, we contend that it represents a distinct phenomenon, albeit with similarities, as both involve the non-breeding movements of birds. We acknowledge that our manuscript did not adequately address this distinction and have considered the reviewer’s feedback. In our response, we clarify the difference between the described phenomenon and itinerancy. Our revised manuscript will include a new section in the Discussion to address this issue comprehensively.

In the first part of the review, the reviewer emphasizes that the pattern we are describing is consistent with itinerancy. Regardless of the terminology used, we want to highlight the existence of two different types of migratory behavior, both of which involve movement in non-breeding areas.

The first type, called itinerancy, was first described by Moreau in 1972 in “The Palaearctic-African Bird Migration Systems.” As noted by the reviewer, this behavior involves an alternation of stopovers and movements between different short-term non-breeding residency areas. They usually occur in response to food scarcity in one part of the non-breeding range, causing birds to move to another part of the same range. These movements typically cover distances of 10 to 100 kilometers but are neither continuous nor directional. Moreau (1972) defined itinerancy as prolonged stopovers, normally lasting several months, primarily in tropical regions. He noted observations of certain species disappearing from his study areas in sub-Saharan Africa in December and others appearing, suggesting they may have multiple home ranges during the non-breeding season. Subsequent research, as mentioned by the reviewer, has confirmed itinerancy in many species, particularly among Palaearctic-African migrants in sub-Saharan Africa. In particular, the Montagu’s Harrier has been extensively studied in this regard. The reviewer rightly points out that our study does not include recent findings on this species. In our revised version, we will include references to recent studies, such as those by Trierweiler et al. (2013, Journal of Animal Ecology, 82:107-120) and Schlaich et al. (2023, Ardea, 111:321-342), which show that Montagu’s Harrier has an average of 3-4 home ranges separated by approximately 200 kilometers. These studies suggest that the species spends approximately 1.5 months at each site, with the most extended period typically observed at the last site before migrating to the breeding grounds.

In the second type, birds undertake a post-breeding migration, arrive in their non-breeding range, and then gradually move in a particular direction throughout the season. This continuous directional movement covers considerable distances and continues throughout the non-breeding period. In our study, this movement covered about 1000 km, comparable to the total migration distance of Rough-legged Buzzards of about 1500 km. As observed in our research, these movements are influenced by external factors such as snow cover. In such cases, the progression of snow cover in a south-westerly direction during winter can prevent birds from finding food, forcing them to continue migrating in the same direction. In essence, this movement represents a prolonged phase of the migration process but at a slower pace. Similar behavior has been documented in buzzards, as reported by Strandberg et al. (2009, Ibis 151:200-206). Although several transmitters in their study stopped working in mid-winter, the authors observed a phenomenon they termed ‘prolonged autumn migration.’

In the second part of the review, the reviewer questions the need to distinguish between the two behaviors we have discussed. However, we believe these behaviors differ in their structure (with the first being intermittent and often non-directional, whereas the second is continuous and directional) and in their causes (with the first being driven by seasonal food resource cycles and the second by advancing snow cover). We therefore argue that it is worth distinguishing between them. To differentiate these forms of non-breeding movement, we propose to use ‘itinerancy’ for the first type, as described initially by Moreau in 1972, and introduce a separate term for the second behavior. Although ‘slow directional itinerancy’ could be considered, we find it too cumbersome.

Moreover, ‘itinerancy’ in the literature refers not only to non-breeding movements but also to the use of different nesting sites, e.g., Lislevand et al. (2020, Journal of Avian Biology: e02595), reinforcing its association with movements between multiple sites within habitats. We, therefore, propose that the second behavior be given a distinct name. We acknowledge the reviewer’s point that we did not adequately address this distinction in the Discussion and plan to include a separate section in our paper’s revised version. In the third part of his review, the reviewer suggests an alternative title. Another reviewer, Dr Theunis Piersma, suggested the current title during the first round of reviewing, and we have chosen his version.

In the fourth part of the review, the reviewer questions whether it is appropriate to discuss the conservation aspect of this study. This type of non-breeding movement raises concerns about accurately determining non-breeding ranges and population dynamics for species that exhibit this behavior. We believe that accurate determination of range and population dynamics is critical to conservation efforts. While this may be less important for species breeding in Europe and migrating to Africa, for which monitoring breeding territories is more feasible, it’s essential for Arctic and sub-Arctic breeding species. Large-scale surveys in these regions have historically been challenging and have become even more so with the end of Arctic cooperation following Russia’s war with Ukraine (Koivurova, Shibata, 2023). For North America and Europe, non-breeding abundance is typically estimated once per season in mid-winter. In North America, these are the so-called Christmas counts (which take place once at the end of December), and in Europe, they are the IWC counts mentioned by the reviewer (as follows from their official website - “The IWC requires a single count at each site, which should be repeated each year. The exact dates vary slightly from region to region, but take place in January or February”). Because of such a single count in mid-winter, non-breeding habitats occupied in autumn and spring will be listed as ‘uncommon’ at best, while south-western habitats where birds are only present in mid-winter will be listed as ‘common.’ However, the situation will be reversed if we consider the time birds spend in these habitats.

The reviewer also highlights the introduction’s unconventional structure and information redundancy at the beginning. We have chosen this structure and provided basic explanations to improve readability for a wider audience, given eLife’s readership. At the same time, we will certainly take the reviewers’ feedback into account in the revised version. We plan to include the references to modern itinerancy research mentioned above and to add a section on itinerancy to the Discussion.

We appreciate the reviewer’s input and sincerely thank them for their time and effort in reviewing our paper. While we may not fully agree on the classification of the behavior we describe, we value the opportunity to engage in discussion and believe that presenting arguments and counterarguments to the reader is beneficial to scientific progress.

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

I much enjoyed reading this manuscript, that is, once I understood what it is about. Titles like "Conserving bird populations in the Anthropocene: the significance of non-breeding movements" are a claim to so-called relevance, they have NOTHING to do with the content of the paper, so once I understood that this paper was about the "Quick quick slow: the foxtrot migration of rough-legged buzzards is a response to habitat and snow" (an alternative title), it was becoming very interesting. So the start of the abstract as well as the introduction is very tedious, as clearly much trouble is taken here to establish reputability. In my eyes this is unnecessary: eLife should be interested in publishing such a wonderful description of such a wonderful migrant in a study that comes to grips with limiting factors on a continental scale!

We sincerely appreciate your time and effort in reviewing our manuscript. Thank you for your appreciation of our study.

We agree that the focus of the article should be changed from conservation to migration patterns. We have rewritten the Introduction and Discussion as suggested. We have added the application of this pattern including conservation at the end of the Discussion by completely changing Figure 5. We have also changed the title to the suggested one.

Not sure that the first paragraph statements that seek to downplay what we know about wintering vs breeding areas are valid (although I see what purpose they serve). Migratory shorebirds have extensively been studied in the nonbreeding areas, for example, including movement aspects (see, as just one example, Verhoeven, M.A., Loonstra, A.H.J., McBride, A.D., Both, C., Senner, N.R. & Piersma, T. (2020) Migration route, stopping sites, and non breeding destinations of adult Black tailed Godwits breeding in southwest Fryslân, The Netherlands. Journal of Ornithology 162, 61-76) and there are very impressive studies on the winter biology of migrants across large scale (for example in Zwarts' Living on the Edge book on the Sahel wetlands). Think also about geese and swans and about seabirds!

We have rewritten the first paragraph and it now talks about patterns of migratory behavior. We have also rewritten the second paragraph, now it is devoted to studies of movements in the non-breeding period. We explain how our pattern differs from those already studied and give references to the papers you mentioned.

Directional movements in nonbreeding areas as a function of food (in this case locusts) have really beautifully been described by Almut Schlaich et al in JAnimEcol for Montagu's harriers.

We have added Montagu's harrier example in the second paragraph of the Introduction and the Discussion. We have added a reference to Schlaich and to Garcia and Arroyo, who suggested that Montagu's harriers have long directional migrations during the non-breeding period.

Once the paper starts talking buzzards, and the analyses of the wonderful data, all is fine. It is a very competent analysis with a description of a cool pattern.

Thank you for your appreciation of our study. We hope the revised version is better and clearer.

However, i would say that it is all a question of spatial scale. The buzzards here respond to changes in food availability, but there is not an animal that doesn't. The question is how far they have to move for an adequate response: in some birds movements of 100s of meters may be enough, and then anything to the scale of rough-legged buzzards.

In the new version of the manuscript, we emphasize that this is a large distance (about 1000 km), comparable to the distance of the fall and spring migrations (about 1400 km) in lines 70-72 of the Introduction and 379-383 of the Discussion.

And actually, several of the shorebirds I know best also do a foxtrot, such as red knots and bar-tailed godwits moulting in the Wadden Sea, then spending a few months in the UK estuaries, before returning to the Wadden Sea before the long migrations to Arctic breeding grounds. The publication of the rough-legged buzzard story may help researchers to summarize patterns such as this too. Mu problem with this paper is the framing. A story on the how and why of these continental movements in response to snow and other habitat features would be a grand contribution. Drop Anthropocene, and rethink whether foxtrot should be introduced as a hypothesis or a summary of cool descriptions. I prefer the latter, and recommend eLife to go with that too, rather than encourage "disconnected frames that seek 'respectability'" Good luck, theunis piersma

We thank the reviewer again for his valuable comments and suggestions. We have changed the framing to the suggested one and removed the Anthropocene from the article.

Reviewer #2 (Recommendations For The Authors):

We sincerely appreciate the time and effort you have taken to review our manuscript. We have carefully considered all of your comments, including both public and author comments, and provided detailed responses to each of them below. In addition, we would like to address the most important public comments.

We agree with the suggestion to shift the focus of the article from conservation to migration patterns. Accordingly, we have rewritten both the Introduction and Discussion sections to focus on migration behavior rather than conservation.

However, we respectfully disagree with the suggestion that the migration patterns we describe are synonymous with itinerancy. We acknowledge that our original presentation may have been unclear and may have hindered full understanding. In the revised version, we provide a detailed analysis of migratory behavior in the Introduction that describes how our pattern differs from itinerancy. We also revisit this distinction in the Discussion section. We have also carefully revised Figure 1 to improve clarity and avoid potential misunderstandings.

Regarding the applicability of the described migration pattern, we acknowledge that the Rough-legged Buzzard is not listed as an endangered species. However, we believe that our findings have practical implications. We have moved our discussion of this issue to the end of the Discussion section and have completely revised Figure 5. While the overall population of Rough-legged Buzzards is not declining, certain regions within its range are experiencing declines. We show that this decline does not warrant listing the species as endangered. Instead, it may represent a redistribution within the non-breeding range - a shift in range dynamics. We use the example of the Rough-legged Buzzard to illustrate this concept and emphasize the importance of considering such dynamics when assessing the conservation status of species in the future.

We also acknowledge that the hypothesis of this form of behavior has been proposed previously for Montagu's Harrier, and we have included this information in the revised manuscript. In addition, we agree that the focus on the Anthropocene is unnecessary in this context and have therefore removed it.

We believe that these revisions significantly improve the clarity and robustness of the manuscript, and we are grateful for your insightful comments and suggestions.

As a general comment, please note that including line numbers (as it is the standard in any manuscript submission) would facilitate reviewers providing more detailed comments on the text.

We apologize for this oversight and have added line numbers to our revised manuscript.

Dataset: unclear what is the frequency of GPS transmissions. Furthermore, information on relative tag mass for the tracked individuals should be reported.

We have included this information in our manuscript (L 157-163). We also refer to the study in which this dataset was first used and described in detail (L 164).

Data pre-processing: more details are needed here. What data have been removed if the bird died? The entire track of the individual? Only the data classified in the last section of the track? The section also reports on an 'iterative procedure' for annotating tracks, which is only vaguely described. A piecewise regression is mentioned, but no details are provided, not even on what is the dependent variable (I assume it should be latitude?).

Regarding the deaths. We only removed the data when the bird was already dead. We have corrected the text to make this clear (L 170).

Regarding the iterative procedure. We have added a detailed description on lines 175-188.

Data analysis: several potential issues here:

(1) Unclear why sex was not included in all mixed models. I think it should be included.

Our dataset contains 35 females and eight males. This ratio does not allow us to include sex in all models and adequately assess the influence of this factor. At the same time, because adult females disperse farther than males in some raptor species, we conducted a separate analysis of the dependence of migration distance on sex (Table S8) and found no evidence for this in our species. We have written a separate paragraph about this. This paragraph can be found on lines 356-360 of the new manuscript.

(2) Unclear what is the rationale of describing habitat use during migration; is it only to show that it is a largely unsuitable habitat for the species? But is a formal analysis required then? Wouldn't be enough to simply describe this?

Habitat use and snow cover determine the two main phases (quick and slow) of the pattern we describe. We believe that habitat analysis is appropriate in this case and that a simple description would be uninformative and would not support our conclusions.

(3) Analysis of snow cover: such a 'what if' analysis is fine but it seems to be a rather indirect assessment of the effect of snow cover on movement patterns. Can a more direct test be envisaged relating e.g. daily movement patterns to concomitant snow cover? This should be rather straightforward. The effectiveness of this method rests on among-year differences in snow cover and timing of snowfall. A further possibility would be to demonstrate habitat selection within the entire non-breeding home range of an individual in relation snow cover. Such an analysis would imply associating presence-absence of snow to every location within the non-breeding range and testing whether the proportion of locations with snow is lower than the proportion of snow of random locations within the entire non-breeding home range (95% KDE) for every individual (e.g. by setting a 1/10 ratio presence to random locations).

The proposed analysis will provide an opportunity to assess whether the Rough-legged Buzzard selects areas with the lowest snow cover, but will not provide an opportunity to follow the dynamics and will therefore give a misleading overall picture. This is especially true in the spring months. In March-April, Rough-legged Buzzards move northeast and are in an area that is not the most open to snow. At this time, areas to the southwest are more open to snow (this can be seen in Figure 4b). If we perform the proposed analysis, the control points for this period would be both to the north (where there is more snow) and to the south (where there is less snow) from the real locations, and the result would be that there is no difference in snow cover.

A step-selection analysis could be used, as we did in our previous work (Curk et al 2020 Sci Rep) with the same Rough-legged Buzzard (but during migration, not winter). But this would only give us a qualitative idea, not a quantitative one - that Rough-legged Buzzards move from snow (in the fall) and follow snowmelt progression (in the spring).

At the same time, our analysis gives a complete picture of snow cover dynamics in different parts of the non-breeding range. This allows us to see that if Rough-legged Buzzards remained at their fall migration endpoint without moving southwest, they would encounter 14.4% more snow cover (99.5% vs. 85.1%). Although this difference may seem small (14.4%), it holds significance for rodent-hunting birds, distinguishing between complete and patchy snow cover. Simultaneously, if Rough-legged Buzzards immediately flew to the southwest and stayed there throughout winter, they would experience 25.7% less snow cover (57.3% vs. 31.6%). Despite a greater difference than in the first case, it doesn't compel them to adopt this strategy, as it represents the difference between various degrees of landscape openness from snow cover.

We write about this in the new manuscript on lines 385-394.

Results: it is unclear whether the reported dispersion measures are SDs or SEs. Please provide details.

For the date and coordinates of the start and end of the different phases of migration, we specified the mean, sd, and sample size. We wrote this in line 277. For the values of the parameters of the different phases of the migration (duration, distance, speed, and direction), we used the mean, the standard error of the mean, and the confidence interval (obtained using the ‘emmeans’ package). We have indicated this in lines 302-303 and the caption of Table 1 (L 315) and Figure 2 (L 293-294). For the values of habitat and snow cover experienced by the Rough-legged Buzzards, we used the mean and the error of the mean. We reported this on lines 322 and 337 and in Figures 3 (L 332-333) and 4 (L 355-356).

Discussion: in general, it should be reshaped taking into account the comments. It is overlong, speculative and quite naive in several passages. Entire sections can be safely removed (I think it can be reduced by half without any loss of information). I provide some examples of the issues I have spotted below. For instance, the entire paragraph starting with 'Understanding....' is not clear to me. What do you mean by 'prohibited management' options? Without examples, this seems a rather general text, based on unclear premises when related to the specific of this study. Some statements are vague, derive from unsubstantiated claims, and unclear. E.g. "Despite their scarcity in these habitats, forests appear to hold significant importance for Rough-legged buzzards for nocturnal safety". I could not find any day-night analysis showing that they actually roost in forests during nighttime. Being a tundra species, it may well be possible that rough-legged buzzards perceive forests as very dangerous habitats and that they prefer instead to roost in open habitats. Analysing habitat use during day and night during the non-breeding period may be of help to clarify this. Furthermore, considering the fast migration periods, what is the flight speed during day and night above forests? Do these birds also migrate at night or do they roost during the night? Perhaps a figure visualizing day and night track segments could be of help (or an analysis of day vs. night flight speed) (there are several R packages to annotate tracks in relation to day and night). This is an example of another problematic statement: "The progression of snow cover in the wintering range of Rough-legged buzzards plays a significant role in their winter migration pattern." The manuscript does not contain any clear demonstration of this, as I wrote in my previous comments. Without such evidence, you must considerably tone down such assertions. But since providing a direct link is certainly possible, I think that additional analyses would clearly strengthen your take-home message.

The paragraph starting with "The quantification of environmental changes that could prove fatal to bird species presents yet another challenge for conservation efforts in an era of rapid global change." is quite odd. Take the following statement "For instance, the presence of small patches of woodland in the winter range might appear crucial to the survival of the Rough-legged buzzard. Elimination of these seemingly minor elements of vegetation cover through management actions could have dire consequences for the species.". It is based on the assumption that minor vegetation elements play a key role in the ecology of the species, without any evidence supporting this. Does it have any sense? I could safely say exactly the opposite and I would believe it might even be more substantiated.

We agree with these comments.

We have completely rewritten this section. As suggested, we have shortened it by removing statements that were not supported by the research. We have completely removed the statements about "prohibited management". We have also removed the statement that "forests appear to be of significant importance to Rough-legged buzzards for nocturnal safety" and everything associated with that statement, e.g. the statement about "small elements of vegetation cover", etc. We do believe that this statement is true in substance, but we also agree that it is not supported by the results and requires separate analysis. At the same time, we believe that this is a topic for a separate study and would be redundant here. Therefore, we leave it for a separate publication.

Conclusion paragraph: I believe this severely overstates the conservation importance of this study. That the results have "crucial implications for conservation efforts in the Anthropocene, where rapidly changing environmental factors can severely impact bird migration" seems completely untenable to me. What is the evidence for such crucial implications? For instance, these results may suggest that climate change, because global warming is predicted to reduce snow cover in the non-breeding areas, might well be beneficial for populations of this species, by reducing non-breeding energy expenditure and improving non-breeding survival. I think statements like these are simply not necessary, and that the study should be more focused on the actual results and evidence provided.

We have completely rewritten this section. We removed the reference to the Anthropocene and focused on migratory behavior and migration patterns.

Possible Problem with Cleavers Analysis of Frankfurt: Marcuse apparently evolved from the position he held in One dimensional man. https://marxandphilosophy.org.uk/reviews/8113_the-frankfurt-school-postmodernism-and-the-politics-of-the-pseudo-left-review-by-javier-sethness/ Eric commits the common fallacy of reducing “the Frankfurt School” to One-Dimensional Man. Please reread my words: ‘by the end of the same decade, he had jettisoned such pessimism. In An Essay on Liberation (1969), Marcuse clarifies his belief that the proletariat retains its revolutionary role, amidst the “historical power of the general strike and the factory occupation, of the red flag and the International” (Marcuse 1969, 51-3, 69).’

Cleaver backs up the view of Marcuse as being pessimist on page 56 using Marcuse's later work Counter Revolution and revolt which came out in 1972. One dimensional man came out in 1964 so it does not seem like he evolved.

But in the comment section from above I found this: I am reading the 1974 Paris Vincennes Lectures. These do indeed show a very different side to Marcuse. “Which are today the social and historical agents of radical change in the United States ? First among the working class… I never said that the working class can be replaced by any other class in the transition from capitalism to socialism.”...In fact the Vincennes Lectures make it seem that class struggle is more than just one current but is THE absolutely essential current (into which the other currents must flow in order to be historically effective). So this is very interesting indeed. It’s very important that these Vincennes Lectures become better known.

reply to u/EquivalentHead3589 at https://www.reddit.com/r/typewriters/comments/1cbzx1n/how_do_you_price_typewriters/

The primary difference is that listing prices don't indicate actual value. That is only determined by actual sales price. Things are worse for the listings which don't indicate much about condition as you're probably more likely to need to have the machine serviced and/or replace or recondition parts. This can often add a few hundred dollars (or significant research and time, tools, and elbow grease) to the bottom line to be able to use a machine.

I do recall a burgundy Olympia SM3 which sold in the last 4 months for right at $300 which was regularly used (loved) and serviced and in excellent condition with some fantastic photos. If you compare it to this Burgundy/Gray machine (https://www.ebay.com/itm/404901285037) for $299, but which has a missing key cap, and a damaged case, and may likely have other hiding issues. If you consider that you'll likely need to put a minimum of another $100 into this to get it up to the fighting shape that the first was in and it's still got damage, you'll start seeing the stark difference. The people with listings at $550-800 know they're not selling and they're just sitting there, so why not email them and ask more specific questions about condition and get a typed typeface sample of all the keys. Then make an offer for $200 +/- with some wiggle room for service costs once you've gotten it to see if they'll sell?

As an example, look at https://www.ebay.com/itm/226016437104 which is a Gray SM3 originally listed for $549 and now on sale for $428. The seller knows it's not moving. They state that they got it at an estate sale (probably for around $25) and they definitely did no work other than quick check of the keys. If you demonstrate that you've savvy enough to know the specific machine (what shape are the rubber washers on the frame next to the feet to prevent the carriage from rubbing against the frame? how what is the durometer measurement on (how hard is) the platen?), the market (in top shape maybe $300), and what servicing/repair costs are, they'd probably accept an offer of $150-200 and you're off to the races and they've made a solid profit.

The biggest issue in the typewriter market at present is the broad lack of information and knowledge about them on both the buyer and seller side. If you can demonstrate you've got more knowledge than the other side, you'll be in a far better position to negotiate, otherwise a seller can sit and wait an undetermined amount of time waiting for a sucker who will likely never show up.

Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go Look, I'm bettin' you like people And I'm bettin' you love freak mode And I'm bettin' you like girls That give love to girls and stroke your little ego I bet you I'm guilty, your honor That's just how we live in my genre Who in the hell done paved the road wider? There's only one Flo and one Rida I'm a damn shame, order more champagne Pull a damn hamstring tryna put it on ya Let your lips spin back around corner Slow it down, baby, take a little longer Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby It's like everywhere I go, my whistle ready to blow Shawty don't even know, she can get any for the low Told me she's not a pro, it's okay, it's under control Show me soprano 'cause, girl, you can handle Baby, we start slow then you come up and park close Girl, I'm the whistle man, my Bugatti the same notes Show me your perfect pitch, you got it, my banjo Talented with your lips like you blew out a candle So amusing (amusing) Now you can make a whistle with the music (music) Hope you ain't got no issues, you can do it (do it) Even if it's no picture, never lose it (lose it) Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go (hey) Whistle baby, whistle baby (hey) Whistle baby, whistle baby Whistle baby, whistle baby Whistle baby, whistle baby Go on, girl, you can twerk it Let me see you whistle while you work it I'ma lay back, don't stop it 'Cause I love it how you drop it, drop it, drop it on me Now, shorty, let that whistle blow, oh-oh-oh Yeah, baby, make that whistle blow, oh-oh Can you blow my whistle baby, whistle baby Let me know Girl I'm gonna show you how to do it And we start real slow You just put your lips together And you come real close Can you blow my whistle baby, whistle baby Here we go You blow my whistle baby Whistle baby, whistle baby You blow my whistle baby Whistle baby, whistle baby You blow my whistle baby Whistle baby, whistle baby You blow my whistle baby Whistle baby, whistle baby

Not sure what to make of this, a combination of Solid Pods and AP, with Solid being the data storage. Meaningless refs to data ownership (you don't own your data on Fedi, you've spread it to 100s of servers around the globe with each message. You don't even have control over the database you use in your client, unless self-hosted. You can move, without your messages.) It's just that nobody afayk mines the stuff for adtech. And data ownership doesn't legally exist in most parts of the world. So what is the purpose of Solid here, if you store recipes and ephemeral socmed messages in it? Just that it's there so you can skip having to build the database part of an AP server / client combo? So that everyone can run their personal instance with something that can also do other things? It doesn't say but that would be a potential step up (assuming people know how to run a solid pod that is).

I like this.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors present a detailed study of a nearly complete Entomophthora muscae genome assembly and annotation, along with comparative analyses among related and non-related entomopathogenic fungi. The genome is one of the largest fungal genomes sequenced, and the authors document the proliferation and evolution of transposons and the presence/absence of related genetic machinery to explore how this may have occurred. There has also been an expansion in gene number, which appears to contain many "novel" genes unique to E. muscae. Functionally, the authors were interested in CAZymes, proteases, circadian clock related genes (due to entomopathogenicity/ host manipulation), other insect pathogenspecific genes, and secondary metabolites. There are many interesting findings including expansions in trahalases, unique insulinase, and another peptidase, and some evidence for RIP in Entomophthoralean fungi. The authors performed a separate study examining E. muscae species complex and related strains. Specifically, morphological traits were measured for strains and then compared to the 28S+ITSbased phylogeny, showing little informativeness of these morpho characters with high levels of overlap.

This work represents a big leap forward in the genomics of non-Dikarya fungi and large fungal genomes. Most of the gene homologs have been studied in species that diverged hundreds of millions of years ago, and therefore using standard comparative genomic approaches is not trivial and still relatively little is known. This paper provides many new hypotheses and potential avenues of research about fungal genome size expansion, entomopathogenesis in zygomycetes, and cellular functions like RIP and circadian mechanisms.

Strengths:

There are many strengths to this study. It represents a massive amount of work and a very thorough functional analysis of the gene content in these fungi (which are largely unsequenced and definitely understudied). Too often comparative genomic work will focus on one aspect and leave the reader wondering about all the other ways genome(s) are unique or different from others. This study really dove in and explored the relevant aspects of the E. muscae genome.

The authors used both a priori and emergent properties to shape their analyses (by searching for specific genes of interest and by analyzing genes underrepresented, expanded, or unique to their chosen taxa), enabling a detailed review of the genomic architecture and content. Specifically, I'm impressed by the analysis of missing genes (pFAMs) in E. muscae, none of which are enriched in relatives, suggesting this fungus is really different not by gene loss, but by its gene expansions.

Analyzing species-level boundaries and the data underlying those (genetic or morphological) is not something frequently presented in comparative genomic studies, however, here it is a welcome addition as the target species of the study is part of a species complex where morphology can be misleading and genetic data is infrequently collected in conjunction with the morphological data.

Thank you for your careful reading of our work. We’re glad that you identified these areas as strengths.

Weaknesses:

The conclusions of this paper are mostly well supported by data, but a few points should be clarified.

In the analysis of Orthogroups (OGs), the claim in the text is that E. muscae "has genes in multi-species OGs no more frequently than Enotomophaga maimaiga. (Fig. 3F)" I don't see that in 3F. But maybe I'm really missing something.

Thank you for catching this. You were, in fact, not missing anything at all. There was a mismatch between the data plotted in F and G and how the caption described these data. We very much apologize for the confusion that this must have caused. We have corrected these plots and also made changes to improve interpretability (see below).

Also related, based on what is written in the text of the OG section, I think portions of Figure 3G are incorrect/ duplicated. First, a general question, related to the first two portions of the graph. How do "Genes assigned to an OG" and "Genes not assigned to an OG" not equal 100% for each species? The graph as currently visualized does not show that. Then I think the bars in portion 3 "Genes in speciesspecific OG" are wrong (because in the text it says "N. thromboides had just 16.3%" species-specific OGs, but the graph clearly shows that bar at around 50%. I think portion 3 is just a duplicate of the bars in portion 4 - they look exactly the same - and in addition, as stated in the text portion 4 "Potentially speciesspecific genes" should be the simple addition of the bars in portion 2 and portion 3 for each species.

As mentioned above, we sincerely regret the error made in the plot and for the confusion that this caused. F now reflects the percentage of orthogroups (OGs) that possess at least one representative from the indicated species (left) and the percentage of OGs that are species-specific (only possess genes from one species; right). The latter is a subset of the former. G now reflects the percentage of annotated genes that were assigned an OG, per species, as well as the inverse of this - genes that were not assigned to any OG. These should, and now do, sum to 100%. The “Within species-specific OG” data summed with the “Not assigned OG” data yields the “Potentially species-specific data” in the rightmost column.

In the introduction, there is a name for the phenomenon of "clinging to or biting the tops of plants," it's called summit disease. And just for some context for the readers, summit disease is well-documented in many of these taxa in the older literature, but it is often ignored in modern studies - even though it is a fascinating effect seen in many insect hosts, caused by many, many fungi, nematodes (!), etc. This phenomenon has evolved many times. Nice discussions of this in Evans 1989 and Roy et al. 2006 (both of whom cite much of the older literature).

You’re right. We have now clarified that this behavior is called “summit disease” and referenced the suggested articles, along with a more recent review.

Reviewer #2 (Public Review):

In their study, Stajich and co-authors present a new 1.03 Gb genome assembly for an isolate of the fungal insect parasite Entomophthora muscae (Entomophthoromycota phylum, isolated from Drosophila hydei). Many species of the Entomophthoromycota phylum are specialised insect pathogens with relatively large genomes for fungi, with interesting yet largely unexplored biology. The authors compare their new E. muscae assembly to those of other species in the Entomophthorales order and also more generally to other fungi. For that, they first focus on repetitive DNA (transposons) and show that Ty3 LTRs are highly abundant in the E. muscae genome and contribute to ~40% of the species' genome, a feature that is shared by closely related species in the Entomophthorales. Next, the authors describe the major differences in protein content between species in the genus, focusing on functional domains, namely protein families (pfam), carbohydrate-active enzymes, and peptidases. They highlight several protein families that are overrepresented/underrepresented in the E. muscae genome and other

Entomophthorales genomes. The authors also highlight differences in components of the circadian rhythm, which might be relevant to the biology of these insect-infecting fungi. To gain further insights into E. muscae specificities, the authors identify orthologous proteins among four Entomophthorales species. Consistently with a larger genome and protein set in E. muscae, they find that 21% of the 17,111 orthogroups are specific to the species. To finish, the authors examine the consistency between methods for species delineation in the genus using molecular (ITS + 28S) or morphological data (# of nuclei per conidia + conidia size) and highlight major incongruences between the two.

Although most of the methods applied in the frame of this study are appropriate with the scripts made available, I believe there are some major discrepancies in the datasets that are compared which could undermine most of the results/conclusions. More precisely, most of the results are based on the comparison of protein family content between four Entomophthorales species. As the authors mention on page 5, genome (transcriptome) assembly and further annotation procedures can strongly influence gene discovery. Here, the authors re-annotated two assemblies using their own methods and recovered between 30 and 60% more genes than in the original dataset, but if I understand it correctly, they perform all downstream comparative analyses using the original annotations. Given the focus on E. muscae and the small sample size (four genomes compared), I believe performing the comparisons on the newly annotated assemblies would be more rigorous for making any claim on gene family variation.

Thank you for this comment. While we did compare gene model predictions for two of these assemblies to assess if this difference could account for discrepancies in gene counts, completely reannotating all non-E. muscae datasets was outside of the scope of this study. In our opinion, the total number of predicted genes in a genome is not a best representation of differences since splitting or fusing gene models can inflate seeming differences; the orthology and domain counts are a more accurate assessment of the content. It’s possible that annotation differences may have inflated some gene family counts, however we will note that similar domain trends were observed between the closest species to E. muscae, Entomophaga maimaiga, suggesting that these differences were not sufficient to prevent us from detecting real biological signals. We look forward to continued improvement of our genome through additional sequencing and more clarity on total gene content of E. muscae.

The authors also investigate the putative impact of repeat-induced point mutation on the architecture of the large Entomophthorales genomes (for three of the eight species in Figure 1) and report low RIP-like dinucleotide signatures despite the presence of RID1 (a gene involved in the RIP process in Neurospora crassa) and RNAi machinery. They base their analysis on the presence of specific PFAM domains across the proteome of the three Entomophthorales species. In the case of RID1, the authors searched for a DNA methyltransferase domain (PF00145), however other proteins than RID1 bear such functional domain (DNMT family) so that in the current analysis it is impossible to say if the authors are actually looking at RID1 homologs (probably not, RID1 is monophyletic to the Ascomycota I believe). Similar comments apply to the analysis of components of the RNAi machinery. A more reliable alternative to the PFAM analysis would be to work with full protein sequences in addition to the functional domains.

While we understand this concern regarding domain vs. full length protein, the advantage of the domain search is that HMM-based searches are sensitive to detecting more distantly related homologs. Entomophthoralean fungi are distantly related from the ascomycetes in which these mechanisms have been characterized, so we chose a broader search approach that may identify proteins with similar domain structure, but are not necessarily homologs. These searches are presented in the manuscript as preliminary, but worth further investigation. However, our RID-based analysis did not identify convincing homologs for RID1 in entomophthoralean fungi included in our investigation, and we reported low homology (i.e., 12-14%) among our orthogroup of interest and RID1. We have further edited this section to clarify our understanding that these candidates are not RID1 homologs. We had hoped to avoid this implication, but we felt this investigation and null result were worth reporting.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Specific points:

Results:

"1.03 Gb genome consisting of 7,810 contigs (N50 = 301.1 kb). Additional... resulted in a final contig count of 7,810 (N50 = 329.6 kb)" So you started and ended with the same contig count but a different N50? Is this a typo?

Yes, this was a typo. Thank you for bringing this to our attention.

Figure 1D.

The colors of Complete1x and Complete2x are too similar to tell them apart.

The colors have been made more distinct.

Figure 4B.

I know C. rosea has been found from insects before, but it's mostly a mycoparasite and occasionally an endophyte, and has bioactivity against a lot of things. I just saw that it's listed as an entomopathogen, and I was surprised. Anyway, leave it as is if you want to, but it's definitely better studied and better known (Google Scholar) as a mycoparasite.

Thanks for this comment. For the sake of including a more diverse representation of entomopathogenic fungi, we have opted to leave this as is.

Full references (from the public comment)

Evans, H.C., 1989. Mycopathogens of insects of epigeal and aerial habitats. Insect-fungus interactions, pp.205-238.

Roy, H.E., Steinkraus, D.C., Eilenberg, J., Hajek, A.E. and Pell, J.K., 2006. Bizarre interactions and endgames: entomopathogenic fungi and their arthropod hosts. Annu. Rev. Entomol., 51, pp.331-357.

Reviewer #2 (Recommendations For The Authors):

I believe the manuscript could largely benefit from restructuring the results section to enhance clarity. The results section reads like a lot of descriptive back and forth, so that the reader lacks a clear rationale. The absence of a consistent dataset used for the different comparisons made all along the manuscript makes it hard to follow.

Minor comments:

(No line numbers were available so I refer to page numbers).

p1

• not sure about the use of "allied" to describe other fungal species in the title and after (sister species?).

We didn’t want to use the word sister because not all of these species could be considered sister.

• Genomic defence against transposable elements rather than "anti"?

We have rephrased to genomic defense.

p3

• Extra parenthesis at Bronski et al.

This is now corrected.

• What does newly-available mean here?

We mean recent. A lot of the datasets we used were very new, and we wanted to emphasize that point.

• The back and forth between genomes and transcriptomes makes it hard to follow, would clarify from the beginning (in addition to the sequencing method - short vs long-read assemblies as in Figure 1B) or perhaps use a consistent dataset for all subsequent comparative analysis in the Entomophthorales.

We have denoted our transcriptomic datasets in Fig 1C using parentheses.

p5

• Perhaps clarify that class II DNA transposons can also "copy" (single-strand excisions can be repaired by the host machinery).

We have now included mention of “copy” as well as “jump” mechanisms of Class II transposons per your suggestion.

p6

• "beginning roughly concurrently", not clear what "began".

This is now corrected.

• "control" rather than "protect against"?

We’ve changed “protect against” to “counter”.

• I believe RIP has only been observed (experimentally) in a handful of fungal species, all from the Ascomycota phylum.

Hood et al, 2005 found signatures of RIP in anther-smut fungus and Horns et al, 2012, found evidence of hypermutability across repeat elements within several Pucciniales species.

• "RID1 contains two DNA_methylase domains", RID1 has one methyltransferase domain according to the reference Freitag et al, 2002.

Thank you for drawing this to our attention. It is true RID1 has one methyltransferase region; however, the sequence deposited by Freitag et al, 2002 (AAM27408) is predicted by HMMer to have two adjacent Pfam DNA_methylase domains (i.e., PF00145). In this exploratory analysis, we tried to leverage this characteristic to identify candidate proteins of interest. We have reworded this section to clarify this.

p8

• Here and after I would use more informative titles for each paragraph.

With the exception of the headings for Pfam, CAZy and MEROPs analyses, we believe the other headings are informative. We appreciate this comment, but opt to leave the heading titles as is.

• I believe presenting the orthology analysis before the more in-depth protein family domain search.

We leveraged the OG analysis mostly as a way to identify potentially unique genes in E. muscae, so we think the current order makes the most sense.

p10

• Figures 3F and G are confusing. The legend for Figure 3F mentions "OGs with >= 2 species" while the figure shows "multi-species OGs", and reads as redundant with the "species-specific" OGs. For the "OGs within species" do I understand it correctly that it represents the number of genes assigned to OGs for each species? If yes, the numbers are in contradiction with Figure 3G. And in Figure 3G shouldn't the sum of "genes assigned in OGs" and "genes nor assigned in OGs" add up to 100? I'm probably missing something here, but I would clarify what the different sets of orthogroups are in the figure and in the text (perhaps adopting a pangenome-like nomenclature).

Thanks for this comment. This legend, unfortunately, reflected an earlier version of the figure and was overlooked prior to submission. We have since amended this and sincerely apologize for the error on our part.

p12

• The whole first paragraph reads more like it should be part of an introduction/discussion.

We’ve moved some of this paragraph to the discussion but left the background information necessary for the reader to understand why we were looking for homologs of wc and frq.

p13

• The last paragraph reads like discussion.

We have revised this paragraph so it now reads: “Because E. muscae is an obligate insect-pathogen only living inside live flies, we investigate the presence of canonical entomopathogenic enzymes in the genome. We find that E. muscae appear to have an expanded group of acid-trehalases compared to other entomopathogenic and non-entomopathogenic Entomophthorales (Fig. 4A), which correlates with the primary sugar in insect blood (hemolymph) being trehalose (Thompson, 2003). The obligate insectpathogenic lifestyle is also evident when comparing the repertoire of lipases, subtilisin-like serine proteases, trypsins, and chitinases in our focal species versus Zoopagomycota and Ascomycota fungi that are not obligate insect pathogens (Fig. 4B). Sordariomycetes within Ascomycota contains the other major transition to insect-pathogenicity within the kingdom Fungi (Araújo and Hughes, 2016). Based on our comparison of gene numbers, Entomophthorales possess more enzymes suitable for cuticle penetration than Sordariomycetes (Fig. 4B). In contrast, insect-pathogenic fungi within Hypocreales possess a more diverse secondary metabolite biosynthesis machinery as evidenced by the absence of polyketide synthase (PKS) and indole pathways in Entomophthorales (Fig. 4C).”

p15 and 16

• This all reads as redundant with the previous protein family domain analysis. I would try to merge them.

Thank you for this comment, however we have opted to maintain the current structure.

p18

• In the first sentence, I'm not sure about what was performed here.

This has been reworded to clarify.

p20

• Regarding the assembly, do I understand it correctly that a nuclear genome can be partially haploid / diploid?

Thanks for your comment. The genome itself is, of course, some integer multiple of n, but based on BUSCO scores our assembly doesn’t appear to have completely collapsed into a haploid genome. We think it makes more sense here to say “partially haploid” than “partially diploid” so have altered this.

p21

• RIP has only been observed in a couple of Ascomycetes. RIP-like genomic signatures (GC bias) have been observed elsewhere.

Hood et al, 2005 found signatures of RIP in anther-smut fungus and Horns et al, 2012, found evidence of hypermutability across repeat elements within several Pucciniales species.

p23

• Interesting that the peptidase A2B domain is found uniquely in E. muscae genome and is associated with Ty3 activity. Does the domain often overlap with annotated Ty3 in E. muscae genome? Or how come the domain is not present in other sister species with large genomes full of Ty3 transposons? Could it relate to a new active transposon in E. muscae specifically?

Thanks for this comment. The domain-based analysis was only performed on the predicted transcriptome of the genome assembly, which does not include the repeat elements (e.g., Ty3). It could be that this peptidase reflects a new active transposon that’s specific to E. muscae, which would certainly be very interesting. We’ve now included this idea in the discussion.

p26

• In the case of fungal genomes, I would not advise masking the assembly for repeated sequences prior to gene annotation (in particular given the current focus on protein family variation).

Thank you for this comment, however we disagree with this assertion as a typical approach for genome annotation in fungi and eukaryotic genomes is to use soft masking of transposable elements before performing gene prediction to avoid over-prediction. While there could be alternative approaches that compare masked or unmasked. This is a recommended protocol for underlying tools like Augustus (10.1002/cpbi.57) and in general descriptions of genome annotation (10.1002/0471250953.bi0401s52). The false positive rate of genes predicted through TE regions is likely to be more a problem than false negatives of missed genes in our experience. Further it seems appropriate to use consistent approach to annotation throughout when including genomes from other sources (e.g., Joint Genome Institute annotated genomes) which also use a repeat masking approach first before annotation. It seems most appropriate to use consistent methods when generating datasets to be used for comparative analyses. It is outside the scope of this project to reannotate all genomes with and without repeat masking.

p27

• Interrupted sentence at "Classification of DNA and LTR .. by similarity The".

This was an unnecessary partial phrase as the information on classification of elements via RepBase was made a few sentences above this.

p28

• Enriched/depleted rather than "significantly different"?

Thank you for this comment, however we have opted to maintain the current phrasing.

Same problem here. B is not an arbitrary Borel set of R^2. It's a subset of the power set of {0,1} cross the Borel sets of R.

Actually, I guess you can get away with just taking a Borel set in R^2, but that might be confusing for people since most of them are "silly" in this context. I don't know, something to think about.

This is actually what I did with RVVMs. Since the power set on {0,1} is a sub-sigma-algebra of the Borel sets on R, you can still define a Bernoulli measure on the Borel sets on R. It's just that it's overkill. But the convenience is that you can just always talk about Borel sets then and not have to switch sigma-algebras. You let the distributions or random variables inform which Borel sets are actually meaningful. Pros and cons to each approach here.

I might consider reversing the order of these items. It is much more natural for people to think of a "sample space" as the set of experimental objects. So start with that. Then, with that notation and concept established, introduce the "universe of possible outcomes" sample space.

Aside: I know it's common to write, but I've never liked the language "universe of possible outcomes." It can be misleading. This comes from my physics training though. Really, the sample space Omega is "possibility space" or "all parallel worlds", etc. It's just a set, but there isn't any distribution or function attached to it yet, so it's always been confusing to me to talk about "outcomes" - outcomes of what? You're likely better off just leaving the language as is, especially since it's so common, but I just wanted to point out the ambiguity.

This method essentially treats each password like its own secret treasure, locked away in a unique encrypted vault. It's like having a separate lock for each door in a massive mansion - even if someone gets inside, they can't just wander around and open any door they please. Instead, they're left with the daunting task of trying every key in existence to find the right one. And let's face it, that's not only time-consuming but also incredibly frustrating. So, by giving each password its own special treatment, we're essentially making it a lot harder for anyone to break in and steal our precious data. It's like having a personalized bodyguard for each piece of sensitive information!

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

The authors build upon prior data implicating the secreted peptidoglycan hydrolase SagA produced by Enterococcus faecium in immunotherapy. Leveraging new strains with sagA deletion/complementation constructs, the investigators reveal that sagA is non-essential, with sagA deletion leading to a marked growth defect due to impaired cell division, and sagA being necessary for the immunogenic and anti-tumor effects of E. faecium. In aggregate, the study utilizes compelling methods to provide both fundamental new insights into E. faecium biology and host interactions and a proof-of-concept for identifying the bacterial effectors of immunotherapy response.

We thank the Reviewers for their positive feedback on our manuscript. We also appreciate their helpful comments/critiques and have revised the manuscript as indicated below.

Public Reviews:

Reviewer #1 (Public Review):

Klupt, Fam, Zhang, Hang, and colleagues present a novel study examining the function of sagA in E. faecium, including impacts on growth, peptidoglycan cleavage, cell separation, antibiotic sensitivity, NOD2 activation, and modulation of cancer immunotherapy. This manuscript represents a substantial advance over their prior work, where they found that sagA-expressing strains (including naturally-expressing strains and versions of non-expressing strains forced to overexpress sagA) were superior in activating NOD2 and improving cancer immunotherapy. Prior to the current study, an examination of sagA mutant E. faecium was not possible and sagA was thought to be an essential gene.

The study is overall very carefully performed with appropriate controls and experimental checks, including confirmation of similar densities of ΔsagA throughout. Results are overall interpreted cautiously and appropriately.

I have only two comments that I think addressing would strengthen what is already an excellent manuscript.

In the experiments depicted in Figure 3, the authors should clarify the quantification of peptidoglycans from cellular material vs supernatants. It should also be clarified whether the sagA need to be expressed endogenously within E. faecium, and whether ambient endopeptidases (perhaps expressed by other nearby bacteria or recombinant enzymes added) can enzymatically work on ΔsagA cell wall products to produce NOD2 ligands?

We mentioned in the main text that peptidoglycan was isolated from bacterial sacculi and digested with mutanolysin for LC-MS analysis. We have now also included “mutanolysin-digested” sacculi in the Figure 3 legend as well.

We have added the following text “We next evaluated live bacterial cultures with mammalian cells to determine their ability to activate the peptidoglycan pattern recognition receptor NOD2” and “our analysis of these bacterial strains” to indicate live cultures were evaluated for NOD2 activation.

We have also added the following text “Our results also demonstrated that while many enzymes are required for the biosynthesis and remodeling of peptidoglycan in E. faecium, SagA is essential for generating NOD2 activating muropeptides ex vivo.”

In the murine experiments depicted in Figure 4, because the bacterial intervention is being performed continuously in the drinking water, the investigators have not distinguished between colonization vs continuous oral dosing of the mice peptidoglycans. While I do not think additional experimentation is required to distinguish the individual contributions of these 2 components in their therapeutic intervention, I do think the interpretation of their results should include this perspective.

We have added the following text “We note that by continuous oral administration in the drinking water, live E. faecium and soluble muropeptides that are released into the media during bacterial growth may both contribute to NOD2 activation in vivo.” and revised the following text “Nonetheless, these results demonstrate SagA is not essential for E. faecium colonization, but required for promoting the ICI antitumor activity through NOD2 in vivo.

Reviewer #2 (Public Review):

Summary:

The gut microbiome contributes to variation in the efficacy of immune checkpoint blockade in cancer therapy; however, the mechanisms responsible remain unclear. Klupt et al. build upon prior data implicating the secreted peptidoglycan hydrolase SagA produced by Enterococcus faecium in immunotherapy, leveraging novel strains with sagA deleted and complemented. They find that sagA is non-essential, but sagA deletion leads to a marked growth defect due to impaired cell division. Furthermore, sagA is necessary for the immunogenic and anti-tumor effects of E. faecium. Together, this study utilizes compelling methods to provide fundamental new insights into E. faecium biology and host interactions, and a proof-of-concept for identifying the bacterial effectors of immunotherapy response.

Strengths:

Klupt et al. provide a well-written manuscript with clear and compelling main and supplemental figures. The methods used are state-of-the-art, including various imaging modalities, bacterial genetics, mass spectrometry, sequencing, flow cytometry, and mouse models of immunotherapy response. Overall, the data supports the conclusions, which are a valuable addition to the literature.

Weaknesses:

Only minor revision recommendations were noted.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

General comments - the number/type of replicates and statistics are missing from some of the figure panels. Please be sure to add these throughout - all main figure panels should have replicates. I've also noted some specific cases below.

Abstract - sagA is non-essential, need to edit text at "essential functions".

This change has been made.

"small number of mutations" - specify how many in the text.

We revised the text. “Small number” is changed to “11”.

"under control of its native promoter" - what was the plasmid copy number? It looks clearly overexpressed in Figure 1d despite using a native promoter, although it's a bit hard to know for sure without a loading control.

pAM401 has p15A origin of replication, therefore the plasmid copy number ~20-30 copies (Lutz R. et al Nucleic Acids Res. 1997). Total protein was visualized by Stain-Free™ imaging technology (BioRad) and serves as protein loading control and has been relabeled accordingly.

"decrease levels of small muropeptides" - the asterisks are missing from Figure 3a.

Green asterisks for peaks 2, 3, 7 and purple asterisks for peaks 13, 14 were added.

The use of "Com 15 WT" in the figures is confusing - just replace it with "wt" and specify the strain in the text. Presumably, all of the strains are on the Com 15 background.

“Com15 WT” was replaced to “WT” in figures and main text.

Change 1d to 1b so that the panels are in order (reading left to right and then top to bottom).

Figure 1 legend is missing a number of replicates and statistics for 1a.

Number of replicates were added.

Figure 1b - it's unclear to me what to look at here, could add arrows indicating the feature or interest and expand the relevant text.

Arrows pointing to cell clusters were added.

Figure 1d - what is "stain free"? It would be preferable to show a loading control using an antibody against a constitutive protein to allow for normalization of the loading control.

Stain-Free Imaging technology (BioRad) utilizes gel-containing trihalo compound to make proteins fluorescent directly in the gel with a short photoactivation, allowing the immediate visualization of proteins at any point during electrophoresis and western blotting. Stain-Free total protein measurement serves as a reliable loading control comparable to Coomassie Blue Staining. This has been relabeled a “Total protein” in the Figure and Stain-free imaging technology is noted in the legend.

ED Figure 1 - representative of how many biological replicates?

Legends are updated.

ED Figure 2a - I would replace this with a table, it's not necessary to show the strip images. Also, please specify the number of replicates per group.

Additional Extended Data Table 2 was added.

ED Figure 2b - This data was not that convincing since the sagA KO has a marked growth defect and the time points are cut off too soon to know if growth would occur later. The MIC definition is potentially misleading. Should specific a % growth cutoff (i.e. <10% of vehicle control) and the metric used (carrying capacity or AUC). Then assign MIC to the tested concentration, not a range. The empty vector also seems to impact MIC, which is concerning and complicates the interpretation. Specify the number of replicates and add statistics. Given these various concerns, I might suggest removing this figure, as it doesn't really add much to the story.

We appreciate this comment from the Reviewer, but believe this data is helpful for paper and have included longer time points for the growth data. The definition of MIC for ED Fig. 2b has been included in the legend.

Figure 2 - specify the type of replicate. Number of cells? Number of slices? Number of independent cultures?

For Cryo-ET experiments single bacterial cultures were prepared. Number of cells and slices for analysis are indicated in the legend. Legends are updated.

Figure 4e - missing the water group, was it measured?

Water (αPD-L1) group was not included in immune profiling of tumor infiltrating lymphocytes (TILs) experiment, as we have previously demonstrated limited impact on ICI anti-tumor activity and T cell activation in this setting (Griffin M et al Science 2021).

Figure 4d - is this media specific to your strains? If not, qPCR may be a better method using strain-specific primers.

Yes, HiCrome™ Enterococcus faecium agar plates (HIMEDIA 1580) are selective for Enterococcus species, moreover the agar is chromogenic allowing to identify E. faecium as yellow colonies among other Enterococcus species.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

RC-2023-02105R: Brunetta et al.,

IF1 is a cold-regulated switch of ATP synthase to support thermogenesis in brown fat

We are happy to submit our revised manuscript after considering the suggestions made by reviewers. The comments were overall positive, and the changes requested were mostly editorial. We have, nevertheless, added new experiments as quality controls. These experiments did not affect the main conclusions of our work. In addition, we also included two in vivo experimental models of gain and loss-of-function, to further address the physiological relevance of IF1 in BAT thermogenesis. We believe with these additional experiments, quality controls as well as in vivo models, our study has improved considerably. We hope our efforts will be appreciated by the reviewers and we make ourselves available to answer any further questions.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: In the present manuscript, the authors present data in support of their primary discovery that "IF1 controls UCP1-dependent mitochondrial bioenergetics in brown adipocytes". The opening figure convincingly demonstrates that IF1 expression is cold-exposure dependent. They then go on to show that loss of IF1 has functional consequences that would be predicted based on IF1's know role as a regulator of ATP hydrolysis by CV. They go on to make a few additional claims, succinctly detailed in the Discussion section. Specific claims include the following: 1) IF1 is downregulated in cold-adapted BAT, allowing greater hydrolytic activity of ATP synthase by operating in the reverse mode; 2) when IF1 is upregulated in brown adipocytes in vitro mitochondria unable to sustain the MMP upon adrenergic stimulation, 3) IF1 ablation in brown adipocytes phenocopies the metabolic adaptation of BAT to cold, and 4) IF1 overexpression blunts mitochondrial respiration without any apparent compensator response in glycolytic activity. The claims described above are well supported by the evidence. The manuscript is very well written, figures are clear and succinct. Overall, the quality of the work is very high. Given that IF1 is implicated across many fields of study, the novel discovery of IF1 as a regulator of brown adipose mitochondrial bioenergetics will be of significance across several fields. That said, a few areas of concern were apparent. Concerns are detailed in the "Major" and "Minor" comments section below. Additional experiments do not appear to be required, assuming the authors adequately acknowledge the limitations of the study and either remove or qualify speculative claims.

Major Comments:

1. The authors convincingly demonstrate that IF1 expression is specifically down-regulated in BAT upon cold-exposure. These data strongly implicate a role for IF1 in BAT bioenergetics, a major claim of the authors and a novel finding herein. Additional major strengths of the paper, which provide excellent scientific rigor include the use of both loss of function and gain of function approaches for IF1. In addition, the mutant IF1 experiments are excellent, as they convincingly show that the effects of IF1 are dependent on its ability to bind CV. RESPONSE: We thank the reviewer for the positive feedback on our work.

Regarding Figure 1 - Did the content of ATP synthase change? In figure 1A-B, the authors show that ATPase activity of CV is higher in cold-adapted mice. While this result could be due to a loss of IF1, it could also be due to a higher expression of CV. To control for this, the authors should consider blotting for CV, which would allow for ATPase activity to be normalized to expression.

RESPONSE: Thank you for this suggestion. We have now determined complex V subunit A in our experimental protocol. We found that cold exposure does not impact complex V protein levels. Given the importance of this control, we have now included it in Figure 1 (Please, see the revised version) alongside the IF1/complex V ratio. In addition, we have now performed WBs in the BAT from mice exposed for 3 and 7 days to thermoneutrality (~28°C). We found that IF1 is not reduced following whitening of BAT by this approach whilst UCP1 and other mitochondrial proteins are reduced. This set of data is now included in Figure 1I,K,L.

Regarding MMP generated specifically by ATP hydrolysis at CV, the reversal potential for ANT occurs at a more negative MMP than that of CV (PMID: 21486564). Because reverse transport of ATP (cytosol to matrix) via ANT will also generate a MMP, it is speculative to state that the MMP in the assay is driven by ATP hydrolysis at CV. It is possible and maybe even likely that the majority of the MMP is driven by ANT flux, which in turn limits the amount of ATP hydrolyzed by CV. Admittedly, it is very challenging to different MMP from ANT vs that from CV, thus the authors simply need to acknowledge that the specific contribution of ATP hydrolysis to MMP remains to be fully determined. That said, the fact that ATP-dependent MMP tracks with IF1 expression does certainly implicate a role for ATP hydrolysis in the process. The authors should consider including a discussion of the ambiguity of the assay to avoid confusion. A role for ANT likely should be incorporated in the Fig. 1J cartoon.

RESPONSE: Thank you for bringing the ANT contribution to MMP to our attention. The effects of ATP in the real-time MMP measurements were totally abolished by the addition of oligomycin in BAT-derived isolated mitochondria, thus suggesting dependency of complex V in this process. However, the assessment of MMP in intact cells is much more challenging given cytosolic vs. mitochondrial contribution to ATP pool, and ATP synthase vs. ANT reversal capacity depending on MMP. Nevertheless, we have addressed these points in the discussion section as well as added to our schematic cartoon in Figure 1m.

Regarding the lack of effect of IF1 silencing on MMP, it is possible that IF1 total protein levels are simply lower in cultured brown fat cells relative to tissue? The authors could consider testing this by blotting for IF1 and CV in BAT and brown fat cells. The ratio of IF1/ATP5A1 in tissue versus cells may provide some amount of mechanistic evidence as to their findings.

RESPONSE: We have now blotted for complex V and IF1 in both differentiated primary brown adipocytes and BAT homogenates derived from mice kept at room temperature (~22°C). We found the levels of complex V in primary brown adipocytes are higher than BAT homogenates. Therefore, IF1/complex V ratio is different between these two systems. This has indeed the potential to influence our gain and loss-of-function experiments. We have added these results alongside their interpretation in the revised manuscript.

The calculation of ATP synthesis from respiration sensitive to oligomycin has many conceptual flaws. Unlike glycolysis, where ATP is produced via substrate level phosphorylation, during OXPHOS, the stoichiometry of ATP produced per 2e transfer is not known in intact brown adipose cells. This is a major limitation of this "calculated ATP synthesis" approach that is beginning to become common. Such claims are speculative and thus likely do more harm than good. In addition to ANT and CV, there are many proton-consuming reactions driven by the proton motive force (e.g., metabolite transport, Ca2+ cycling, NADPH synthesis). Although it remains unclear how much proton conductance is diverted to non-ATP synthesis dependent processes, it seems highly likely that these processes contribute to respiratory demand inside living cells. Moreover, just as occurs with UCP1 in response to adrenergic stimuli, proton conductance across the various proton-dependent processes likely changes depending on the cellular context, which is another reason why using a fixed stoichiometry to calculate how much ATP is produced from oxygen consumption is so highly flawed. Maximal P/O values that are often used for NAD/FAD linked flux are generated using experimental conditions that favor near complete flux through the ATP synthesis system (supraphysiological substrate and ADP levels). The true P/O value inside living cells is likely to be lower.

RESPONSE: We agree with the reviewer regarding the limitations on calculating ATP production in intact cells based on respiration and proton flux. However, this was only one experiment on which we based our conclusions, as these were also supported by i.e. ATP/ADP ratio measurements and oxygen consumption using different substrates. Therefore, we do not rely exclusively on the ATP production estimative, rather we use this experiment to support complementary methodologies. Nevertheless, we have now better detailed our experimental protocol as well as acknowledged the limitations of the method, so the reader is aware of our procedure and its limitations. We hope the reviewer understands our motivation to perform these experiments and the contribution to our study.

Why are the results in Figure 3K expressed as a % of basal? Could the authors please normalize the OCR data to protein and/or provide a justification for why different normalization strategies were used between 3K and 3M?

RESPONSE: We apologize for the lack of consistency. We have now updated Figure 3 to show all the data in absolute values divided by protein content. This change does not affect the overall interpretation of the findings.

The authors claim that IF1 overexpression lowers ATP production via OXPHOS. However, given the major limitations of this assay (ass discussed above), these claims should be viewed as speculation. This needs to be addressed by the authors as a major limitation. The fact that the ATP/ADP levels did not change do not support of reduction in ATP production, as claimed in the title of Figure 4.

RESPONSE: The reduction in ATP levels and mitochondrial respiration (independent of the substrate offered) suggests a reduction in ATP production rather than an increase in ATP consumption. Moreover, the maintenance of ATP/ADP ratio suggests the existence of a compensatory mechanism to avoid cellular energy crises, which we interpreted as reduced metabolic activity of the cells. Nevertheless, we have now reworded our statements to address the limitations of the methods and our interpretation of the data.

In the discussion, the authors state "However, considering that IF1 inhibits F1-ATP synthase in a 1:1 stoichiometric ratio, the relatively higher expression of IF1 in BAT at room temperature could represent an additional inhibitory factor for ATP synthesis in this tissue." This does not appear to be correct. Although IF1 has been suggested to partially lower maximal rates of ATP synthesis rates, most of this evidence comes from over-expression experiments. According to the current understanding of IF1-CV interaction, the protein is expelled from the complex during rotation in favor of ATP synthesis (PMID: 37002198). It is far more likely that ATP synthesis is low in BAT mitochondria due to the low CV expression. Relative to heart and when normalized to mitochondrial content, CV expression in BAT mitochondria is about 10% that of heart (PMID: 33077793).

RESPONSE: We agree with the reviewer and removed this sentence.

The last sentence of the manuscript states, "Given the importance of IF1 to control brown adipocyte energy metabolism, lowering IF1 levels therapeutically might enhance approaches to enhance NST for improving cardiometabolic health in humans." This sentence seems at odds with the evidence that IF1 levels go up, not down, in human BAT upon cold exposure.

RESPONSE: In light of our new experiments, we have now updated our conclusions.

Minor Comments:

The term "anaerobic glycolysis" is used throughout. All experiments were performed under normoxic conditions, thus the correct term is "aerobic glycolysis.

RESPONSE: Thank you for this comment and we have replaced this term as suggested.

Only male mice were used in the study, could the authors please provide a justification for this?

RESPONSE: Given we devoted most of our efforts to the manipulation of IF1 in vitro, we have used the mouse model as a proof-of-principle on the impact of IF1 in adrenergic-induced thermogenesis. We have now included IF1 KO male and female mice to address the role of IF1 in adrenergic-induced thermogenesis. However, due to the limitation of material, we could only perform AAV in vivo gain-of-function in male mice, therefore, our results cannot be immediately transferred to both sexes, unfortunately.

Reviewer #1 (Significance (Required)):

Overall, the quality of the work is very high. Given that IF1 is implicated across many fields of study, the novel discovery of IF1 as a regulator of brown adipose mitochondrial bioenergetics will be of significance across several fields.

My expertise is in mitochondrial thermodynamics; thus, I do not feel there are any parts of the paper that I do not have sufficient expertise to evaluate.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary

The manuscript by Brunetta and colleagues conveys the message that the ATPase inhibitory factor 1 (IF1) protein, a physiological inhibitor of mitochondrial ATP synthase, is expressed in BAT of C57BL/6J mice. Moreover, upon cold-adaption of mice they report that the content of IF1 in BAT is downregulated to sustain the mitochondrial membrane potential (MMP) as a result of reverse functioning of the enzyme. In experiments of loss and gain of function of IF1 in cultured brown adipocytes and WT cells they further stress that IF1 silencing promotes metabolic reprogramming to an enhanced glycolysis and lipid oxidation, whereas IF1 overexpression blunts ATP production rendering a quiescent cellular state of the adipocytes.

RESPONSE: We appreciate the time the reviewer invested in our work. Please, see our responses below in a point-by-point manner.

Reviewer #2 (Significance (Required)):

Claims and conclusions:

I have been surprised by the claim that IF1 protein is expressed in BAT under basal conditions and that its expression is downregulated in the cold-adapted tissue. In a previously published work by Forner et al., (2009) Cell Metab 10, 324-335 (reference 43), using a quantitative proteomic approach, it is reported that the mitochondrial proteome of mouse BAT under basal conditions contains a low content of IF1 (at level comparable to the background of the analysis). Remarkably, in the same study they show that there is roughly a 2-fold increase in the content of IF1 protein in mitochondria of BAT at 4d and 24d of cold-adaptation of mice. In other words, just the opposite of what is being reported in the Brunetta study.

RESPONSE: We are aware of the inconsistencies between our findings and Forner et al. (2009). We would like to point out that we have determined IF1 levels in BAT in two separate cohorts with the same findings, and in a third cohort, we observed IF1 mRNA levels to be downregulated in a much shorter timeframe. Our functional analysis is line with this pattern of regulation. A closer look at the supplementary table provided by Forner et al. (2009), shows that the increase in IF1 content following cold exposure is not supported and since we do not have further insight into the methods and analysis employed by the Forner et al. group, we believe a direct comparison should be avoided at the moment. Regarding the baseline levels of IF1 in BAT, the relatively high abundance of IF1 in BAT was also found by another independent group (https://doi.org/10.1101/2020.09.24.311076).

Importantly, the last paragraph of the discussion needs to be amended when mentioning the work of Forner et al. (ref.43). The mentioned reference studied changes in the mouse mitochondrial proteome not in human mitochondria, as it is stated in the alluded paragraph.

RESPONSE: We apologize for this overlook; we have now reworded our statement.

More puzzling are the western blots in Figures 1E, 1H, Supp. Fig. 1C, D were IF1 (ATP5IF1) is identified by a 17kDa band. However, in other Figures (Fig. 2, Fig. 3, Fig. 4, Supp Fig. 2) IF1 is identified by its well-known 12kDa band. What is the reason for this change in labeling of the IF1 band? The reactivity of the anti-IF1 antibody used? It has been previously documented that liver of C57BL/6J and FVB mouse strains do not express IF1 to a significant level when compared to heart IF1 levels (Esparza-Molto (2019) FASEB J. 33, 1836-1851). However, in Fig. 1E they show opposite findings, much higher levels of IF1 in liver than in heart as reveal by the 17kDa band. Moreover, in Fig. 1H they show the vanishing of the 17 kDa band under cold adaptation, which is not the migration of IF1 in gels as shown in their own figures (see Fig. 2, Fig. 3, Fig. 4, Supp Fig. 2). I am certainly reluctant to accept that the 17kDa band shown in Figures 1E, 1H, Supp. Fig. 1C, D is indeed IF1. Most likely it represents a non-specific protein recognized by the antibody in the tissue extracts analyzed. Cellular overexpression experiments of IF1 in WT1 cells (Fig. 2E) and primary brown adipocytes (Fig. 4B) also support this argument. Overall, I do not support publication of this study for the reasons stated above.

RESPONSE: We understand the concerns raised by the reviewer and apologize for the lack of details in our experimental procedures. While we used the same antibody in the study (Cell Sig. cat. Num. 8528, 1:500), we used two different types of gels. The difference in the molecular weight appearance of IF1 is likely through the migration of the protein in the agarose gel. By using custom-made gels, we observe the protein ~17kDa (Fig. 1 and 5), whereas by using commercial gels (Fig. 2, 3, and 4), we observe the protein closer to the predicted molecular weight (i.e. ~12kDa). Of note, gain and loss-of-function experiments, both in vivo as well as in vitro confirm this statement and the specificity of the antibody (Fig. 2, 3, 4, 5, Fig. EV2). In addition, when we ran a custom-made gel with primary BAT cells, we observed again the ~17kDa band (see Figure for the reviewer below). These experiments alongside the absence of other bands in the gels (see uncropped membranes in Supplementary Figure 1) make us conclude that the band we observe is indeed IF1. Nevertheless, we have now updated our methods section, so the reader is aware of our approaches. We hope the reviewer is satisfied with our additional experiments and editions throughout the manuscript.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary:

In this manuscript, Brunneta et al describe the role of IF1 in brown adipose tissue activation using in vivo and in vitro experimental models. They observed that cold adaptation promotes a reduction in IF1 expression and an increase in the reverse activity of mitochondrial ATPase or Complex V. Based on these results, the authors explore the contribution of IF1 in this metabolic pathway by modeling the thermogenic process in differentiated primary brown adipocytes. They silenced and overexpressed IF1 in culture and studied their adrenergic stimulation under norepinephrine.

Major comments:

The experiments are well explained and the manuscript flows very well. There are several comments that should be addressed.

RESPONSE: We thank the reviewer for the kind words regarding our work.

1. The authors measure ATP hydrolysis in isolated mitochondria from BAT in Figure 1. They observed that IF1 is decreased upon cold exposure and that ATP hydrolysis is increased. They assess protein levels of different OXPHOS proteins, including IF1 but not other proteins of Complex V (ATP5A) as they do in Figures 3 and 4. It is important to see that cold exposure only affects IF1 levels but not other proteins from Complex V. Does IF1/Complex V ratio change? RESPONSE: We thank the reviewer for this suggestion which was also raised by Reviewer #1. We have now measured complex V subunit A in our experimental protocol. We found that cold exposure does not impact complex V protein levels. Given the importance of this information, we have now included it in Figure 1 (Please, see the revised version) alongside the IF1/complex V ratio. In addition, we have now performed WBs in the BAT exposed for 3 and 7 days to thermoneutrality (~28°C) where we found that IF1 is not reduced following whitening of BAT by this approach whilst UCP1 and other mitochondrial proteins are reduced.

This set of data is now included in Figure 1I,K,L.

In Figure 2J, the drop in MMP is lower upon adrenergic stimulation than in Figure 2E. The same observation applies to other results when the reduction in MMP after NE addition is minimal. Why do the authors remove TMRM for the measurements of membrane potential? TMRM imaging is normally done in the presence of the dye in non-quenching mode. Treatments should be done prior to the addition of the dye and then TMRM should be added and left during the imaging analysis and measure in non-quenching mode. This might explain some of the above-mentioned points regarding the MMP data. Alternatively, if the dye is removed before the measurements, they should let the cells to adapt and so the dye equilibrates between mitochondria and cytosol. A more elegant method to measure membrane potential could be live-cell imaging. In addition, authors propose that mitochondrial membrane potential upon NE stimulation is maintained by reversal of ATP synthase. If this is the case, one would expect that addition of oligomycin in NE treated adipocytes would cause depolarization. However, in FigS2A this is not the case. Authors should comment on this in addition to considering more elegant approach to measure MMP.

RESPONSE: We apologize for the lack of details in the methods. All treatments (i.e., transfection and norepinephrine stimulation) were performed before the addition of TMRM. Indeed, this approach does not have the resolution compared to safranine in isolated mitochondria (Fig. 1D), which limits our interpretation regarding the dynamic role of IF1 on MMP in brown adipocytes. We have taken care to state the limitations of our method throughout the entire paper to avoid overinterpretation of our data. Regarding the removal of the dye before the measurements, our internal controls indicate that this procedure does not change the ability of our method to detect fluctuations in MMP (i.e., oligomycin and FCCP as internal controls). Nevertheless, as suggested by the reviewer, to test the time effect of the probe equilibrium (i.e., mitochondria versus cytosol) in our method, we loaded cells with TMRM 20 nM for 30 min and measured the fluorescence right after the removal of the probe/washing steps for another 10 min. We were not able to detect differences in the fluorescence in a time-dependent manner (see below). Therefore, we conclude the removal of TMRM does not influence the fluorescence of the probe in differentiated brown adipocytes.

+NE

-NE

In addition, we performed a similar experiment using TMRM in the quenching mode (200 nM), however, after the removal of TMRM, we added FCCP (1 mM) to the cells for 10 min under constant agitations at 37°C. This approach aimed to expel all TMRM that accumulated within the mitochondria in an MMP-dependent manner. Therefore, excluding the dynamic Brownian movement that we could have caused by the removal of the dye before the measurement mentioned by the reviewer. By doing this, we found the same effect of IF1 overexpression in the reduction of MMP in the presence of norepinephrine.

Protocol:

• Transfection (24h) on day 4 of differentiation + 24h just normal media

• 30 min norepinephrine 10 µM

• 200 nM TMRM on top of NE

• Washing step

• Add FCCP 1 µM for 10 min, and read (The aim here was to release all TMRM accumulated inside of mitochondria in a MMP-dependent manner)

In summary, the data suggests the removal of the dye from the cells does not influence the fluorescence of TMRM, therefore, enabling us to make conclusions regarding the biological effects of IF1 manipulation in the MMP of brown adipocytes. Regarding the reverse mode of ATP synthase and the absence of effects with oligomycin, given oligomycin inhibits both rotation of ATP synthase and even uncoupled brown adipocytes respond to oligomycin (i.e. reduction in O2 consumption), the prediction of lowering MMP in the presence of oligomycin due to inhibition of the reserve mode of ATP synthase is more complicated than anticipated. Nevertheless, we have now addressed this topic in the discussion section. Lastly, we generally observe a reduction in MMP around 10-25% in differentiated adipocytes upon NE treatment (30 minutes, 10mM). However, due to the differentiation state of the cells, MMP response from norepinephrine fluctuated from experiment to experiment. Therefore, we did not compare experiments performed on different days or batches, but only within the same differentiation batch to reduce variability.

In Figure 2, in the model of siIF1, there is baseline more phosphorylation of AMPK than in the scramble control (pAMPK). However, this is not the case of p-p38MAPK. Do the authors have any explanation for those differences in baseline activation of the stress kinases when IF1 is silenced? In the same experimental group, addition of NE seems to have more effect in the scrambled than in siIF1, but the plotted data does not reflect these differences. In contrast, increase in pAMPK upon NE is higher in IF1 overexpressing cells compared to EV (Figure 2H), but again this is not reflected in western blot quantification (Figure 2I).

RESPONSE: Although some differences in pAMPK in the treatments were observed as gathered by the representative blots, these changes were not confirmed later in different biological replicates, therefore, the overall effect of IF1 manipulation in pAMPK does not change. Given we used this approach as quality control for our experiments to guarantee norepinephrine treatment works, we removed the pAMPK data from the study and kept p38 as a marker of adrenergic signaling activation (please see revised Fig. 2 in the main file).

Does NE promote decrease of IF1 expression in control (siScramble and EV) adipocytes? The authors should test it and see whether it goes in the same direction as the observations derived from the experiments in cold exposed mice. This is very important point, as it could explain the lack of an additional effect of IF1 silencing on NE-induced depolarization (Figure 2E).

RESPONSE: We thank the reviewer for this suggestion. In line, with the in vivo data, acute NE treatment in differentiated brown adipocytes does not change IF1 mRNA and protein levels. We have now added this information and the corresponding interpretation to the updated manuscript.

Does NE promote decrease of IF1 expression in the scramble and EV adipocytes? The authors should test it and see whether it goes in the same direction as the observations derived from the experiments in cold exposed mice.

RESPONSE: As this question is the same as #4, we believe the reviewer may have erroneously pasted this here.

For MMP data in Fig2, they should include significance between non treated and NE-treated groups. They say: "While UCP1 ablation did not cause any effect on MMP upon adrenergic stimulation...", but NE caused (probably significant) depolarization in siUCP1, which seems even stronger than depolarization in EV. This is opposite to what you would expect. They also didn't confirm UCP1 silencing with western blot.

RESPONSE: We thank the reviewer for this suggestion. We have now included the expected statistical main effect of NE upon MMP. Although the effects of IF1 overexpression were blunted when Ucp1 was silenced, we indeed still observed the same degree of reduction in MMP in brown adipocytes. This finding has two possible explanations, one is the effectiveness of the silencing protocol, therefore, residual Ucp1 expression may still play a role in this experiment; second, other ATP-consuming processes are able to lower MMP in a UCP1-independent manner. We have added this information to the updated manuscript to make the reader aware of our findings as well as the limitations of the method. Unfortunately, we were not able to detect UCP1 protein levels due to technical issues. Given the effects of IF1 overexpression were blunted when Ucp1 was silenced, we believe this functional outcome is sufficient, alongside mRNA levels, to demonstrate the effectiveness of our silencing protocol.

It has been established that decreased expression of IF1 promotes increase in the reverse activity of Complex V, ATP hydrolytic activity. Increase in ATP hydrolysis also affects ECAR. The authors should consider this when calculating the contribution of ATP glycolysis versus ATP OXPHOS since the ATP hydrolysis is also playing a role in the ECAR increase. The data should be reinterpreted. ATP hydrolysis should be measured in the situation where IF1 is silenced and overexpressed. These measurements can be done in cells using the seahorse.

RESPONSE: The only differences we observed in MMP are in the presence of norepinephrine (i.e. UCP-1-dependent proton conductance), which is not present during the estimation of ATP production by Seahorse analysis. Nevertheless, we have now improved the description of our experimental protocol and limitations to estimate ATP production to make it as clear as possible to the reader. Lastly, given the addition of in vivo gain-of-function experiments, we have now determined the ATP hydrolytic activity in this model, which offers a better understanding of the in vivo modulation of IF1 levels affecting ATP synthase activity (reverse mode). We hope the reviewer understands our motivation to focus on the in vivo model of gain-of-function regarding ATP synthase activity.

The authors use GAPDH as loading control in western blots. They should use another protein since GAPDH is part of the intermediary metabolism and plays a role in glycolysis.

RESPONSE: We understand the concern of the reviewer regarding the use of GAPDH as a loading control for the studies of metabolism. However, as can be observed by the western blot images, GAPDH levels do not change in our experimental models, therefore, we feel confident that our loading is homogeneous throughout our gels.

The authors show that reduction of IF1 involves more lipid utilization. They should include more experiments showing the connection of the metabolic adaptation in the absence of IF1 and some lipid imaging.

RESPONSE: We appreciate this suggestion. We have now performed Oil Red O staining in differentiated adipocytes following ablation of IF1. However, we did not observe any effect on lipid accumulation in primary brown adipocytes following IF1 knockdown. Therefore, the effects of IF1 ablation on lipid mobilization are not due to lipid content or reflected in lipid accumulation. We have now added this new information to the manuscript (please, see the revised form Fig. EV3).

In the text, "Despite this adjustment of experimental conditions, we did not detect any effect of IF1 ablation on mitochondrial oxygen consumption (Supplementary Fig. 3A,B)", this is true for baseline, NE-driven and ATP-linked respiration, but what about maximal respiration? There is a huge increase in IF1 knockdown... They should explain these results.

RESPONSE: We perform this experiment to address the question of whether the lipid mobilization induced by norepinephrine would uncouple mitochondria in a UCP1-independent manner. Given the absence of effect between scrambled and IF1 ablated cells in mitochondrial respiration in the presence of norepinephrine and following the addition of oligomycin, we concluded no effect of lipolysis-induced UCP1-independent uncoupling. However, as observed by the reviewer and consistent with other data within the study, the interaction between lipid metabolism and IF1 knockdown seems to affect maximal electron transport chain activity, which although interesting, was not the focus of the present study. Nevertheless, we have now acknowledged these findings and a possible explanation for them in the revised manuscript.

In Figure 3K they present OCR as % of baseline, but in a similar experiment in Figire 4G it is OCR/protein, they should make the Y axis consistent across experiments.

RESPONSE: We apologize for this overlook. We have now edited all the axes and labels for consistency.

The graphical abstract is confusing. In BAT there are two populations of mitochondria, the cytosolic and the mitochondria attached to the lipid droplet, peridroplet mitochondria (PDM). Upon adrenergic stimulation, PDM leave the lipid droplet and lipolysis takes place. The authors propose that upon adrenergic stimulation, IF1 is reduced and there is lipid mobilization. The part of the scheme where it says "fully recruited" should be removed or rewritten, since adrenergic stimulation is not compatible with mitochondria recruitment around the lipid droplet.

RESPONSE: Thank you for this input. Given the addition of new experiments and interpretation, we have now redrawn the graphical abstract and addressed this topic in the discussion section.

The title should be rewritten to better reflect the research presented in the manuscript.

RESPONSE: Thank you for this input. Given the addition of new experiments, we have now rewritten the title accordingly.

Minor comments:

Some of the Y axis should be corrected. For example, in Figure 2J, L and M should say % of EV untreated, Similarly, in Figure 2E, it should say % of scramble untreated. In Figure 3N, the Y axis is misspelled. All the Y axis referring to percentages should have the same scale for comparison purposes.

RESPONSE: Thank you for the proofreading. We have now edited the scales and labels to keep consistency.

The authors should describe better the results corresponding to Figure 2. There is a lot of information and they should improve the description pertaining the connection between the different pieces of data relating the different signaling pathways that are shown. For westerns in this Figure, they should provide some rationale (one to two sentences in the results section) as to why they are checking the expression of pAMPK and p38-MAPK.

RESPONSE: We have now edited the description of our results to make them as clear as possible.

Here are some comments referring to the methods section:

For Complex V hydrolytic activity, the reaction buffer contains 10mM Na-azide. I guess this is to inhibit respiration, but wouldn't azide also inhibit complex V at this concentration?

RESPONSE: We thank the reviewer for this question. To test that, we performed complex V activity in buffers containing or not 10 mM sodium azide. As demonstrated below, the presence of sodium azide in the buffer does not influence complex V activity in two different tissues with low and high complex V activity (BAT and heart, respectively).

Table 1. ATP synthase hydrolytic activity in the presence or absence of Na-azide.

BAT

Heart

+Na-azide

100 ± 43.01

100 ± 39.36

-Na-azide

82.6 ± 4.33

111.3 ± 43.32

+Na-azide + oligomycin

15.3 ± 4.32*

13.8 ± 14.01*

-Na-azide + oligomycin

14.2 ± 3.53*

11.9 ± 2.88*

Data presented as % of control (i.e. presence of Na-azide and absence of oligomycin) for both tissues independently. N = 2-3/condition. Statistical test: two-way ANOVA. * main effect of oligomycin (p In the mitochondrial isolation protocol, they say "mitochondria were centrifuged at 800g for 10min..." Will this speed pellet the mitochondria? I think this is a mistake in writing.

RESPONSE: We apologize for the lack of clarity. What was centrifuged at 800 g was the whole-tissue homogenate to discard cellular debris, before pelleting mitochondria at 5000 g. We have now corrected this mistake in the methods section.

For the safranin-O experiment, they don't mention mitochondrial substrate used, probably it's in the reference that they provide, but I think it should be included in the text.

RESPONSE: We did not use any substrate because our goal was to test the contribution of ATP synthase to mitochondrial membrane potential. For that, we inhibited proton movement within the ETC with antimycin A and through UCP1 with GDP (see Methods). We have now edited our Method’s description to make sure the reader is aware of our approach.

Reviewer #3 (Significance (Required)):

The manuscript is well written, and it flows well when reading. However, there are some additional experiments that need to be performed to reach the conclusions the authors claim.

RESPONSE: We thank the reviewer for the positive commentaries regarding our work and hope to have answered the open questions with the edits and new experiments.

The role of ATP hydrolysis in BAT thermogenesis is novel and interesting as it can sed some light onto potential approaches to promotes BAT activation.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

This is an interesting investigation into the activity of IF1 in brown adipocytes. The findings are innovative and the conclusion is well-supported by the data. The conclusion is in line with previous reports on IF1 activities in other cell types, particularly in terms of its regulation of FoF1-ATPase. The authors have executed an exceptional job in designing the study, preparing the figures, and writing the manuscript. Overall, this study significantly contributes to the understanding of IF1 activity in brown adipocytes and its role in thermogenesis.

RESPONSE: We thank the reviewer for the kind words. Please, find below our answers in a point-by-point manner.

Reviewer #4 (Significance (Required)):

The study demonstrates involvement of IF1 in regulating thermogenesis in brown adipocytes, which is a unique aspect not covered in existing literature. Advantage of the study is well-designed cellular studies. The major weakness is lack of proof of conclusion in vivo. There are a few minor concerns that should be addressed to further enhance quality of the manuscript.

RESPONSE: We have now included two in vivo models, whole-body IF1 KO mice and BAT-injected IF1 overexpression to test the role of IF1 in BAT biology. The whole dataset is included in the main manuscript, where we conclude the BAT IF1 overexpression partially suppresses b3-adrenergic induction of thermogenesis alongside a reduction (overall and UCP1 dependent) in mitochondrial oxygen consumption. Also, similar to our in vitro experiments, IF1 KO mice did not present any difference in adrenergic-stimulated oxygen consumption.

1. Current discussion does not mention the regulation of IF1 protein by the cAMP/PKA pathway. This point should be included to provide a comprehensive understanding of the regulatory mechanisms of IF1 protein. RESPONSE: Thank you for this suggestion. We have now added this topic to the discussion.

It has been reported that IF1 also influences the structure of mitochondrial crista. Considering the observed changes with IF1 knockdown, it would be valuable to discuss this activity in relation to the findings of the study.

RESPONSE: We discussed the implications of IF1 modulation in mitochondrial morphology in the revised manuscript.

And the more egregious something arbitrary is, the more likely that there are other even more egregious things out there – so it's no sign that that's the one thing that should be top priority!

That would be better for user to decide.

I think a good default is to express semantics explicitly.

I.e., for Bob to not automatically state his utterance as important just because it's atop of Alice's, that she considers important.

If Bob tries to reword - ok. If Bob want to add - no.

2 comments on the same text

This is a comment about this

Author Response

The following is the authors’ response to the original reviews.

eLife assessment

This work provides a valuable contribution and assessment of what it means to replicate a null study finding, and what are the appropriate methods for doing so (apart from a rote p-value assessment). Through a convincing re-analysis of results from the Reproducibility Project: Cancer Biology using frequentist equivalence testing and Bayes factors, the authors demonstrate that even when reducing 'replicability success' to a single criterion, how precisely replication is measured may yield differing results. Less focus is directed to appropriate replication of non-null findings.

Reviewer #1 (Public Review):

Summary:

The goal of Pawel et al. is to provide a more rigorous and quantitative approach for judging whether or not an initial null finding (conventionally with p ≥ 0.05) has been replicated by a second similarly null finding. They discuss important objections to relying on the qualitative significant/non-significant dichotomy to make this judgment. They present two complementary methods (one frequentist and the other Bayesian) which provide a superior quantitative framework for assessing the replicability of null findings.

Strengths:

Clear presentation; illuminating examples drawn from the well-known Reproducibility Project: Cancer Biology data set; R-code that implements suggested analyses. Using both methods as suggested provides a superior procedure for judging the replicability of null findings.

Weaknesses:

The proposed frequentist and the Bayesian methods both rely on binary assessments of an original finding and its replication. I'm not sure if this is a weakness or is inherent to making binary decisions based on continuous data.

For the frequentist method, a null finding is considered replicated if the original and replication 90% confidence intervals for the effects both fall within the equivalence range. According to this approach, a null finding would be considered replicated if p-values of both equivalences tests (original and replication) were, say, 0.049, whereas would not be considered replicated if, for example, the equivalence test of the original study had a p-value of 0.051 and the replication had a p-value of 0.001. Intuitively, the evidence for replication would seem to be stronger in the second instance. The recommended Bayesian approach similarly relies on a dichotomy (e.g., Bayes factor > 1).

Thanks for the suggestions, we now emphasize more strongly in the “Methods for assessing replicability of null results” and “Conclusions” sections that both TOST p-values and Bayes factors are quantitative measures of evidence that do not require dichotomization into “success” or “failure”.

Reviewer #2 (Public Review):

Summary:

The study demonstrates how inconclusive replications of studies initially with p > 0.05 can be and employs equivalence tests and Bayesian factor approaches to illustrate this concept. Interestingly, the study reveals that achieving a success rate of 11 out of 15, or 73%, as was accomplished with the non-significance criterion from the RPCB (Reproducibility Project: Cancer Biology), requires unrealistic margins of Δ > 2 for equivalence testing.

Strengths:

The study uses reliable and shareable/open data to demonstrate its findings, sharing as well the code for statistical analysis. The study provides sensitivity analysis for different scenarios of equivalence margin and alfa level, as well as for different scenarios of standard deviations for the prior of Bayes factors and different thresholds to consider. All analysis and code of the work is open and can be replicated. As well, the study demonstrates on a case-by-case basis how the different criteria can diverge, regarding one sample of a field of science: preclinical cancer biology. It also explains clearly what Bayes factors and equivalence tests are.

Weaknesses:

It would be interesting to investigate whether using Bayes factors and equivalence tests in addition to p-values results in a clearer scenario when applied to replication data from other fields. As mentioned by the authors, the Reproducibility Project: Experimental Philosophy (RPEP) and the Reproducibility Project: Psychology (RPP) have data attempting to replicate some original studies with null results. While the RPCB analysis yielded a similar picture when using both criteria, it is worth exploring whether this holds true for RPP and RPEP. Considerations for further research in this direction are suggested. Even if the original null results were excluded in the calculation of an overall replicability rate based on significance, sensitivity analyses considering them could have been conducted. The present authors can demonstrate replication success using the significance criteria in these two projects with initially p < 0.05 studies, both positive and non-positive.

Other comments:

• Introduction: The study demonstrates how inconclusive replications of studies initially with p > 0.05 can be and employs equivalence tests and Bayesian factor approaches to illustrate this concept. Interestingly, the study reveals that achieving a success rate of 11 out of 15, or 73%, as was accomplished with the non-significance criterion from the RPCB (Reproducibility Project: Cancer Biology), requires unrealistic margins of Δ > 2 for equivalence testing.

• Overall picture vs. case-by-case scenario: An interesting finding is that the authors observe that in most cases, there is no substantial evidence for either the absence or the presence of an effect, as evidenced by the equivalence tests. Thus, using both suggested criteria results in a picture similar to the one initially raised by the paper itself. The work done by the authors highlights additional criteria that can be used to further analyze replication success on a case-by-case basis, and I believe that this is where the paper's main contributions lie. Despite not changing the overall picture much, I agree that the p-value criterion by itself does not distinguish between (1) a situation where the original study had low statistical power, resulting in a highly inconclusive non-significant result that does not provide evidence for the absence of an effect and (2) a scenario where the original study was adequately powered, and a non-significant result may indeed provide some evidence for the absence of an effect when analyzed with appropriate methods. Equivalence testing and Bayesian factor approaches are valuable tools in both cases.

Regarding the 0.05 threshold, the choice of the prior distribution for the SMD under the alternative H1 is debatable, and this also applies to the equivalence margin. Sensitivity analyses, as highlighted by the authors, are helpful in these scenarios.

Thank you for the thorough review and constructive feedback. We have added an additional “Appendix C: Null results from the RPP and EPRP” that shows equivalence testing and Bayes factor analyses for the RPP and EPRP null results.

Reviewer #3 (Public Review):

Summary:

The paper points out that non-significance in both the original study and a replication does not ensure that the studies provide evidence for the absence of an effect. Also, it can not be considered a "replication success". The main point of the paper is rather obvious. It may be that both studies are underpowered, in which case their non-significance does not prove anything. The absence of evidence is not evidence of absence! On the other hand, statistical significance is a confusing concept for many, so some extra clarification is always welcome.

One might wonder if the problem that the paper addresses is really a big issue. The authors point to the "Reproducibility Project: Cancer Biology" (RPCB, Errington et al., 2021). They criticize Errington et al. because they "explicitly defined null results in both the original and the replication study as a criterion for replication success." This is true in a literal sense, but it is also a little bit uncharitable. Errington et al. assessed replication success of "null results" with respect to 5 criteria, just one of which was statistical (non-)significance.

It is very hard to decide if a replication was "successful" or not. After all, the original significant result could have been a false positive, and the original null-result a false negative. In light of these difficulties, I found the paper of Errington et al. quite balanced and thoughtful. Replication has been called "the cornerstone of science" but it turns out that it's actually very difficult to define "replication success". I find the paper of Pawel, Heyard, Micheloud, and Held to be a useful addition to the discussion.

Strengths:

This is a clearly written paper that is a useful addition to the important discussion of what constitutes a successful replication.

Weaknesses:

To me, it seems rather obvious that non-significance in both the original study and a replication does not ensure that the studies provide evidence for the absence of an effect. I'm not sure how often this mistake is made.

Thanks for the feedback. We do not have systematic data on how often the mistake of confusing absence of evidence with evidence of absence has been made in the replication context, but we do know that it has been made in at least three prominent large-scale replication projects (the RPP, RPEP, RPCB). We therefore believe that there is a need for our article.

Moreover, we agree that the RPCB provided a nuanced assessment of replication success using five different criteria for the original null results. We emphasize this now more in the “Introduction” section. However, we do not consider our article as “a little bit uncharitable” to the RPCB, as we discuss all other criteria used in the RPCB and note that our intent is not to diminish the important contributions of the RPCB, but rather to build on their work and provide constructive recommendations for future researchers. Furthermore, in response to comments made by Reviewer #2, we have added an additional “Appendix B: Null results from the RPP and EPRP” that shows equivalence testing and Bayes factor analyses for null results from two other replication projects, where the same issue arises.

Reviewer #1 (Recommendations For The Authors):

The authors may wish to address the dichotomy issue I raise above, either in the analysis or in the discussion.

Thank you, we now emphasize that Bayes factors and TOST p-values do not need to be dichotomized but can be interpreted as quantitative measures of evidence, both in the “Methods for assessing replicability of null results” and the “Conclusions” sections.

Reviewer #2 (Recommendations For The Authors):

Given that, here follow additional suggestions that the authors should consider in light of the manuscript's word count limit, to avoid confusing the paper's main idea:

2) Referencing: Could you reference the three interesting cases among the 15 RPCB null results (specifically, the three effects from the original paper #48) where the Bayes factor differs qualitatively from the equivalence test?

We now explicitly cite the original and replication study from paper #48.

3) Equivalence testing: As the authors state, only 4 out of the 15 study pairs are able to establish replication success at the 5% level, in the sense that both the original and the replication 90% confidence intervals fall within the equivalence range. Among these 4, two (Paper #48, Exp #2, Effect #5 and Paper #48, Exp #2, Effect #6) were initially positive with very low p-values, one (Paper #48, Exp #2, Effect #4) had an initial p of 0.06 and was very precisely estimated, and the only one in which equivalence testing provides a clearer picture of replication success is Paper #41, Exp #2, Effect #1, which had an initial p-value of 0.54 and a replication p-value of 0.05. In this latter case (or in all these ones), one might question whether the "liberal" equivalence range of Δ = 0.74 is the most appropriate. As the authors state, "The post-hoc specification of equivalence margins is controversial."

We agree that the post hoc choice of equivalence ranges is a controversial issue. The margins define an equivalence region where effect sizes are considered practically negligible, and we agree that in many contexts SMD = 0.74 is a large effect size that is not practically negligible. We therefore present sensitivity analyses for a wide range of margins. However, we do not think that the choice of this margin is more controversial for the mentioned studies with low p-values than for other studies with greater p-values, since the question of whether a margin plausibly encodes practically negligible effect sizes is not related to the observed p-value of a study. Nevertheless, for the new analyses of the RPP and EPRP data in Appendix B, we have added additional sensitivity analyses showing how the individual TOST p-values and Bayes factors vary as a function of the margin and the prior standard deviation. We think that these analyses provide readers with an even more transparent picture regarding the implications of the choice of these parameters than the “project-wise” sensitivity analyses in Appendix A.

4) Bayes factor suggestions: For the Bayes factor approach, it would be interesting to discuss examples where the BF differs slightly. This is likely to occur in scenarios where sample sizes differ significantly between the original study and replication. For example, in Paper #48, Exp #2 and Effect #4, the initial p is 0.06, but the BF is 8.1. In the replication, the BF dramatically drops to < 1/1000, as does the p-value. The initial evidence of 8.1 indicates some evidence for the absence of an effect, but not strong evidence ("strong evidence for H0"), whereas a p-value of 0.06 does not lead to such a conclusion; instead, it favors H1. It would be interesting if the authors discussed other similar cases in the paper. It's worth noting that in Paper #5, Exp #1, Effect #3, the replication p-value is 0.99, while the BF01 is 2.4, almost indicating "moderate" evidence for H0, even though the p-value is inconclusive.

We agree that some of the examples nicely illustrate conceptual differences between p-values and Bayes factors, e.g., how they take into account sample size and effect size. As methodologists, we find these aspects interesting ourselves, but we think that emphasizing them is beyond the scope of the paper and would distract eLife readers from the main messages.

Concerning the conceptual differences between Bayes factors and TOST p-values, we already discuss a case where there are qualitative differences in more detail (original paper #48). We added another discussion of this phenomenon in the Appendix C as it also occurs for the replication of Ranganath and Nosek (2008) that was part of the RPP.

5) p-values, magnitude and precision: It's noteworthy to emphasize, if the authors decide to discuss this, that the p-value is influenced by both the effect's magnitude and its precision, so in Paper #9, Exp #2, Effect #6, BF01 = 4.1 has a higher p-value than a BF01 = 2.3 in its replication. However, there are cases where both p-values and BF agree. For example, in Paper #15, Exp #2, Effect #2, both the original and replication studies have similar sample sizes, and as the p-value decreases from p = 0.95 to p = 0.23, BF01 decreases from 5.1 ("moderate evidence for H0") to 1.3 (region of "Absence of evidence"), moving away from H0 in both cases. This also occurs in Paper #24, Exp #3, Effect #6.

We appreciate the suggestions but, as explained before, think that the message of our paper is better understood without additional discussion of more general differences between p-values and Bayes factors.

6) The grey zone: Given the above topic, it is important to highlight that in the "Absence of evidence grey zone" for the null hypothesis, for example, in Paper #5, Exp #1, Effect #3 with a p = 0.99 and a BF01 = 2.4 in the replication, BF and p-values reach similar conclusions. It's interesting to note, as the authors emphasize, that Dawson et al. (2011), Exp #2, Effect #2 is an interesting example, as the p-value decreases, favoring H1, likely due to the effect's magnitude, even with a small sample size (n = 3 in both original and replications). Bayes factors are very close to one due to the small sample sizes, as discussed by the authors.

We appreciate the constructive comments. We think that the two examples from Dawson et al. (2011) and Goetz et al. (2011) already nicely illustrate absence of evidence and evidence of absence, respectively, and therefore decided not to discuss additional examples in detail, to avoid redundancy.

7) Using meta-analytical results (?): For papers from RPCB, comparing the initial study with the meta-analytical results using Bayes factor and equivalence testing approaches (thus, increasing the sample size of the analysis, but creating dependency of results since the initial study would affect the meta-analytical one) could change the conclusions. This would be interesting to explore in initial studies that are replicated by much larger ones, such as: Paper #9, Exp #2, Effect #6; Goetz et al. (2011), Exp #1, Effect #1; Paper #28, Exp #3, Effect #3; Paper #41, Exp #2, Effect #1; and Paper #47, Exp #1, Effect #5).

Thank you for the suggestion. We considered adding meta-analytic TOST p-values and Bayes factors before, but decided that Figure 3 and the results section are already quite technical, so adding more analyses may confuse more than help. Nevertheless, these meta-analytic approaches are discussed in the “Conclusions” section.

8) Other samples of fields of science: It would be interesting to investigate whether using Bayes factors and equivalence tests in addition to p-values results in a clearer scenario when applied to replication data from other fields. As mentioned by the authors, the Reproducibility Project: Experimental Philosophy (RPEP) and the Reproducibility Project: Psychology (RPP) have data attempting to replicate some original studies with null results. While the RPCB analysis yielded a similar picture when using both criteria, it is worth exploring whether this holds true for RPP and RPEP. Considerations for further research in this direction are suggested. Even if the original null results were excluded in the calculation of an overall replicability rate based on significance, sensitivity analyses considering them could have been conducted. The present authors can demonstrate replication success using the significance criteria in these two projects with initially p < 0.05 studies, both positive and non-positive.

Thank you for the excellent suggestion. We added an Appendix B where the null results from the RPP and EPRP are analyzed with our proposed approaches. The results are also discussed in the “Results” and “Conclusions” sections.

9) Other approaches: I am curious about the potential impact of using an approach based on equivalence testing (as described in https://arxiv.org/abs/2308.09112). It would be valuable if the authors could run such analyses or reference the mentioned work.

Thank you. We were unaware of this preprint. It seems related to the framework proposed by Stahel W. A. (2021) New relevance and significance measures to replace p-values. PLoS ONE 16(6): e0252991. https://doi.org/10.1371/journal.pone.0252991

We now cite both papers in the discussion.

10) Additional evidence: There is another study in which replications of initially p > 0.05 studies with p > 0.05 replications were also considered as replication successes. You can find it here: https://www.medrxiv.org/content/10.1101/2022.05.31.22275810v2. Although it involves a small sample of initially p > 0.05 studies with already large sample sizes, the work is currently under consideration for publication in PLOS ONE, and all data and materials can be accessed through OSF (links provided in the work).

Thank you for sharing this interesting study with us. We feel that it is beyond the scope of the paper to include further analyses as there are already analyses of the RPCB, RPP, and EPRP null results. However, we will keep this study in mind for future analysis, especially since all data are openly available.

11) Additional evidence 02: Ongoing replication projects, such as the Brazilian Reproducibility Initiative (BRI) and The Sports Replication Centre (https://ssreplicationcentre.com/), continue to generate valuable data. BRI is nearing completion of its results, and it promises interesting data for analyzing replication success using p-values, equivalence regions, and Bayes factor approaches.

We now cite these two initiatives as examples of ongoing replication projects in the introduction. Similarly as for your last point, we think that it is beyond the scope of the paper to include further analyses as there are already analyses of the RPCB, RPP, and EPRP null results.

Reviewer #3 (Recommendations For The Authors):

I have no specific recommendations for the authors.

Thank you for the constructive review.

Reviewing Editor (Recommendations For the Authors):

I recognize that it was suggested to the authors by the previous Reviewing Editor to reduce the amount of statistical material to be made more suitable for a non-statistical audience, and so what I am about to say contradicts advice you were given before. But, with this revised version, I actually found it difficult to understand the particulars of the construction of the Bayes Factors and would have appreciated a few more sentences on the underlying models that fed into the calculations. In my opinion, the provided citations (e.g., Dienes Z. 2014. Using Bayes to get the most out of non-significant results) did not provide sufficient background to warrant a lack of more technical presentation here.

Thank you for the feedback. We added a new “Appendix C: Technical details on Bayes factors” that provides technical details on the models, priors, and calculations underlying the Bayes factors.

This is interesting because it's about more than just cultural differences. It includes how societal expectations and norms influence behavior, not just for boys more than girls, but for everyone.

It's crazy to me that etiquette about posting pictures of other people has become so lax. As a kid, parents and teachers would thoroughly impress on me that posting pictures that reveal personal information like your face or location was a bad idea. Over time, people have just accepted that it's fine or even choose to take pictures of strangers.

I didn't know about the metadata either. I'll definitely keep that in mind in the future too.

I think it's crucial, especially in academic research and data analysis, to be aware of this phenomenon and to look beyond just surface-level correlations. We need to dig deeper, consider potential confounding variables or alternative explanations, and really scrutinize whether there is a logical, causal mechanism that could explain the apparent correlation. Just because two things appear related in the data doesn't necessarily mean one is causing the other.

I agree with this statement and feel deeply concerned. When we use most social media platforms, they usually select user preference content for us when registering an account in order to push content to users that they are more interested in. And this is actually a way to obtain information and find ways to attract the user's attention. Moreover, as we use the software, we are also using different algorithms to infer how our interests have changed. At the same time, we may also cooperate with shopping software to directly push the items of interest we just mentioned in the video or forum social media so that we can purchase them. I think it’s a bit scary how much big data knows about us.

I think it's very productive to have your name mispronounced. There are students from different countries at the university, and each country has a different culture and pronunciation. My name is pronounced in Mandarin, and I recognize that a lot of Chinese is very difficult to pronounce, just as I sometimes have trouble pronouncing other people's names, so it's important to understand each other.

Age is Just a Number in Relationship - Related Pages

• Age is just a number: it's not a barrier to me - <q>I didn't think age should be a problem in marriage or relationships; love should be given the chance to express itself. One of my aunts wanted to get married …</q>
• Age Is Just A Number - <q>… Photo by Innoh Khumbuza: Relationships are sometimes a sensitive topic which when we engage In at times arguments abound… by marriot5464.</q>
• Is age just a number in relationships? - <q> … Whichever is your choice, your decision should be based on the love, connection, and understanding that exist between you both. With this, you …</q>
• Is age really just a number in a relationship? - <q>… Over the years, one of the most popular pieces of relationship advice you will get from people is that age is just a number… by vickoly</q>
• When Age Is Just A Number In Marriage - <q>Having common interest matters a lot in a relationship, it keeps the marriage alive. By nature, men's strength stays for a longer time and that's the reason …</q>
• Anything Worth Doing, is Worth Doing Well - <q>… relationship,and school, and just everything in general. … She would just smile and tell me that age is just a number … just had to do it anyhow , just because …</q>
• My Age Gap Relationship - <q>… age gap relationship. … age really is but a number. When I look at … If weight, height, social status, bank account totals, and distance are just numbers in a …</q>

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Dear Dr. Sara Monaco,

Thank you very much for your kind e-mail dated 1-Feb-2024. Please find enclosed our revised version and our point-by-point reply to the comments from the reviewers. We have answered all major and minor points raised by the reviewers. Original Figures and Supplementary Figures were revised and renamed as follows.

Figure 1 -> Figure 1 and Revised Figure 2

Figure 2 -> Revised Figure 3 and Supplementary Figure 7

Figure 3 -> Revised Figure 4

Figure 4 -> Revised Figure 5 and 6

Figure 5 -> Revised Figure 7

Figure 6 -> Revised Figure 8

Supplementary Figure 1 -> Supplementary Figure 1

Supplementary Figure 2 -> Revised Supplementary Figure 3

Supplementary Figure 3 -> Revised Supplementary Figure 4

Supplementary Figure 4 -> Revised Figure 2

Supplementary Figure 5 -> Revised Supplementary Figure 6

Supplementary Figure 6 -> Revised Supplementary Figure 9

Supplementary Figure 7 -> Revised Supplementary Figure 10

Supplementary Figure 8 -> Revised Supplementary Figure 11

Supplementary Figure 9 -> Revised Supplementary Figure 12

We believe that our revised manuscript has been significantly improved thanks to your help. Thank you very much again for your help.

Yours sincerely,

Yosuke Mai and Ken Natsuga

*Reviewer #1 (Evidence, reproducibility and clarity (Required)):

SUMMARY In this study, the researchers investigate the spontaneous patterning of keratinocytes. As model they use HaCaT cells, an immortalized keratinocyte line. The cells exhibit a self-organized pattern of high and low cell density, which is disrupted by medium changes but reappear over time. The researchers find that serum starvation and high calcium concentration are crucial for the formation of these keratinocyte patterns. RNA sequencing analysis of regions of high vs low density indicates enrichment in gene ontology terms related to cell-cell adhesion, mainly adherens junctions (AJs), and keratinocyte differentiation. Experimental manipulations, such as inhibiting E-cadherin- or α-catenin-mediated adhesion, and disrupting myosin-II activity, all interfer with the formation of keratinocyte patterns, emphasizing the importance of AJs. Mathematical modeling suggests that cell-cell adhesion alone is sufficient for the emergence of density patterns. Keratinocyte patterns have spatial regulation of keratinocyte differentiation and proliferation. Differentiated cells are abundant in areas of high cell density, while proliferative cells are in areas of low cell density. The authors verify that YAP activity regulates pattern-dependent differentiation and proliferation. The role of serum starvation and cell-cell adhesion through AJs in the differentiation of keratinocytes are supported by epidermal stratification experiments in 3D culture, and ex vivo experiments on mouse skin suction blister wounds. In conclusion, the study provide insights into the spatial regulation of differentiation and proliferation in epidermal cells. MAJOR COMMENTS Although not novel, given that it has been already demonstrated with several other epithelial cell monolayers and in vivo in Drosophila, the conclusions that serum starvation facilitates epidermal stratification through cell-cell adhesion is convincing. It is unclear whether the cell patterning the authors are describing is a real patterning, defined in biology as any regularly repeated cell or structural arrangement or simply an inhomogeneous distribution of cell densities.*

We have addressed this issue by analyzing our images with the autocorrelation function (see Fig. 1g, 1h, and Supplementary Fig. 5) and confirmed that the distribution of high/low cell density is patterned with the average nearest neighbor distance between areas of high cell density being approximately 300 µm. We have incorporated these new data into the revised manuscript.

The conclusion that the cell-cell adhesion signaling pathway identified in the paper "might promote wound healing in clinical settings" (last sentence of the abstract) is not substantiated by the results.

We agree with the reviewer's point and have deleted the sentence in the abstract, accordingly.

It would be opportune to better describe the type of "cell patterning" that the authors are seeing in their experiments. In my opinion the effect seen in the described experiment is not a "patterning" but a difference in cell density which can be less or more homogeneous in an HaCat monolayer.

Please see the answer above on our analysis using the autocorrelation function.

Importantly, it is unclear whether the "cell patterning" is a subsequent consequence or proceed stratification.

As the mathematical modeling indicated patterning without the need for stratification steps, we believe that cell patterning is not a direct consequence of stratification. However, it is technically difficult to differentiate whether patterning developed prior to stratification in our experimental settings. We have added this limitation to the Discussion of the revised manuscript.

It is unclear how starvation relates to the increased adhesions and YAP signaling.

As the reviewer pointed out, we could not address what molecules in the serum are responsible because the serum is a complex mixture of biomolecules that includes hormones, growth factors, vitamins, and other nutrients. We have added this limitation of our study to the revised manuscript.

The authors conclude the discussion section proposing "that molecules involved in cell-cell adhesion-induced patterning are suitable target candidates to facilitate wound healing". None the experiments done in the wound healing setting are addressing the role of any molecules described in the paper. I would suggest the authors to remove this last claim from the manuscript. Alternatively, the authors should provide evidence that targeting some of the molecules described in the manuscript are accelerating wound healing in a clinically relevant model of wound healing.

We agree with the reviewer's point and have deleted the passage in the revised manuscript, accordingly.

I would request the authors to provide the following essential data to substantiate their experiments: - Provide a full gene list related to Figure 2a.

We have provided the gene list (Supplementary Table 1), accordingly.

- In relation to Figure 2c, stain for a-catenin and quantify the intensity ration of a-catenin vs a-18-catenin as proper readout of adhesion strength (see Yonemura et al., Nat Cell Biol 2010).

As the reviewer pointed out, the intensity ratio of α-catenin vs. α18 is a general readout of cell adhesion strength. However, this ratio should be based on similar intensity of alpha catenin between two groups for comparison. In contrast, the intensity of α-catenin itself was weaker in the area with low cell density compared with in that with high cell density in our experimental setting (Supplementary Fig. 8d, e, g), which could greatly affect the ratio. To overcome this problem, we have reanalyzed line plots of α-catenin immunofluorescence, picked up the α18 intensity at the peaks (corresponding to cell-cell adhesion) of α-catenin, and compared that of high and low cell density area. As expected, α18 was more pronounced in the area with high cell density. We have added the data to Supplementary Fig. 8d-h in the revised manuscript.

- Properly quantify nuclear vs cytoplasmic localization of YAP in low vs high density areas in Figure 4f.

According to the reviewer's suggestion, we have quantified nuclear/cytoplasmic YAP and added the data (Revised Fig. 6b (original Fig. 4)) to the revised manuscript.

• The nuclear localization of YAP is not sufficient to demonstrate activation of the YAP signaling. The authors should provide evidence of YAP activity in low vs high density areas looking for example at known downstream target genes in epithelial cells (see Zhao et al., Genes Dev 2007; Yu et al., Cell 2012; Aragona et al., Cell 2013).

We have analyzed ANKRD1 (Yu et al., Cell 2012) as a YAP readout molecule and confirmed that, in line with YAP dynamics, ANKRD1 was localized in the nucleus of high cell density area. We have provided the data (Revised Fig. 6c, d (original Fig. 4)) for the revised manuscript.

• The activity of PY-60 in Figure 4g and XAV939 in Figure 4i as YAP activator and repressor respectively, should be controlled against YAP localization and activity.

We have quantitatively analyzed YAP and ANKRD1 localization upon chemical treatment and added the data (Supplementary Fig. 1a-d, g-j (original Supplementary Fig. 8)) to the revised manuscript.

• In Figure 5a a quantification of the numbers of cell layers should be used instead of the thickness and a staining and quantification of K14 and K10 should be added to formally address stratification.

As expected, the number of K10-positive cell layers was larger in serum-starved conditions than in serum-rich conditions, while the number of K14-positive cell layer was comparable between the two groups. We have provided the quantification data (Supplementary Fig. 12 c-e (original Supplementary Fig. 9)) to the revised manuscript accordingly.

*Most of the proposed experiments are simply additional quantifications of images or adjustments of data that are already available to the authors. I estimate that the remaining experiments can be done in less than a month and will not require additional expertise.

The methods, figures presentation and legends, and the statistical analysis are adequate, clear and accurate.

MINOR COMMENTS There are three fundamental studies that the authors should discuss: - Saw, Doostmohammadi et al., Nature 2017. Topological defects in epithelia govern cell death and extrusion. Here, the role of topological defects (see also Bonn et al., Phys Res E 2022) and a-catenin-dependent cell-cell interactions are connected to cell extrusion and Yap activity in epithelial monolayers including HaCat cells. - Miroshnikova et al., Nat Cell Biol 2018. Adhesion forces and cortical tension couple cell proliferation and differentiation to direct epidermal stratification. Here, the authors demonstrated that the increase of cell-cell adhesion couples with a decrease of cortical tension triggers stratification in the skin epidermis. - Boocock et al., Nature Physics 2021. Theory of mechanochemical patterning and optimal migration in cell monolayers. Here, cell density and ERK activity are formalized to be key players in patterning formation in a cell monolayer. In addition, several components of the Hippo-YAP pathway are known regulators of cell-cell adhesion (e.g. AMOT and NF2) and should be discussed (for reference see reviews on the topic Zheng & Pan, Dev Cell 2019; Karaman & Halder Cold Spring Harb Perspect Biol 2018; Gumbiner & Kim, J Cell Sci 2014) as important molecules implicated in the biological phenomena described in the manuscript.0*

We appreciate the reviewer's suggestion and have cited and discussed these seminal papers in the revised manuscript.

Reviewer #1 (Significance (Required)):

The study aims at understanding spontaneous patterning of keratinocytes. The authors nicely employ various experimental approaches, including cell imaging, RNA sequencing, cell manipulation by genetic engineering and pharmacological treatments, and mathematical modeling, to elucidate the underlying cellular and molecular mechanisms regulating this proces. However, several of the conclusions presented in the manuscript do not present any conceptual advance to the field of self-organization of cell density patterns or epithelial biology.

The role of starvation in effecting epithelial growth is very well known. The role of AJ in pattern formation has been described previously in epithelial monolayers (Saw, Doostmohammadi et al., Nature 2017) and in vivo in Drosophila (Mao et al., Genes Dev 2011; Mao et al., EMBO J 2013). The effect of cell density on YAP signaling is known (Zhao et al., Genes Dev 2007; Aragona et al., Cell 2013). The importance of AJ for keratinocytes differentiation and stratification has been demonstrated in vitro and in vivo (Miroshnikova et al., Nat Cell Biol 2018). The role of a-catenin upstream of YAP activity in regulating interfollicular epidermis stem cells self-renewal and wound healing has been demonstrated in vitro and in vivo by the group of Fernando Camargo in Cell 2011.

The manuscript could be of interest for researchers interested in basic cell biology and a specialised audience in cell self-organisation.

My field of expertise: epithelial biology, stem cell biology, skin homeostasis and wound healing, mechanobiology, YAP signaling. I do not have sufficient expertise to evaluate the mathematical modelling.

We appreciate the reviewer's constructive comments.

*Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary:

Mai et al. reported an interesting observation that serum starvation induced the keratinocytes, a type of epithelial cells, to form a pattern characterized by regions with high and low densities. They showed that this patterning processing depends on cell-cell adhesion using a series of pharmacological treatment and a CRISPR knockout of alpha-catenin. They used mathematical modeling to demonstrate that cell-density dependent stress can sufficiently generate patterns of high and low cell densities, but the interpretation of the modeling is questionable (see below). They showed correlation of a differentiated keratinocyte marker, keratin 10, with the high-density region, but over claimed this result as patterning modulates differentiation. They also showed correlation of YAP activity (cytoplasmic to nuclear ratio) to the high vs. low-density regions. Interestingly, treatment with a YAP activator PY-60 disrupted pattern formation, while the YAP inhibitor XAV939 barely affected pattern formation. Finally, the authors demonstrated that serum starvation increased the thickness of keratinocytes cultured in a trans-well system (which they called 3D culture), and a mouse back skin explant compared to serum-rich culture conditions. In the former system, they showed dependence on alpha-catenin using the CRISPR knockout.

Major comments:

The conclusion that "mathematical modeling indicates that cell-cell adhesion alone is sufficient to form regions with high/low cell density" is misleading. The key assumption of the modeling is that the time derivative of stress (d_sigma/dt) is proportional to the cell density (rho), where the proportion parameter (beta) was interpreted as cell adhesion strength. However, beta could be interpreted as any general attractor proportional to the cell density, such as a chemoattractant.*

Our purpose here is to demonstrate that the model based on the assumption of cell-cell adhesion as a mere source of attractive forces can reproduce the experimentally observed spatial patterning. As the referee rightly points out, the term beta*rho in the second equation allows different interpretations such as the effect of attractant proportional to cell density. Therefore, our mathematical model cannot be used as a proof of the existence of cell-cell adhesion. We have reduced the tone in the revised manuscript.

In addition, it is unclear why the time derivative of stress (d_sigma/dt) instead of stress itself (sigma) proportional to the cell density. The authors should further clarify the meanings of modeling parameters and be more careful with their conclusions.

If the system is in the steady state (d_sigma/dt = 0) with no spatial variations (nabla^2 \sigma = 0), then the second equation reduces to sigma = (beta/alpha) rho, namely that the cell density is proportional to stress, as pointed out by the referee.

Our model, which describes temporal and spatial variations, generalizes this situation. The spatial dependence represented by nabla^2 sigma was introduced according to the Reference 72 (original Reference 51). Furthermore, we introduced the time derivative d_sigma/dt to account for the fact that the system should relax into the steady state described above. We have included these into the revised manuscript.

Related to above, the authors should revise the title to reflect that the patterning depends on cell-cell adhesion instead of claiming that cell-cell adhesion drives patterning. This would require experimentally demonstrating sufficiency, for example, showing that increasing adhesion in a cell line with low adhesion that does not show patterning can sufficiently induce patterning.

We agreed with the reviewer and have revised the title into "Patterning in stratified epithelia depends on cell-cell adhesion" and reduce the tone of the final sentence of the Discussion section accordingly.

The conclusion that "patterning modulates differentiation" is not supported by evidence. Differentiation as evidenced by the presence of keratin 10 occurred as early as day 2 before any signs of patterning (Fig. 4A). When patterning was completely disrupted by alpha-catenin KO, there are still many keratin 10 positive cells. The apparent higher proportion of keratin 10+ cells in the wild type seems to be merely reflecting the higher cell density - if the quantification were normalized by the cell number, they are probably comparable. Overall, the presented data only supports a correlation of the differentiation marker keratin 10 with high-density regions.

According to the reviewer's suggestion, we have reduced the tone of the title of Revised Fig. 4 (original Fig. 3) and changed it into "Patterning correlates with differentiation and proliferation markers in keratinocytes".

The choice of RNA-seq comparison groups (high-density vs. low-density culture) is puzzling, since the effects caused by culture density changes may not be related to the high vs. low-density regions in the patterned cultures. There are so many changes there and the rationale of following up on cell adhesion was unclear. In fact, it seems that the RNA-seq data didn't help the logic flow of the paper at all.

Although we believe that comparison between high-density and low-density culture partly recapitulates high/low cell density regions in our study, the comparison is not identical to patterned cultures as the reviewer pointed out. We have moved RNA-seq data to the Supplementary Information (Supplementary Fig. 7) and added more analysis to address that cell adhesion and differentiation are major differences between high-density and low-density culture, supporting further analysis on this matter in our study.

The claim of 3D culture of keratinocytes is confusing. The culture in the trans-well insert is still on the flat 2D surface, why should it be called 3D culture? If the point is to culture at air/liquid interface, that should instead be emphasized instead of calling it 3D.

We have changed "3D culture" into air-liquid interface culture, accordingly.

Reviewer #2 (Significance (Required)):

The observation that serum starvation and replenishment induced reversible patterning of the keratinocytes is quite interesting. However, the biological relevance is unclear - isn't all skin stratified? The evidence supporting the dependence of this patterning on adherens junction by disrupting E-cadherin, myosin, or alpha-catenin is convincing, although not surprising. The involvement of YAP in differentiation vs. proliferation is interesting, but it's in line with the known functions of YAP. The modeling part, with some clarification, can be quite insightful. Overall, this research could be interesting to those working in epithelial morphogenesis, if further developed.

My expertise is in epithelial tissue morphogenesis, mechanobiology, and extracellular matrix biology.

We appreciate the reviewer's constructive and thoughtful comments.

*Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript the authors aim to understand the signals that coordinate spatial patterns of keratinocyte proliferation and differentiation. To address this question the authors use the HaCaT keratinocyte cell line that upon serum starvation forms spatially separated domains of proliferation and differentiation. The data presented in this manuscript potentially suggest that serum starvation works through adherens junctions to create differentially dense fields within the cultures which determines whether cells proliferate or differentiate. The authors then perform experiments to show that junction formation with starvation drive keratinocyte differentiation potentially through YAP signaling. However, these experiments are rather loosely connected and their results often do not support the conclusions drawn by the authors. However, the not well supported conclusion the form the basis for a fact statement, but their data really did not show that. For example, the authors state: "By contrast, YAP inhibition by a tankyrase inhibitor, XAV939, suppressed pattern-dependent proliferation (Fig. 4i, j)," . However, their data do not show that proliferation is pattern-dependent but is nevertheless used to connect to and draw a conclusion about YAP signaling. The data itself appear to be of high quality, figures are well organized and statistics of quantification seem appropriate, but it somewhat problematic that throughout the manuscript it remains unclear if certain statements are hypotheses or conclusions on real data. Pattern formation as a requirement for differentiation is an interesting concept. However, the presented study lacks proper conclusive data how these patterns may contribute to proliferation and differentiation and remains rather short on what exactly is the instructive nature of these patterns, as they only use high density and are not generating own patterns with defined cues that explore what cues contribute. Major points: The statement "According to the RNA-seq data, AJ molecules, such as E-cadherin and actin, were localized at intercellular junctions in areas of high cell density" is not correct. RNA-seq does not allow conclusions about protein localization. Instead, the GO-Term analysis shown in Figure 2b shows downregulation of "cell-adhesion" in dense areas. *

According to the reviewer's suggestion, we have corrected the sentence. We have reanalyzed our RNA-seq data and confirmed that GO-term "cell-adhesion" was in the top list of both high and low cell density regions. We have provided more data to the revised manuscript (Supplementary Fig. 7 and Supplementary Table 1).

*Consistently the E-cadherin staining presented in Figure2c suggest lower intercellular E-cadherin levels in the most dense areas. However, any statement about junctional localization of adhesion components requires e.g. intensity quantification at junctions vs. cytoplasm or else to discriminate from intense overall staining due to high cell density and thus high overall junction numbers. *

Actually, junctional E-cadherin was more pronounced in the high cell density area. We have provided line plot data to confirm this and also added a quantification data (Supplementary Fig. 8).

Hence, even though potentially true, the statement: "These data suggest that cells in regions of high cell density form AJs in response to intercellular forces" is not fully supported by the data shown so far.

Please see the answer to the comments of Reviewer 1 on the quantification of α18. We believe that, as α18 intensity is more pronounced in the cell-cell junction of high cell density area compared with low cell density regions, our claim is experimentally supported.

The authors suggest a pattern of high and low density that is formed over time. However, at the same time high density areas show formation of a second layer. Hence, "denser" areas as observed by phase contrast images or DAPI positive nuclei may either represent dense or stratified cells. What is missing is an analysis of cell density before cells started to stratify making sure only cells in the basal layer are analyzed. Otherwise, density and stratification which are perhaps interdependent in this system cannot be discriminated.

As the reviewer pointed out, the patterning was analyzed at the level of basal layer. In addition to Figure 1c, we have provided another plane cut immunofluorescence data (Supplementary Fig. 2) to the revised manuscript to address this issue.

*The mathematical model does not include stratification and it is thus not clear to what extend it may explain the observed patterns. *

It is true that the model does not account for stratification. It focuses solely on the patterning of cell density in the basal layer. We have incorporated this notion into the revised manuscript as a limitation of this study.

*Moreover, the model appears to assume variables that have not been determined or cited. This reviewer is not an expert in modeling and thus cannot fully judge the math behind the model. However, the model appears to be biased if it assumes, as mentioned, that cell-adhesion increases with density. *

The first equation, describing the time evolution of rho (the cell density), incorporates diffusion, collective cell movement due to stress from adjacent cells, and random fluctuations. Each of these terms comes from a general consideration of density dynamics. The second equation, describing stress balance, is a generalization of Reference 72 (originally Reference 51). The crucial assumption here is that the cell-cell adhesion increases with density, which corresponds to the experimental findings (Revised Fig. 3a, b, Supplementary Figure 8a-h).

What we have demonstrated here is that we only need cell-cell adhesion as a source of attractive interactions for cells to form the density patterning as observed in the experiment. Since it is not self-evident whether the assumption of the density-dependent adhesion entail the emergence of density patterns, we do not believe that our model is begging the question or biased.

*If low adhesion forces do not produce patterns, what is the counterforce in the model? Are cells allowed to change size to enable low density areas or do cells lose contact with neighbors despite high adhesion strength? *

Our model does not have a variable corresponding to cell-shape change, which is considered only implicitly: Cells in the low density region (small rho) are regarded as flattened, whereas those in the high density region (large rho) as compressed (though not stratified) (Fig 1c).

The behavior of the model is controlled by the parameter beta: a smaller beta means that density variations have little effect on stress, whereas a larger beta leads to significant stress changes with density variation. Since stress increases as beta*rho (in the second equation), stress in the low density region remains low even when the parameter beta is large.

Overall, it appears that the model is set up such, that it tends to reproduce what was observed in experiment. This conclusion, however, may result of an incomplete understanding of the model parameters.

The model setup, the assumption on the relationship between density and cell-cell adhesion in particular, does not inherently dictate the emergence of high/low density patterns: It might be the case that cell density is uniformly distributed everywhere with uniformly strong adhesion among cells. What our computer simulations have shown, however, is that the model exhibits spatially heterogeneous density patterns for sufficiently high beta values. The emergence of such spatial patterns is not a predefined aspect of the mathematical model itself.

In the revised manuscript, the non-triviality of the spatial patterning has been made clear in the Results, and more explanations on the mathematical model to address the above points have been added to the Methods section.

If dense areas do actually represent stratified areas it may not be surprising that the GO analysis indicates an increase in differentiation. A requirement for AJ or intercellular junctions in general is less surprising as stratification requires cell-cell adhesion. The observation that AJ are essential for intercellular junction formation in keratinocytes or in other epithelial cells is not new (e.g. Michels et al. JID 2009).

We agree with the reviewer in the point that the role of AJ is not new. We have incorporated the notion into the Discussion of the revised manuscript and cited the paper the reviewer indicated.

The part of the paper addressing the role of YAP suffers from a number of potentially mislead assumptions/conclusions based on a previous experiment which then did not properly supported that conclusion (see also overall comments). For example, the statement "YAP inhibition by a tankyrase inhibitor, XAV939, suppressed pattern-dependent proliferation" contains interdependencies that have not been show [sic]. XAV939 may just inhibit proliferation which is not necessarily pattern dependent. Too much speculation confuses data and hypotheses.

We agree with the reviewer to point out that pattern-dependency was not supported by our results. We have reduced the tones and corrected these terms in the revised manuscript.

The 3D HaCaT cultures are performed on transwell filters with medium supply above and below cells, with the assumption that organizing patterns are also formed under these conditions. However, this has not been shown by the authors. Their suggestion that serum starvation may increases thickness of cultures through alterations in the organization of [sic]

We showed the patterning in air-liquid interface culture in the Supplementary Data (Supplementary Fig. 12a, b, original Supplementary Fig. 9a, b), which presents starvation-induced pattering even in such condition.

Reviewer #3 (Significance (Required)):

The mechanisms that drive self-organization of epithelial cells to spatially separate domains of proliferation and differentiation is in principle a very interesting topic of high interest to the cell and mechanobiology community, [sic]

We appreciate the reviewer's constructive and thoughtful comments.

Summary of "So you want to build an ECS-backed GUI framework"

• Introduction to ECS and Bevy for GUI: The authors discuss using the Entity-Component-System (ECS) framework within Rust, specifically Bevy, to build a UI framework. They highlight the unconventional nature of ECS-based GUIs but reference prior implementations like flecs and various Bevy experiments demonstrating the feasibility and potential of this approach.

• "It's a type-safe, trendy solution for state management and most importantly: it'll be blazing fast" and "existing experiments like belly, bevy_lunex, bevy_ui_dsl, cuicui_layout and kayak_ui show a ton of promise using Bevy's ECS."

• Challenges of Developing bevy_ui: The text outlines that the issues with bevy_ui stem not from its ECS or Rust foundations, but rather from the inherent complexity and labor intensity of GUI framework development, which involves numerous components and meticulous coordination.

• "most of the problems that plague bevy_ui aren't driven by the decision to use an ECS, or even to use Rust. They're the boring, tedious and frustrating ones: writing GUI frameworks is a lot of work with many moving parts."

• Authors’ Background and Disclaimer: Alice is a maintainer (not the lead) of Bevy and Rose uses Bevy professionally, indicating their deep involvement but also stressing that their opinions are personal and not official.

• "Alice is a maintainer of Bevy, but not the project lead or even a UI subject-matter-expert. Rose is an employee at the Foresight Spatial Labs."

• Vision for bevy_ui: The post aims to clarify the rationale behind using ECS for GUI, addressing common critiques and misconceptions, and outlining necessary improvements for making bevy_ui competitive and functional.

• "This post aims to record how you might make a GUI framework, why we're using an ECS at all, and what we need to fix to make bevy_ui genuinely good."

• Common Misconceptions and Arguments: The discussion addresses several typical arguments against developing a native Bevy GUI framework, like the feasibility of a single framework satisfying diverse application requirements and the tendency to prefer existing solutions to avoid 'Not Invented Here' syndrome.

• "One GUI framework to rule them all?" and "Bevy should just use an existing GUI framework."

• Technical and Social Reasons for a Bevy-Specific GUI: The authors argue for a Bevy-specific GUI to ensure consistency, ease of maintenance, integration with Bevy’s core features, and avoiding dependency risks which can complicate maintenance and updates.

• "Consistency with the rest of the engine is valuable in its own right. It makes for an easier and more consistent learning experience for new users."

• Implementation Challenges: The article details specific technical challenges involved in building bevy_ui, such as managing a UI tree structure, input collection, text rendering, and integrating with Bevy's system for rendering, state management, and data transfer.

• "Storing a tree of nodes... In bevy_ui, this is stored in the World: each node is an entity with the Node component."

• Strategic Plan for Improvement: The authors propose a mix of straightforward, controversial, and research tasks to progressively refine and enhance bevy_ui. These include enhancing documentation, adopting new layout strategies, creating a styling abstraction, and improving integration with accessibility features.

• "We can split the work to be done into three categories: straightforward, controversial and research."

• Conclusion: The text concludes with optimism about the future of bevy_ui, encouraging the Bevy community to engage in its development to realize its potential fully.

• "But critically, none of it is impossible. If we (the Bevy developer community) can come together and steadily fix these problems, one at a time, we (Alice and Rose) genuinely think bevy_ui will one day live up to the standard for quality, flexibility and ergonomics that we expect out of the rest of the engine."

It's interesting that the result of this deregulation was the formation of just a few companies that have do much power. It doesn't really make sense that those in power would not have seen this coming, which does support the idea that this was a heavily political move that was aiming to enrich a few companies.

Although I don't really mind disclosing my information to some apps, after all it's just my name and so on. But Facebook would shock me how much they can collect information about non-users. Facebook collects data on non-users in two main ways, from their browsing history and from their friends. Although this data will not be stored permanently, many people will consider it an invasion of their privacy. Sometimes I also feel that too many advertisements on many software or web pages will bore me. But we have to admit that Facebook's use of big data is very successful from a certain perspective. When we think of social software, we think of Facebook.

The extent of consciousness, and the extent of accommodation: cis women, more accommodated without effort than before, perhaps also less conscious?

delete the session on your best fit occupations--that info goes into your Career Ready Portfolio, not the SLJ.

Wow, I had no idea that energy access was so low in like this.

you write the notes on it and you're faced with a dilemma because you don't know which folder to use it's problems like these that makes the use of the system cumbersome and makes the users eventually abandon their system altogether. On the contrary zedl casting is bottom up you start with a hodgepodge of nodes each indicating an idea and you link them with each other there are no folders nothing but as you keep adding more and more notes into the system into this primary soup you can see the emergent structure you'll see that some nodes form clusters like they become the central hubs around which many other ideas and concepts revolve so they must be crucial and over time the system becomes more structured despite the initial thought that it will just turn into a confusing hairball of notes

^ middle member send his third event to his left and right member. So it's not just "send to one random". How is that specified?

Just an observation here but I remember my godchild using You tube kids whilst they stayed here and we had to double check because it wasn't all good content, you tube is kind of notorious with their bad content checks and algorithms. Elsa Gate Scandal comes to mind.

That's interesting. Block may refer to many blocks at once.

Curios whether Hashgraph's consensus can hold with it. It'll give inacurate timestamps. I.e., accuracy of how events been received will suffer. Perhaps we can only receive immediately events that contain ops that requrire a point of order. But overall, receiving and propagating further events right away seems to be a good strategy, since it captures more accurately what happens over-the-network. And it's more constricting, meaning it'll be easier to detect byzantine behaviour. Nodes are required to accept any valid events right away. Given we want to propagate events by gossip fast, we'd need nodes to accept and propagate their newly created event right away.

I.e., the approach of multiple blocks is not gossip-about-gossip.

There's a downside of having to accept right away. That is if we strictly comply to gossip-about-gossip, then event must point to other-parent. But if there's no other-parent, how do we get our local ops packaged and sent?

Perhaps a way is to ease the requirement of other-parent ref, then nodes can add local ops with ref to self-parent alone, and propagate them, will be alright even for a single keystroke op in text editors, although the metadata cost is somewhat high.

Perhaps metadata can be reduced down to signature. So whenever one receives an event with just signature - we know that it's an event with only self-parent ref. A well-known special case.

And when you want to accrete atop other-parent, you include a hash of it.

So the metadata cost is signature and, optionally, 1 hash.

Processing cost of deriving author out of signature, and verification is to be measured.

Is this the tablet Bianca mentioned? It sounds great but an entire tablet? I want to reach consumers on already owned devices. Edit no it's the one who expanded - they just launched english lit en are still launching afrikaans this year.

Variety is the spice of life. I don't think there is room for only one application, personally I wouldn't have enjoyed those graphics as a child, not all children like the same things and learn the same way, so to address that we would need many different applications carering to different tastes.

Somewhere in this paragraph, it might be helpful to relate these strategies or an additional strategy to the concepts of growth and fixed mindset, to really solidify. You may not need to explain how it fits that term (if examples are provided in previous section), but it would be helpful to just have it as another way of viewing the strategies of polyglots

It's important to not have assumptions control our perceptions of students and why they may struggle in school. As educators, we have to take considerations of the experiences students had in their early life and how it impacts their academic and grit development. That way, we can be properly informed and help students succeed both academically and emotionally.

The Hart and Risley study’s revelation about the vast differences in the number of words children hear from their parents based on socioeconomic status is both fascinating and disheartening. It underscores the critical role of early language exposure in cognitive development and future academic success. This research highlights not just an educational disparity but a profound societal issue, emphasizing the importance of addressing educational inequalities from an early age. It’s a compelling argument for the value of interventions aimed at enriching the linguistic environments of all children, regardless of their family’s income.

This passage about the educational income gap captivates me because it offers an in-depth look into an issue I deeply care about: social equity. Understanding how income influences children’s performance in school is crucial for someone concerned with social justice, like myself. It helps in pinpointing the root causes and pondering possible solutions. The widening of this gap not only exposes the inequalities within the educational system but also underscores the urgency of addressing this issue. It’s clear that tackling these disparities is not just about fairness but also about shaping a more inclusive future where every child has the chance to succeed.

While different situations require different solutions, I'm inclined to accept "don't feed the trolls" as a general rule. Responding to a general trolling post takes up time and mental energy. It's just not worth it.

That being said, "don't feed the trolls" can't be used on all situations. Moderators and admins should still moderate their online spaces, and targets of harassment campaigns need different strategies. Either way though, responding to and engaging with the trolls won't make the situation better.

This particular part stuck out to me because it’s important that we are able to differentiate real life and life over the internet. It’s important to note that whatever is portrayed is what the person wants you to see. So, even though the content was deemed unauthentic, people still watched because they understood that it’s just pure entertainment and not something that should be taken seriously.

Reviewer #3 (Public Review):

This manuscript reports a novel pedigree with four intact copies of RHO on a single chromosome which appears to lead to overexpression of rhodopsin and a corresponding autosomal dominant form of RP. The authors generate retinal organoids from patient- and control-derived cells, characterize the phenotypes of the organoids, and then attempt to 'treat' aberrant rhodopsin expression/mislocalization in the patient organoids using a small molecule called photoregulin 3 (PR3). While this novel genetic mechanism for adRP is interesting, the organoid work is not compelling. There are multiple problems related to the technical approaches, the presentation of the results, and the interpretations of the data. I will present my concerns roughly in the order in which they appear in the manuscript and will separate them into 'major' and 'minor' categories:

Major concerns:<br /> (1) Individual human retinal organoids in culture can show a wide range of differentiation phenotypes with respect to the expression of specific markers, percentages of given cell types, etc. For this reason, it can be very difficult to make rigorous, quantitative comparisons between 'wild-type' and 'mutant' organoids. Despite this difficulty, the author of the present manuscript frequently present results in an impressionistic manner without quantitation. Furthermore, there is no indication that the investigator who performed the phenotypic analyses was blind with respect to the genotype. In my opinion, such blinding is essential for the analysis of phenotypes in retinal organoids.

To give an example, in lines 193-194 the authors write "we observed that while the patient organoids developing connecting cilium and the inner segments similar to control organoids, they failed to extend outer segments". Outer segments almost never form normally in human retinal organoids, even when derived from 'wild-type' cells. Thus, I consider it wholly inadequate to simply state that outer segment formation 'failed' without a rigorous, quantitative, and blinded comparison of patient and control organoids.

(2) The presentation of qPCR results in Fig. 3A in very confusing. First, the authors normalize expression to that of CRX, but they don't really explain why. In lines 210-211 they write "CRX, a ubiquitously expressing photoreceptor gene maintained from development to adulthood." Several parts of this sentence are misleading or incomplete. First, CRX is not 'ubiquitously expressed' (which usually means 'in all cell types') nor is it photoreceptor-specific: CRX is expressed in rods, cones, and bipolar cells. Furthermore, CRX expression levels are not constant in photoreceptors throughout development/adulthood. So, for these reasons alone, CRX is a poor choice for normalization of photoreceptor gene expression.

Second, the authors' interpretation of the qPCR results (lines 216-218) is very confusing. The authors appear to be saying that there is a statistically significant increase in RHO levels between D120 and D300. However, the same change is observed in both control and patient organoids and is not unexpected, since the organoids are more mature at D300. The key comparison is between control and patient organoids at D300. At this time point, there appears to be no difference control and patient. The authors don't even point this out in the main text.

Third, the variability in number of photoreceptor cells in individual organoids makes a whole-organoid comparison by qPCR fraught with difficulty. It seems to me that what is needed here is a comparison of RHO transcript levels in isolated rod photoreceptors.

(3) I cannot understand what the authors are comparing in the bulk RNA-seq analysis presented in the paragraph starting with line 222 and in the paragraph starting with line 306. They write "we performed bulk-RNA sequencing on 300-days-old retinal organoids (n=3 independent biological replicates). Patient retinal organoids demonstrated upregulated transcriptomic levels of RHO... comparable to the qRT-PCR data." From the wording, it suggests that they are comparing bulk RNA-seq of patient and control organoids at D300. However, this is not stated anywhere in the main text, the figure legend, or the Methods. Yet, the subsequent line "comparable to the qRT-PCR data" makes no sense, because the qPCR comparison was between patient samples at two different time points, D120 and D300, not between patient and control. Thus, the reader is left with no clear idea of what is even being compared by RNA-seq analysis.

Remarkably, the exact same lack of clarity as to what is being compared plagues the second RNA-seq analysis presented in the paragraph starting with line 306. Here the authors write "We further carried out bulk RNA-sequencing analysis to comprehensively characterize three different groups of organoids, 0.25 μM PR3-treated and vehicle-treated patient organoids and control (RC) organoids from three independent differentiation experiments. Consistent with the qRT-PCR gene expression analysis, the results showed a significant downregulation in RHO and other rod phototransduction genes." Here, the authors make it clear that they have performed RNA-seq on three types of sample: PR3-treated patient organoids, vehicle-treated patient organoids, and control organoids (presumably not treated). Yet, in the next sentence they state "the results showed a significant downregulation in RHO", but they don't state what two of the three conditions are being compared! Although I can assume that the comparison presented in Fig. 6A is between patient vehicle-treated and PR3-treated organoids, this is nowhere explicitly stated in the manuscript.

(4) There are multiple flaws in the analysis and interpretation of the PR3 treatment results. The authors wrote (lines 289-2945) "We treated long-term cultured 300-days-old, RHO-CNV patient retinal organoids with varying concentrations of PR3 (0.1, 0.25 and 0.5 μM) for one week and assessed the effects on RHO mRNA expression and protein localization. Immunofluorescence staining of PR3-treated organoids displayed a partial rescue of RHO localization with optimal trafficking observed in the 0.25 μM PR3-treated organoids (Figure 5B). None of the organoids showed any evidence of toxicity post-treatment."

There are multiple problems. First, the results are impressionistic and not quantitative. Second, it's not clear that the investigator was blinded with respect to treatment condition. Third, in the sections presented, the organoids look much more disorganized in the PR3-treated conditions than in the control. In particular, the ONL looks much more poorly formed. Overall, I'd say the organoids looked considerably worse in the 0.25 and 0.5 microM conditions than in the control, but I don't know whether or not the images are representative. Without rigorously quantitative and blinded analysis, it is impossible to draw solid conclusions here. Lastly, the authors state that "none of the organoids showed any evidence of toxicity post-treatment," but do not explain what criteria were used to determine that there was no toxicity.

(5) qPCR-based quantitation of rod gene expression changes in response to PR3 treatment is not well-designed. In lines 294-297 the authors wrote "PR3 drove a significant downregulation of RHO in a dose-dependent manner. Following qRT-PCR analysis, we observed a 2-to-5 log2FC decrease in RHO expression, along with smaller decreases in other rod-specific genes including NR2E3, GNAT1 and PDE6B." I assume these analyses were performed on cDNA derived from whole organoids. There are two problems with this analysis/interpretation. First, a decrease in rod gene expression can be caused by a decrease in the number of rods in the treated organoids (e.g., by cell death) or by a decrease in the expression of rod genes within individual rods. The authors do not distinguish between these two possibilities. Second, as stated above, the percentage of cells that are rods in a given organoid can vary from organoid to organoid. So, to determine whether there is downregulation of rod gene expression, one should ideally perform the qPCR analysis on purified rods.

(6) In Fig. 4B 'RM' panels, the authors show RHO staining around the somata of 'rods' but the inset images suggest that several of these cells lack both NRL and OTX2 staining in their nuclei. All rods should be positive for NRL. Conversely, the same image shows a layer of cells sclerad to the cells with putative RHO somal staining which do not show somal staining, and yet they do appear to be positive for NRL and OTX2. What is going on here? The authors need to provide interpretations for these findings.

Minor concerns:

(1) The writing is poor in many places. Problems include: poor word choice (e.g., 'semi-occasional' is used three times where 'occasional' or 'infrequent' would be better); superfluous use of the definite article in many places (e.g., lines 189-190 "by the light microscopy" should be "by light microscopy"); awkward sentence structures (e.g., lines 208-209: "To equilibrate the data to equivalent the number of photoreceptors in organoids"), opaque expressions (e.g., line 217 "there was a significant ~3 log2 fold change (log2FC)"; why not just say "an ~8-fold change"?); poor proof-reading (Abstract says that 40% of adRP cases are due to mutation in RHO, then the Introduction says the figure is 25%) etc.

(2) The figures are not numbered, which makes it painful for the reviewer to correlate main text call-outs, figure legends, and actual figures. I had to repeatedly count down the list of figures to determine which figure I should be looking at.

(3) In the abstract, the authors suggest that the patient's disease "develops from a dominant negative gain of function" mechanism. I don't agree with this interpretation. Typically 'dominant-negative' refers to an aberrant protein which directly interferes with the function of the normal protein, for example by forming non-functional heterodimers. In the present patient, the disease can be explained by a simple overexpression mechanism, as it has been previously demonstrated in mice that even minimal overexpression of rhodopsin (e.g., ~25% more than normal levels) can led to progressive rod degeneration: PMID: 11222515.

(4) In line 85 the word 'Morphologically' is superfluous and can be deleted.

(5) In the Introduction the authors should more clearly articulate the rationale for using PR3 to treat this patient: because it leads to downregulation of multiple rod genes including RHO. This isn't clearly explained until the Discussion.

(6) The authors mention in several places that PR3 may act via inhibition of NR2E3. Although this was the conclusion of the original publication, the evidence that PR3 acts via Nr2e3 in mice is not solid. The original study (PMID: 29148976) showed that the main effect of PR3 application on mouse retinas is downregulation of numerous rod genes. However, knockout of Nr2e3 in mouse has been shown to have very little effect on rod gene expression, and Nr2e3 mutant rods have largely preserved rod function as demonstrated by scotopic ERGs PMIDs: 15634773, 16110338, 15689355). The primary gene expression defect in Nr2e3 mutant mouse rods is upregulation of a subset of cone genes, a change not observed upon application of PR3 to mouse retinas. For these reasons, I am skeptical that PR3 acts via inhibition of Nr2e3 activity, and I would suggest that the present authors qualify that interpretation.

(7) This mechanistic speculation presented in lines 274-278 is not warranted. Ectopic localization of opsin to the cytoplasmic membrane occurs in a wide range of genetic forms of rod degeneration.

I think the authenciticity of the Tweets that come from Trump himself is doubtful. Because there is no authoritative way to tell if he wrote these himself. And his statements as a public figure are made with multiple considerations. So it's quite possible that what he posted was just to make people think he was a qualified candidate. And it's possible that what he posted was just something his team came up with, not his own ideas.

This is very true. You don't have to do much; you just let AI write everything down for you, and you really don't have to stress about anything. But still, teachers know what's AI and what's not AI. It's not easy fooling a teacher with robotic writing they've been doing this for a long time and prboably would know whats AI and whats human. It is very easy to access ChatGpt like it said "you toggle over to OPENAI, log in to ChatGPT, and type in your query." it's really that simple.

In 1983, at the dawn of the personal computer age, Apple Inc. in promotional film entitled "Lisa Soul Of A New Machine" touted their new computer, a 16-bit dual disk drive "personal office system", as something that would do away with "the cluttered desk, index cards, file folders, the in-out basket, [and] the calculator." (00:01)

Some of these things moved to the realm of the computer including the messy desk(top) now giving people two messy desks, a real one and a virtual one. The database-like structure of the card index also moved over, but the subjective index and its search power were substituted for a lower level concordance search.

30 years on, for most people, the value of the database idea behind the humble "index card" has long since disappeared and so it seems here as if it's "just" another piece of cluttery paper.

Appreciate the rosy framing of the juxtaposition of "past" and "future" jumping over the idea of the here and now which includes the thing they're selling, the Lisa computer. They're selling the idealized and unclear future even though it's really just today.

(#14)

*N1 (14) (Lily): There are many different phrases discussing “likelihood.” Can we go over the difference between them (i.e., log likelihood versus maximum likelihood)? (Osamudia)

Response: Sure. Likelihood (L) = the probability that a specific event (or combination of events will occur), given the set of coefficients. Log likelihood is just the log of L. Maximum likelihood is the process of solving a likelihood equation.

*N3 (#14)(Savannah): Does the MLE get reported in a typical logit regression table in R, or do we have to do an extra command to see it? Additionally, can we compare MLEs across different model specifications to see which model has the best fit (like we do with R^2 in OLS)?

Response: The MLE would mean the maximum likelihood estimates--those are the coefficients. The log likelihood gets reported--or the deviance--which is just another way of reporting it.

*N4 (#14) (Osamudia)--could we contextualize this against probit and logit models? I’m having trouble understanding the scenarios for which this sort of analyses would be useful.

Response: Maximum likelihood estimation is the process by which all models get estimated (aside from OLS). You can’t avoid it. The results (i.e. the coefficients) are what we interpret.

• N5 (#14) (Syl): Can you explain to me like I’m 5 what you mean by “we’re working backwards” on slide 4? I’ve never thought about logit models like that and for some reason it’s not clicking and maybe even confusing me a little.

Response: This gets at Q2:

*N6 (#14) (Mia): I’m trying to wrap my head around calculating the best probability to give us similar data observations. Are there no implications to research when our predicted probability is a better fit for some demographic groups then others? How important is sample size in accurately getting to maximized likelihoodness?

Response: Note that this is what the program does given the variables (and the paramaterization--i.e., do you want interaction terms? Do you want to stratify by race? Do you want to include age squared etc.--> those are the choice you make. Then, given those choices, the algorithm finds the best fitting coefficients.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews

Reviewer #1 (Public Review):

Summary:

Dormancy/diapause/hibernation (depending on how the terms are defined) is a key life history strategy that allows the temporal escape from unfavorable conditions. Although environmental conditions do play a major role in inducing and terminating dormancy (authors call this energy limitation hypothesis), the authors test a mutually non-exclusive hypothesis (life-history hypothesis) that sex-specific selection pressures, at least to some extent, would further shape the timing of these life-history events. Authors use a metanalytic approach to collect data (mainly on rodents) on various life-history traits to test trade-offs among these traits between sexes and how they affect entry and termination of dormancy.

Strengths:

I found the theoretical background in the Introduction quite interesting, to the point and the arguments were well-placed. How sex-specific selection pressures would drive entry and termination of diapause in insects (e.g. protandry), especially in temperate butterflies, is very well investigated. Authors attempt to extend these ideas to endotherms and trying to find general patterns across ectotherms and endotherms is particularly exciting. This work and similar evidence could make a great contribution to the life-history theory, specifically understanding factors that drive the regulation of life cycle timing.

Weaknesses:

(1) I felt that including 'ectotherms' in the title is a bit misleading as there is hardly (in fact any?) any data presented on ectotherms. Also, most of the focus of the discussion is heavily mammal (rodent) focussed. I believe saying endotherms in the title as well is a bit misleading as the data is mammalfocused.

We change the title to : "Evolutionnary trade-offs in dormancy phenology". This is a hybrid article comprising both a meta-analysis and a literature review. Each of these parts brings new elements to the hypotheses presented. The statistical analyses only concern mammals and especially rodent species. But the literature review highlighted links between the evolution of dormancy in ectotherms and endotherms that have not been linked in previous studies. We feel it is important for readers to know that much of the discussion will focus on the comparison of these two groups. But we understand that placing the term ectotherms in the title might suggest a meta-analysis including these two groups.

In addition, we indicated more specifically in the abstract and at the end of the introduction that the article includes two approaches associated with different groups of animals.

We also specified in the section « review criteria » that:

Only one bird species is considered to be a hibernator, and no information is available on sex differences in hibernation phenology (Woods and Brigham 2004, Woods et al. 2019).

We have also added a "study limitations" section, which explains that although the meta-analysis is limited by the data available in the literature, the information available for the species groups not studied seems to support our results.

(2) I think more information needs to be provided early on to make readers aware of the diversity of animals included in the study and their geographic distribution. Are they mostly temperate or tropical? What is the span of the latitude as day length can have a major influence on dormancy timings? I think it is important to point out that data is more rodent-centric. Along the line of this point, is there a reason why the extensively studied species like the Red Deer or Soay Sheep and other well-studied temperate mammals did not make it into the list?

We specified in the abstract and at the end of the introduction that the species studied in the metaanalysis are mainly Holarctic species. We have also added a map showing all the study sites used in the meta-analysis. Finally, we've noted in the methods and added a "study limitation" section at the end of the discussion an explanation for those species that were not studied in the meta-analysis and the consequences for the interpretation of results

The hypotheses developed in this article are based on the survival benefits of seasonal dormancy thanks to a period of complete inactivity lasting several months. The Red Deer or Soay Sheep remain active above ground throughout the year.

The effect of photoperiod on phenology is one of the mechanisms that has evolved to match an activity with the favorable condition. In this study, we are not interested in the mechanisms but in the evolutionary pressures that explain the observed phenology. Interspecific variation in the effect of photoperiod results from different evolutionary pressures, which we are trying to highlight. It is therefore not necessary to review mechanisms and effects of photoperiod, themselves requiring a lengthy review.

We also tested the “physiological constraint hypothesis” on several variables. Temperature and precipitation are factors correlated with sex differences in phenology of hibernation. These factors allow consideration of the geographical differences that influence hibernation phenology.

(3) Isn't the term 'energy limitation hypothesis' which is used throughout the manuscript a bit endotherm-centric? Especially if the goal is to draw generalities across ectotherms and endotherms. Moreover, climate (e.g. interaction of photoperiod and temperature in temperatures) most often induces or terminates diapause/dormancy in ectotherms so I am not sure if saying 'energy limitation hypothesis' is general enough.

We renamed this hypothesis the "physiological constraint hypothesis" and we have made appropriate changes in the text so as not to focus physiological constraints solely on energy aspects.

(4) Since for some species, the data is averaged across studies to get species-level trait estimates, is there a scope to examine within population differences (e.g. across latitudes)? This may further strengthen the evidence and rule out the possibility of the environment, especially the length of the breeding season, affecting the timing of emergence and immergence.

For a given species, data on hibernation phenology are averaged for different populations, but also for the same population when measurements are taken over several years. To test these hypotheses on a population scale, precise data on reproductive effort would be needed for each population tested, but this concerns very few species (less than 5).

Testing the effects of temperature and precipitation allows us to take into account the effects of climate on phenology.

(5) Although the authors are looking at the broader patterns, I felt like the overall ecology of the species (habitat, tropical or temperate, number of broods, etc.) is overlooked and could act as confounding factors.

Yes, that's why we also tested the physiological constraints hypothesis, including the effect of temperature and precipitation. For the life-history hypothesis, we also tested reproductive effort, which takes into account the number of offspring per year.

(6) I strongly think the data analysis part needs more clarity. As of now, it is difficult for me to visualize all the fitted models (despite Table 1), and the large number of life-history traits adds to this complexity. I would recommend explicitly writing down all the models in the text. Also, the Table doesn't make it clear whether interaction was allowed between the predictors or not. More information on how PGLS were fitted needs to be provided in the main text which is in the supplementary right now. I kept wondering if the authors have fit multiple models, for example, with different correlation structures or by choosing different values of lambda parameter. And, in addition to PGLS, authors are also fitting linear regressions. Can you explain clearly in the text why was this done?

To simplify the results, we reduced the number of models to just three: one for emergence and two for immergence. In place of Table 1, we have written the structure of the models used. We have added a sentence to the statistics section: “each PGLS model produces a λ parameter representing the effect of phylogeny ranging between 0 (no phylogeny effect) and 1 (covariance entirely explained by co-ancestry)”. We have tested only three PGLS models and the estimated lambda value for these models is 0.

(7) Figure 2 is unclear, and I do not understand how these three regression lines were computed. Please provide more details.

We tested new models and modified existing figures.

Reviewer #2 (Public Review):

Summary:

An article with lots of interesting ideas and questions regarding the evolution of timing of dormancy, emphasizing mammalian hibernation but also including ectotherms. The authors compare selective forces of constraints due to energy availability versus predator avoidance and requirements and consequences of reproduction in a review of between and within species (sex) differences in the seasonal timing of entry and exit from dormancy.

Strengths:

The multispecies approach including endotherms and ectotherms is ambitious. This review is rich with ideas if not in convincing conclusions.

Weaknesses:

The differences between physiological requirements for gameatogenesis between sexes that affect the timing of heterothermy and the need for euthermy during mammalian hibernator are significant issues that underlie but are under-discussed, in this contrast of selective pressures that determine seasonal timing of dormancy. Some additional discussion of the effects of rapid climate change on between and within species phenologies of dormancy would have been interesting.

Reviewer #2 (Recommendations For The Authors):

This review provides a very interesting and ambitious among and within-species comparison of the seasonal timing of entry and exit from dormancy, emphasizing literature from hibernating mammals (sans bats and bears) and with attention to ectotherms. The authors test hypotheses related to the timing of food availability (energy) versus life history considerations (requirements for reproduction, avoiding predation) while acknowledging that these are not mutually exclusive. I offer advice for clarifications and description of the limitations of the data (accuracy of emergence and immergence times), but mainly seek more emphasis for small mammalian hibernators on the contrast for requirements for significant periods of euthermy prior to the emergence in males versus females, a contrast that has energetic and timing consequences in both the active and hibernation seasons.

A consideration alluded to but not fully explained or discussed is the differences in mammals between species and sexes in the timing of what can be called ecological hibernation, which is the seasonal duration that an animal remains sequestered in its burrow or den, and heterothermic hibernation, between the beginning and end of the use of torpor. The two are not synonymous. When "emergence" is the first appearance above ground, there is a significant missing observation key to the energetic contrasts discussed in this review, that of this costly pre-emergence behavior.

To explain the difference between heterothermic hibernation and ecological hibernation, we've added a section in review Criteria from materials and methods :

“In this study, we addressed what can be called ecological hibernation, i.e. the seasonal duration that an animal remains sequestered in its burrow or den, which is assumed to be directly linked to the reduced risk of predation. In contrast, we did not consider heterothermic hibernation, which corresponds to the time between the beginning and end of the use of torpor. So when we mention hibernation, emergence or immergence, the specific reference is to ecological hibernation.”

In arctic and other ground squirrel species, males remain at high body temperatures after immerging and remaining in their burrows in the fall for several days to a week, and more consistently and importantly, males that will attempt to breed in the spring end torpor but remain constantly in their burrows for as much as one month at great expense whilst undergoing testicular growth, spermatogenesis, spemiation, and sperm capacitation, processes that require continuous euthermy. Female arctic ground squirrels and non-breeding males do not and typically enter their first torpor bout 1-2 days after immergence and first appear above ground 1-3 days after their last arousal in spring.

The weeks spent euthermic in a cold burrow in spring by males while undergoing reproductive maturation require a significant energetic investment (can equate to the cost of the previous heterothermic period) that contrasts profoundly with the pre-mating energetic investment by females.

Males cache food in their hibernacula and extend their active season in late summer/fall in order to do so and feed from these caches in spring after resuming euthermy, often emerging at body weights similar to that at immergence. Similar between-sex differences in the timing of hibernation and heterothermy occur in golden-mantled and Columbian ground squirrels and likely most other Urocitellus spp., though less well described in other species. These differences are related to life histories and requirements for male vs. female gameatogenesis and, at the same time, energetic considerations in the costs to males for remaining euthermic while undergoing spermatogenesis and the cost related to whether males undergo gonadal development being dependent on individual body mass and cache size. These issues should be better discussed in this review.

It is the time required to complete spermatogenesis, spermiation, and maturation of sperm not the time for growth of different sizes of testes that drives the preparation time for males. This is relatively constant among rodents. I challenge the assumption that larger testes take longer to grow than smaller ones.

We took this comment into account. As we found little evidence of an increase in testicular maturation time with relative testicular size (apart from table 4 in Kenagy and Trombulak, 1986), we no longer tested the effect of relative testicular size on protandry.

We examined whether the ability to store food before hibernation might reduce protandry. Although food storage in the burrow may be favored for overcoming harsh environments or predation, model selection did not retain the food-storing factor. Thus, the ability to accumulate food in the burrow was not by itself likely to keep males of some species from emerging earlier (e.g. Cricetus cricetus, protandry : 20 day, Siutz et al., 2016). Early emerging males may benefit from consuming higher quality food or in competition with other males (e.g., dominance assertion or territory establishment, Manno and Dobson 2008).

We developed these aspects in the discussion

While it is admirable to include ectotherms in such a broad review and modelling, I can't tell what data from how many ectothermic species contributed to the models and summary data included in the figures.

Too few data on ectotherms were available to include ectotherms in the meta-analysis

Some consideration should be made to the limitations of the data extracted from the literature of the accuracy of emergence and immergence dates when derived from only observations or trapping data. The most accurate results come from the use of telemetry for location and data logging reporting below vs. above ground positioning and body temperature.

We added a "study limits" section to the discussion to address all the limits in this commentary.

L64 "favor reproduction", better to say "allow reproduction", since there is strong evolutionary pressure to initiate reproduction early, often anticipating favorable conditions for reproduction, to maximize the time available for young to grow and prepare for overwintering themselves.

Also, generally, it is not how "harsh" an environment is but rather how short the growing season is.

We took this comment into account.

L80 More simply, individuals that have amassed sufficient energy reserves as fat and caches to survive through winter may opt to initiate dormancy. This may decrease but not obviate predation, since hibernating animals are dug from their burrows and eaten by predators such as bears and ermine.

In this sentence, we indicated a gap between dormancy phenology and the growing season, which suggests survival benefits of dormancy other than from a physiological point of view. We've changed the sentence to make it clearer : “However, some animals immerge in dormancy while environnemental conditions would allow them (from a physiological point of view) to continue their activity, suggesting other survival benefits than coping with a short growing season”

L88 other physiological or ecological factors.... (gameatogenesis).

In this study, we examine possible evolutionary pressures and therefore the environmental factors that may influence hibernation phenology. We focus on reproductive effort because, assuming predation pressure, we would expect a trade-off between survival and reproduction.

L113 beginning early to afford long active seasons to offspring while not compromising the survival of parents.

We added to the sentence:

“For females, emergence phenology may promote breeding and/or care of offspring during the most favorable annual period (e.g., a match of the peak in lactational energy demand and maximum food availability, Fig. 1) or beginning early to afford long active seasons to offspring while not compromising the survival of parents.”

L117 based on adequate preparation for overwintering and enter dormancy....

We modified the sentence as follows :

recovering from reproduction, and after acquiring adequate energy stores for overwintering”

L123 given that males outwardly invest the least time in reproduction yet generally have shorter hibernation seasons would seem to reject this hypothesis. This changes if you overtly include the time and energy that males expend while remaining euthermic preparing for hibernation, a cost that can be similar to energy expended during heterothermy.

Males invest a lot of time in reproduction before females emerge (whether for competition or physiological maturation) and some males seem to be subject to long-term negative effects linked to reproductive stress (see Millesi, E., Huber, S., Dittami, J., Hoffmann, I., & Daan, S. (1998). Parameters of mating effort and success in male European ground squirrels, Spermophilus citellus. Ethology, 104(4), 298-313). Both processes may contribute to reducing the duration of male hibernation.

L125 again, costs to support euthermy in males undergoing reproductive development is an investment in reproduction.

You're right, but it's difficult to quantify. We tested a model that takes into account the reproductive effort during reproduction and prior to reproduction. We also considered the hypothesis that species living in a cold climate might have a low protandry while having a high reproductive effort due to their ability to feed in the burrow (interaction effect between reproductive effort and temperature). We think these changes answer your comment.

L134 It isn't growing large testes that takes time, but instead completing spermatogenesis and maturation of sperm in the epdidymides.

We removed this part.

L140 Later immergence in male ground squirrels is related to accumulation and defense of cached food, activities that are related to reproduction the next spring. An experimental analysis that would be revealing is to compare immergence times in females that completed lactation to the independence of their litters vs. females that did not breed or lost their litters. Who immerges first?

Body mass variation from emergence to the end of mating in males seems to explain the delayed immergence of males in species that don't hide food in their burrows for hibernation. For example, in spermophilus citellus, males immege on average more than 3 weeks after females, yet they do not hide food in their burrows for the winter.

Such a study already exists and shows that non-breeding females immerge earlier than breeding females. We refer to it

L386: “In mammals, males and females that invest little or not at all in reproduction exhibit advances in energy reserve accumulation and earlier immergence for up to several weeks, while reproductive congeners continue activity (Neuhaus 2000, Millesi et al. 2008a).”

L164 So you examined literature from 152 species but included data from only 29 species? Did you include data from social hibernators (marmots) that mate before emergence?

With current models, we have 28 different species. We have few species because very few have data on both sex difference data and information on reproductive effort data (especially for males).

Data on sex differences in hibernation were not available for social hibernating species.

L169 Were these data from trapping or observation results? How reliable are these versus the use of information from implanted data loggers or collars that definitively document when euthermy is resumed and/or when immergence and first emergence occurs (through light loggers)?

We did not focus heterothermic hibernation, but in ecological hibernation. We have no idea of the margin of error for these types of data, but we have discussed these limitations in the "Study limitations" section.

L180, again, it is the time required to complete spermatogenesis and spermiation not the time for the growth of different sizes of testes that drives the preparation time for males. This is relatively constant among rodents. I challenge the assumption that larger testes take longer to grow than smaller ones.

We removed this part.

L200 Males that accumulate caches in fall and then feed from those during the spring pre-emergence euthermic interval and after will often be at their seasonal maximum in body mass. Declining from that peak may not be stressful.

It has been suggested that reproductive effort in Spermophilus citellus might induce long-term negative effects that delay male immergence.

Millesi, E., Huber, S., Dittami, J., Hoffmann, I., & Daan, S. (1998). Parameters of mating effort and success in male European ground squirrels, Spermophilus citellus. Ethology, 104(4), 298-313.

L210 How about altitude, which affects the length of the growing season at similar latitudes?

We extracted the location of each study site to determine the temperature and precipitation at that precise location (based on interpolated climate surface). We therefore take into account differences in growing season (based on temperature) in altitude between sites.

L267 How did whether males cache food or not figure into these comparisons? Refeeding before mating occurs during the pre-emergence euthermic interval.

We removed this part.

L332, 344 not a "proxy" but functionally related to advantages in mating systems with multiple mating males.

We removed this part.

L353 The need for a pre-emergence euthermic interval in male ground squirrels requires costs in the previous active season in accumulating and defending a cache and the proximal costs in spring while remaining at high body temperatures prior to emergence with resulting loss in body mass or devouring of the cache.

You're right, but in this section, we quickly explain the benefits of food catching compared with other species that don't do so.

L385 This review should discuss why females are not known to cache and contrast as "income breeders" from "capital breeder" males. What advantages of caches are females indifferent to (no need for a prolonged pre-emergence period) and what costs of accumulating caches do they avoid (prolonged activity period and defense of caches).

We clarified the case of female emergence.

L321 : “Thus, an early emergence of males may have evolved in response to sexual selection to accumulate energy reserve in anticipation of reproductive effort. Females, on the contrary, are not subject to intraspecific competition for reproduction and may have sufficient time before (generally one week after emergence) and during the breeding period to improve their body condition.”

L388 I don't understand the logic of the conclusion that "did not ...adequately explain the late male immergence" in this section. The greater mass loss in males over the mating period is afforded by the presence of a cache that requires later immergence.

We removed this part.

L412 Not just congeners that invest less in reproduction, but within species individuals that do not attempt to breed in one or more years and thus have no reproductive costs should be an interesting comparison for differences in phenology from individuals that do breed. Non-breeders are often yearlings but can be a significant overall proportion of males that fail to fatten or cache enough to afford a pre-emergence euthermic period.

L385: “In mammals, males and females that invest little or not at all in reproduction exhibit advances in energy reserve accumulation and earlier immergence for up to several weeks, while reproductive congeners continue activity (Neuhaus 2000, Millesi et al. 2008a).”

The sentence refers to individuals who reproduce little or not at all.

L445 Males that gain weight between emergence and mating may do so by feeding from a cache regardless of how "harsh" an environment is.

We observe this phenomenon even in species that are not known to hoard food

“Gains in body mass observed for some individuals, even in species not known to hoard food, may indicate that the environment allows a positive energy balance for other individuals with comparable energy demands.”

L492 Some insects retreat to refugia in mid-summer to avoid parasitism (Gynaephora).

Escape from parasites is also a benefit of dormancy.

Fig 1 - It is difficult to see the differences in black and green colors, esp if color blind.<br /> Maternal effort is front-loaded within the active season (line for "optimal period" shown in midseason).

Add "energy" underneath c) Prediction (H1) and "reproduction" underneath d) "Prediction (H2). Explain the orange vs black, green colors of triangles.

We made the necessary changes

Fig 2 - I don't buy the regression lines as significant in this figure. The red line, cannot have a regression with two sample points and without the left-hand most dot, nothing is significant.

We deleted this graph.

Fig 3 - females only?

We deleted this graph.

By fascinating contrast, Donne was frequently underemployed, and perhaps a bit desperate just a decade and change prior and had applied for a job in Virginia

The years between 1607 and 1610 are biographically murky. The letters are hard to date and hard to decipher, and the best historical records we have are of jobs that didn’t happen. He failed to get a position in the Queen’s household in 1607, and there are references in the letters to his application to jobs in Ireland or, even more remotely, Virginia, but neither came to anything, if they were ever serious prospects to begin with. It’s equally likely that they were an attempt on his part to look industrious, both to his friends and to himself; neither Ireland nor Virginia were at all desirable places at the time.

quote via: Rundell, Katherine. Super-Infinite: The Transformations of John Donne. Farrar, Straus and Giroux, 2022. with excerpt linked at https://hypothes.is/a/M3Ma0PXdEe6Lk-doNS30CQ

not sure i would recommend framing this in terms of cancel culture. it's become one of these terms (like "woke") that gets used ad nauseam and means different things to different people. it's imprecise. if you're worried that deepfake could be used to make someone appear guilty of bad behavior, just say that -- it could be used to frame or blackmail people

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

(1) More explanation/description of Fig 3C and 3D would be helpful for readers, including the color code of 3D and black lines shown in both panels.

We have added more description to the legend of Figure 3, and we have used the same color code as in Figure 2, which we now specifically note in the figure legend as well.

(2) Differences between cranial and trunk NCC could be experimentally shown or discussed. Fig 4C shows some differences between these two populations, but in situ, results using Dlc1/Sp5/Pak3 probes in the trunk region may be informative, like Fig 5 supplement 2 for cranial NCCs.

This is an important point. The focus of our study was on cranial neural crest cells, and the single cell sequencing data is therefore truly reflective of only cranial neural crest cells. We have not functionally tested for the roles of Dlc1/Sp5/Pak3 in trunk neural crest cells, however, based on the expression and loss-of-function phenotypes of Sp5 or Pak3 knockout mice, we predict they individually may not play a significant role. It remains plausible that Dlc1 could play an important role in the delamination of trunk neural crest cells, but we have not tested that definitively. Nonetheless, Sabbir et al 2010 showed in a gene trap mouse mutant that Dlc1 is expressed in trunk neural crest cells. Regarding the similarities and differences between cranial and trunk neural crest cells as noted by the reviewer with respect to Figure 4, it’s important to recognize the temporal differences illustrated in Figure 4. Neural crest cell delamination proceeds in a progressive wave from anterior to posterior, but also that the analysis was designed to quantify cell cycle status before and during neural crest cell delamination. We have compared cranial and trunk neural crest cells in more detail in the discussion and also speculate what might happen in the trunk based on what we know from other species.

(3) Discussion can be added about the potential functions of Dlc1 for NCC migration and/or differentiation based on available info from KO mice.

We have added specific details regarding the published Dlc1 knockout mouse phenotype to the discussion, particularly with respect to the craniofacial anomalies which included frontonasal prominence and pharyngeal arch hyperplasia, and defects in neural tube closure and heart development. Although the study didn’t investigate the mechanisms underpinning the Dlc1 knockout phenotype, the craniofacial morphological anomalies would be consistent with a deficit in neural crest cell delamination reducing the number of migrating neural crest cells, as we observed in our Dlc1 knockdown experiments.

Reviewer #2 (Recommendations For The Authors):

The authors used the (Tg(Wnt1-cre)11Rth Tg(Wnt1-GAL4)11Rth/J) line but work from the Bush lab (see Lewis et al., 2013) has demonstrated fully penetrant abnormal phenotypes that affect the midbrain neuroepithelium, increased CyclinD1 expression and overt cell proliferation as measured by BrdU incorporation. The authors should explain why they used this mouse line instead of the Wnt1-Cre2 mice (129S4-Tg(Wnt1-cre)1Sor/J) in the Jackson Laboratory (which lacks the phenotypic effects of the original Wnt1-Cre line), or a "Cre-only" control, or at a minimum explain the steps they took to ensure there were no confounding effects on their study, especially since cell proliferation was a major outcome measure.

This is an important point, and we thank the reviewer for raising it. Yes, it has been reported that the original Wnt1Cre mice exhibit a midbrain phenotype (Ace et al. 2013). However, it has also been noted that Wnt1Cre2 can exhibit recombination in the male germline leading to ubiquitous recombination (Dinsmore et al., 2022). Therefore, to avoid any potential for bias, we used an equal number of cells derived from the Wnt1 and F10N transgenic line embryos in our scRNA-seq, and this included multiple non-Cre embryos. Our scRNA-seq analysis was therefore not dependent upon Wnt1-Cre, but also because we used whole heads not fluorescence sorted cells. However, Wnt1-Cre lineage tracing was advantageous from a computational perspective to help define cells that were premigratory and migratory in concert with Mef2c-lacZ ¬based on their expression of YFP, LacZ or both. We note these specifics more clearly in the methods.

The Results section (line 122) states that scRNA-seq was performed on dissociated cranial tissues but the Methods section (lines 583-584) implies that whole E8.5 mouse embryos were dissociated. Which was dissociated, whole embryos or just cranial tissues? Obviously, the latter would be a better strategy to enrich for cranial neural crest, but the authors also examine the trunk neural crest. This should be clarified in the text.

We apologize that some of the details regarding the tissue isolation were confusing and we have clarified this in the methods and the text. For the record, after isolating E8.5 embryos, we then dissected the head from those embryos, and performed scRNA-seq on dissociated cranial tissues. As the reviewer correctly noted, this approach strategically enriches for cranial neural crest cells.

The authors do not justify why they chose a knockdown strategy, which has its limitations including its systemic injection into the amniotic cavity, its likely global and more variable effects, and its need to be conducted in culture. Why the authors did not instead use a Wnt1-Cre-mediated deletion of Dlc1, which would have been "cleaner" and more specific to the neural crest, is not clear (maybe so they could specifically target different Dcl1 isoforms?). Also, the authors use Sox10 as a marker to count neural crest cells, but Sox10 may only label a subset of neural crest cells and thus some unaffected lineages may not have been counted. The authors should mention what is known about the regulation of Dcl1 by Sox10 in the neural crest. Although the data are persuasive, a second marker for counting neural crest cells following knockdown would make the analysis more robust. Can the authors explain why they did not simply use the Mef2c-F10N-LacZ line and count LacZ-positive cells (if fluorescence signal was required for the quantification workflow, then could they have used an anti-beta Galactosidase antibody to label cells)?

We thank the reviewer for raising these important considerations. It has previously been noted that although Wnt1-Cre is the gold standard for conditional deletion analyses in neural crest cell development, especially migration and differentiation, it is not a good tool for functional studies of the specification and delamination of neural crest cells due to the timing of Wnt1 expression and Cre activation and excision (see Barriga et al., 2015). Therefore, we chose a knockdown strategy instead, and also because it allows us to more rapidly evaluate gene function. We agree that there are limitations to the approach with respect to variability, however, this is outweighed by the ability to repeatedly perform the knockdown at multiple and more relevant temporal stages such as E7.5 (which is prior to the onset of Wnt1-Cre activity), as well as target different isoforms, and also treat large numbers of embryos for quantitative analyses. The advantage of using Sox10 as a marker for counting neural crest cells is that at the time of analysis, cranial neural crest cells are still migrating towards the frontonasal prominences and pharyngeal arches, and the overwhelming majority of these cells are Sox10 positive. Moreover, we can therefore assay every Dlc1 knockdown embryo for Sox10 expression and count the number of migrating neural crest cells. The limitation of using the Mef2c-F10N-LacZ line is that this transgenic line is maintained as a heterozygote, and thus only half the embryos in a litter could reasonably be expected to be lacZ+. But combining Sox10 and Mef2c-F10N-LacZ fluorescent immunostaining for similar analyses in the future is a great idea.

Reviewer #3 (Recommendations For The Authors):

The putative intermediate cells differentially express mRNAs for genes involved in cell adhesion, polarity, and protrusion relative to bona fide premigratory cells (Fig. 2E). This is persuasive evidence, but only differentially expressed genes are shown. Discussing those markers that have not yet changed, e.g. Cdh1 or Zo1 (?), would be instructive and help to clarify the order of events.

We thank the author for this suggestion and we have provided more detail about adherens junction and tight junctions. Cdh1 is not expressed, and although Myh9 and Myh10 are expressed, we did not detect any significant changes. ZO1 is a tight junction protein encoded by the gene Tjp1, which along with other tight junctions protein encoding genes, is downregulated in intermediate NCCs as shown in the Figure 2E.

It is unclear whether the two putative intermediate state clusters differ other than their stage of the cell cycle. Based on the trajectory analysis in Fig. 3C-D, the authors state that these two populations form simultaneously and independently but then merge into a single population. However, without further differential expression, it seems more plausible that they represent a single population that is temporarily bifurcated due to cell cycle asynchrony.

We have addressed the cell cycle question in the discussion by noting that while it is possible the transition states represent a single population that is temporarily bifurcated due to cell cycle asynchrony, if this were true, then we should expect S phase inhibition to eliminate both transition state groups. Instead, our trajectory analyses suggest that the transition states are initially independent, and furthermore, S phase inhibition did not affect delamination of the other population of neural crest cells.

The authors do not present an in-depth comparison of these neural crest intermediate states to previously reported cancer intermediate states. This analysis would reveal how similar the signatures are and thus how extrapolatable these and future findings in delaminating neural crest are to different types of cancer.

We have also added more detail to the discussion to address the potential for similarities and differences in neural crest intermediate states compared to previously reported cancer intermediate states. The challenge, however, is that none of the cancer intermediate states have been characterized at a molecular level. Nonetheless, with the limited molecular markers available, we have not identified any similarities so far, but our datasets are now available for comparison with future cancer EMP datasets.

The reduction in SOX10+ cells may be in part or wholly attributable to inhibition of proliferation AFTER delamination. Showing that there are premigratory NCCs in G2/M at ~E8.0 would bolster the argument that this population is present from the earliest stages.

The presence of premigratory neural crest cells in G2/M is shown by the scRNA-seq data and cell cycle staining data in the neural plate border.

Lines 248-249: The pseudo-time analysis in Fig 3C/D does indicate that the two most mature cell clusters (pharyngeal arch and frontonasal mesenchyme) may arise from common or similar migratory progenitors. However, given the decades of controversy about fate restriction of neural crest cells, the statement that "EMT intermediate NCC and their immediate lineages are not fate restricted to any specific cranial NCC derivative at this timepoint" should be toned down so as to not give the impression that they have identified common progenitors of ectomesenchyme and neuro/glial/pigment derivatives.

We appreciate this comment, because as the reviewer noted, there has been considerable literature and debate about the fate restriction and plasticity of neural crest cells, and indeed we did not intend to imply we have identified common progenitors of ectomesenchyme and neuro/glial/pigment derivatives. That can only be truly functionally demonstrated by clonal lineage tracing analyses. Rather, we interpret our pseudo-time analyses to indicate that irrespective of cell cycle status at the time of delamination, these two populations come together with equivalent mesenchymal and migratory properties, but in the absence of fate determination in the collective of cells. This does not mean that individual cells are common progenitors of both ectomesenchyme and neuro/glial/pigment derivatives. The nuance is important, and we address this more carefully in the text.

Lines 320-321: "...this overlap in expression was notably not observed in older embryos in areas where EMT had concluded". It is unclear whether the markers no longer overlap in older embryos (i.e. segregate to distinct populations) or are simply no longer expressed.

The data in Figure 5 demonstrates the dynamic and overlapping expression of Dlc1, Sp5 and Pak3 in the different clusters of cells as they transition from being neuroepithelial to mesenchymal. In contrast to Sp5 and Pak3, Dlc1 is not expressed by premigratory neural crest cells but is expressed at high levels in all EMT intermediate stage neural crest cells. Later as Dlc1 continues to be expressed in migrating neural crest cells, Pak3 and Sp5 are downregulated. But the absence of overlapping expression in the dorsolateral neural plate at the conclusion of EMT coincides with their downregulation in that territory.

In the final results section on Dlc1, the previously published mutant mouse lines are referenced as having "craniofacial malformation phenotypes". The lack of detail given on what those malformations are (assuming descriptions are available) makes the argument that they may be related to insufficient delamination less persuasive. The degree of knockdown correlates so well with the percentage reduction in migratory neural crest (Fig. 6) that one would imagine a null mutant to have a very severe phenotype.

The inference from the reviewer is correct and indeed Dlc1 null mutant mice do have a severe phenotype. We have added more specific details regarding the craniofacial and other phenotypes of the Dlc1 mutant mice to the discussion. Of note the frontonasal prominences and the pharyngeal arches are hypoplastic in E10.5 Dlc1 mutant embryos, which would be consistent with a neural crest cell deficit. Although a deficit in neural crest cells can be caused my multiple distinct mechanisms, our Dlc1 knockdown analyses suggest that the phenotype is due to an effect on neural crest cell delamination which diminishes the number of migrating neural crest cells.

Use the same y-axis for Fig. 4C/D

This has been corrected.

Fig. 6C: Please note in the panel which gene is being measured by qPCR

This has been corrected to denoted Dlc1.

Lines 108-117: More concise language would be appropriate here.

As requested, we were more succinct in our language and have shortened this section.

The SABER-FISH images are very dim. I realize the importance of not saturating the pixels, but the colors are difficult to make out.

We thank the reviewer for pointing this out and have endeavored to make the SABER-FISH images brighter and easier to see.

I've watched this movie multiple of times it's honestly one of my favorites. One thing that stuck with me is the character and pursuit of success even though the movie raises questions about what cost him to get there. The movie is deeper than just the origins of Facebook but it touches up on things such as loyalty and such which all just comes together perfectly.

Wow, you can already tell how proficiency in math is already different in just four paragraphs. Chantelle didn't get ahead in math for various reasons, which meant that she was going to retake pre algebra over and over again until she was comfortable enough to move onto algebra; this doesn't seem like the most efficient way to move up the ranks in math. However, Jennifer, through the use of higher quality education in private school and talented private tutors, was able to move up the ranks quicker and with much higher confidence.

This makes me wonder, is the reason why the standard based grading system still exist is because we haven't found a better alternative for it. It's also about which one is more time consuming, because giving individual feedback to hundreds of students is a lot more time consuming for teachers than just handing out grades.

Not all high-achieving students actually have a passion for learning in a sense that ungrading really only works if a pre-existing drive to want to learn and grow in a holistic sense exists. If not all the classes are ungraded and the ungraded class allows the student to assign themselves a grade, they will put less effort towards the class with the self-assigned grade because they know its not being directly measured to a high benchmark in the way that other traditionally graded classes are.

People show up at my work without them, and they learn them

oof, too relatable. Maybe good annotations ought to be enough for a bit of this?

Reviewer #3 (Public Review):

In this study, authors used the Drosophila model to characterize molecular details underlying traumatic brain injury (TBI). Authors used the transcriptomic analysis of astrocytes collected by FACS sorting of cells derived from Drosophila heads following brain injury and identified upregulation of multiple genes, such as Pvr receptor, Jun, Fos, and MMP1. Additional studies identified that Pvr positively activates AP-1 transciption factor (TF) complex consisting of Jun and Fos, of which activation leads to the induction of MMP1. Finally, authors found that disruption of endocytosis and endocytotic trafficking facilitates Pvr signaling and subsequently leads to induction of AP-1 and MMP1.

Overall, this study provides important clues to understanding molecular mechanisms underlying TBI. The identified molecules linked to TBI in astrocytes could be potential targets for developing effective therapeutics. The obtained data from transcriptional profiling of astrocytes will be useful for future follow-up studies. The manuscript is well-organized and easy to read.

However, the connection suggested by the authors between Pvr and AP-1, potentially mediated through the JNK pathway, lacks strong experimental support in my view. It's important to recognize that AP-1 activity is influenced by multiple upstream signaling pathways, not just the JNK pathway, which is the most well-characterized among them. Therefore, assuming that AP-1 transcriptional activity solely reflects the activity of the JNK pathway without additional direct evidence is unwarranted. To strengthen their argument, the study could benefit from direct evidence implicating the JNK pathway in linking Pvr to AP-1. This could be achieved through genetic studies involving mutants or transgenes targeting key components of the JNK pathway, such as Bsk and Hep, the Drosophila homologues of JNK and JNKK, respectively. Alternatively, employing p-JNK antibody-based techniques like Western blotting, while considering the potential challenges associated with p-JNK immunohistochemistry, could provide further validation. This important criticism regarding the molecular link between Pvr and AP-1 has been overlooked.

Editors try to make the transitions ismooth, that way you cant tell it has been edited. They call this Continuity editing or invisible editing.

The lens has a major role in filmmaking. The lens takes light to the film or digital sensor.

Using different lighting techniques will allow the illusion of dimension and shadows.

When the film is complete ,it is made up of various shots. While filming every shot they take it is called a "take".

Cinematography is helping tell a story through a single image. The cinematographer or the director of photography is in charge of helping the director transform his vision for the production.

Indeed

Also, while a causal history includes every event, it's by a non-hash-based id. So no actual content is available.

Using hashes to refer to content would allow to be context-free. Any replica would know the stuff they're talking about.

Not compact, yes. Just two hashes are enough, in fact. ;)

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1

(1) Since you only included patients with early-onset preeclampsia in the study, I suggest revising the title to "Identification of novel syncytiotrophoblast membrane extracellular vesicle derived protein biomarkers in early-onset preeclampsia...."

We have changed our title to early-onset preeclampsia.

(2) Under methods, you state that placenta was obtained from women undergoing elective cesarean section. Was this because all the study patients were delivered before the onset of labor? Or were laboring patients specifically excluded from the study?

Indeed, labor influences the extracellular vesicles (EVs) generated. To ensure consistency in our samples and avoid this variable, we chose placentas obtained from elective cesarean sections (CS) for our study.

(3) In Table 1 on page 10, the 8th row (Birth weight grams) needs to be reformatted. The mean birthweights for normal pregnancy and preeclampsia should be the same.

We have reformatted the table and using ranges instead of brackets.

(4) In the legend for Table 1, the sentence beginning on page 10, line 227, and continuing onto page 11, line 228, does not make sense. Part of the sentence was omitted inadvertently.

We have modified this sentence to :

Detergent treatment, which could break down EVs, with NP-40 confirmed that the majority (99%) of our samples were largely vesicular since only 0.1 ± 0.12% of BODIPY FL N-(2-aminoethyl)-maleimide and PLAP double-positive events were detected (a reduction of 99%) (Figure 1E and 1H).'

(5) As you acknowledge, the sample size (12 patients) was small. This is understandable because early-onset preeclampsia occurs in <1% of parturients. You could collaborate with other centers in future studies to increase the sample size.

Thank you very much for your comment. We are willing to cooperate on future research and will try to expand our sample size in subsequent studies.

Reviewer #2 (Recommendations For The Authors):

(1) This is one of the many "catalogue" papers where placental exosome proteins in preeclampsia are profiled. Thus, the manuscript lacks novelty. The only novelty factor is the authors have isolated exosomes by a different method and even separated the small and large exosomes. However, there is no mention of how these exosomes differ from each other in terms of their functionality. Thus it is hard to judge the biological significance of this work.

We appreciate your insights regarding the novelty of our study. While numerous papers have profiled placental exosome proteins in preeclampsia, our methodology for enriching sSTB-EVs (exosomes) offers a distinct perspective. We believe that the separation of sSTB-EVs (exosomes) and medium/large STB-EVs (microvesicles) introduces a differentiation that extends beyond mere profiling, with implications for their functionality. There are previous studies showed that the different sizes of placenta EVs have distinct characteristics (Zabel RR, et al. Enrichment and characterization of extracellular vesicles from ex vivo one-sided human placenta perfusion. Am J Reprod Immunol. 2021 Aug;86(2)). Furthermore, the way cells internalize and respond to EVs may depend on the size of the EV (Zhuang X et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol Ther. 2011 Oct;19(10).) Therefore, it would be important for future studies to distinguish different sizes of EVs for the research.

(2) The authors must demonstrate that these two types of EVs are also produced in vivo by detecting them in the serum of women.

Thank you for the comment. Many previous studies have shown the two types of placental EVs in women's blood. Nakahara et al.'s (PMCID: PMC7755551) extensive review compiles studies that have specifically isolated various subtypes of placenta-derived EVs from maternal circulation. We have also readdressed it in the introduction.

(3) The authors must compare the proteomes of serum-derived placental exosomes and the proteome of the STBs isolated from the perfusion experiments to judge how overlapping the outcomes are from those produced naturally and those produced under ex vivo conditions.

We appreciate the reviewer's suggestion to compare the proteomes of serum-derived placental sSTB-EVs (exosomes) with those from STBs isolated through perfusion experiments. Indeed, such a comparison would provide valuable insights into the similarities and differences between naturally produced and ex vivo-generated sSTB-EVS (exosomes). However, isolating placental EVs from maternal circulation for comprehensive proteomic profiling presents challenges. It requires a significant amount of serum or plasma sample that will be sufficient to enable the isolation of placenta-specific EVs amongst numerous EVs in the circulation. In addition, it will require multiple intricate steps such as ultracentrifugation followed by immunoprecipitation. Each of these steps can potentially lead to the loss of EVs. Additionally, given the high concentration of lipoproteins in plasma relative to EVs, there's a significant risk of obtaining low-purity isolates from the outset. These challenges might compromise the comparability of results between placenta-specific EVs from maternal circulation and those from ex vivo perfusion. Nevertheless, we acknowledge the value of such an endeavor and will consider incorporating this aspect in future studies as the EV and proteomic methodology and technology improve and become more sensitive.

(4) I have a major issue with the chosen study subjects. While the study title and the manuscript mention preeclampsia, as per the inclusion criteria mentioned in lines 88-90, the patients will be HELLP syndrome. Please clarify what was used and modify the manuscript accordingly.

Thank you very much for finding this error. Our patients had none of the features that would qualify them for HELLP syndrome. We have edited to:

PE was defined as new (after 20 weeks) systolic blood pressure of 140 mmHg or diastolic pressure of 90 mmHg, proteinuria (protein/creatinine ratio of 30 mg/mmol or more). None of our patients had maternal acute kidney injury, liver dysfunction, neurological features, hemolysis, or thrombocytopenia.

(5) It is hard to reconcile how only 15 proteins were identified in the placental extract while 300+ in EVs. There is a methodological issue in the mass spec or extraction. With such widely different denominators in the total proteins identified, it is hard to compare the outcomes in terms of the three sample types.

We acknowledge the reviewer's concerns regarding the disparity in protein counts between the placental extract and the EVs. Ultimately, more is not necessarily better. Several factors might contribute to this discrepancy. Firstly, it is plausible that certain proteins exhibit selective affinity to varying sizes of EVs, leading to a more diverse range of proteins than the placental extract. We were also stringent in our analysis to enable us to select proteins whose biological differences are more likely to be reproducible with a different validatory method like a western blot. Additionally, although the placental extract might contain a higher total protein concentration, it doesn't necessarily translate to a richer diversity of disease-specific proteins. Considering these nuances when comparing protein outcomes across sample types is helpful.

(6) I am unable to understand the terms least differentially expressed and most differentially expressed. Do the authors mean upregulated and downregulated? Please clarify and use the terms appropriately by providing fold change values.

We appreciate the reviewer's request for clarification. We intended to provide a relative measure of expression for the terms 'least differentially expressed' and 'most differentially expressed'. The terms are roughly equitable to down- and upregulated. Regarding EVs, we avoid using the terms 'upregulated' and 'downregulated' as EVs act as transporters and do not possess regulatory functions per se. However, for the placenta, we recognize the relevance of these terms.

(7) The data presented is very superficial and lacks methodological details. The authors should provide the total number of targets achieved after mass spec. The cutoffs used the FDRs and other details.

We apologize for the omission. We have added these details to the method section.

(8) It is not clear how were these differentially abundant proteins identified. What was the cutoff used? Was it identified in all the replicates?

We apologize for the omission. We have added these details to the method section.

(9) How many samples were subjected to the discovery cohort, and how many were in the validation cohort? Were they the same or different? If the samples were different, how many PE samples had differentially abundant proteins by both methods?

The study utilized 12 samples for initial discovery and another 12 for western blot validation. The validation samples specifically targeted proteins of interest, rather than undergoing another comprehensive mass spectrometry analysis.

(10) It is striking that the authors report the expression of prostatic acid phosphatase in the placenta. In my understanding of placental biology, this gene or protein is not known to be expressed by the placenta. Please perform immunofluorescence to demonstrate that this protein is indeed produced in the STBs

Research has revealed that even though it's called prostate-specific antigen, it's created in tissues other than the prostate, such as the placenta. Here are a couple of references to support this claim: PMID: 10634405, PMID: 7533063, PMID: 8939403, and PMID: 8945610. Hence it is likely not beneficial to demonstrate what many researchers have already demonstrated.

(11) Please validate the differential abundance of these proteins in the exosomes isolated from the plasma of women with and without preeclampsia. A serial measurement will be of high value to determine how early as compared to hypertension, these biomarkers can predict preeclampsia.

We are validating each EV-carried marker individually in the circulation (plasma or serum), localizing them in the placenta, and performing downstream functional analysis. This article is already lengthy and would likely be too cumbersome to include the details of all individual proteins in this manuscript. However, we have already published papers on Siglec 6 (PMID: 32998819) and Neprilysin (PMID: 30929513), and others will be published soon. We agree that there will be a lot of value to serial measurement, not just in terms of how early as compared to hypertension, these biomarkers can predict preeclampsia but also as potentially a more sensitive or specific test. This would be the subject of subsequent papers.

(12) The authors are recommended to carry out immunofluorescence to localize the differentially abundant proteins in the placental sections and show that they are specific to STBs.

We have already provided a similar response earlier (see response to point 11). In addition, while it is preferable, the biomarkers don't necessarily need to be specific to STB. Not all biomarkers are mechanistic agents/targets, and not all mechanistic agents are biomarkers. However, mechanistic agents should preferably be placental-specific. For example, the total sFLT1, the most studied biomarker, is not exclusively synthesized in the placenta, even though the placental-specific isoform represents a small fraction of the total sFLT-1. For example, in the non-placental world, alkaline phosphatase (ALP) is not exclusively produced by the liver but is a ‘biomarker’ of cholestatic disease.

(13) Table 1 should give the range and SD could be given as + instead of the bracket.

Thank you for your suggestion. We have edited it accordingly.

(14) It is necessary to provide the gestational age of the onset of hypertension to get a judgment of how long these women were preeclamptic, culminating in HELLP.

We want to emphasize that none of our patients experienced HELLP syndrome. In the results section, we have included the gestational age at the time of diagnosis in the table for preeclampsia. It's crucial to understand that the gestational age at diagnosis is distinct from the gestational age when hypertension initially appeared. Detecting the exact gestational age of hypertension onset would be challenging, and it would likely require a prospective or randomized clinical trial with continuous monitoring, possibly on a daily basis. However, our study is retrospective. Thus we can only comment on the gestational age at diagnosis

(15) For newborns the term Sex is used and not gender

Thank you for your suggestion. We have edited it accordingly.

(16) Figure 2 is stretched and hard to read

Thank you for your suggestion. We have edited it accordingly by creating two separate images to promote readability.

(17) Line 278 change the sentence "there fifteen (15) proteins in the placenta" to "there were fifteen (15) proteins in the placenta"

Thank you for your suggestion. We have edited it accordingly.

(18) Line 288 you mean least and not lease

Thank you for your suggestion. We have edited it accordingly.

This is something that feels like it's a given but I wouldn't consider it until someone else points it out for me. Some biases or assumptions are just ingrained in my thinking, which would complicated things a lot.

While watching the " ANIMAL" clip i did notice a few differences from the script. For example; in the script the woman is said to be coming out of her room tying her robe ,heading to the kitchen to fill a kettle.But in the clip we just see her standing looking out the window. At the end of the script it ends with will hugging the woman as she "caresses him softly" but in the clip the scene changes. We now see the ending as a little boy looking at a woman getting into her car.

Reviewer #2 (Public Review):

Summary:

Here the authors describe a model for tracking time-varying coupling between neurons from multi-electrode spike recordings. Their approach extends a GLM with static coupling between neurons to include dynamic weights, learned by a long-short-term-memory (LSTM) model. Each connection has a corresponding LSTM embedding and is read out by a multi-layer perceptron to predict the time-varying weight.

Strengths:

This is an interesting approach to an open problem in neural data analysis. I think, in general, the method would be interesting to computational neuroscientists.

Weaknesses:

It is somewhat difficult to interpret what the model is doing. I think it would be worthwhile to add some additional results that make it more clear what types of patterns are being described and how.

Major Issues:

Simulation for dynamic connectivity. It certainly seems doable to simulate a recurrent spiking network whose weights change over time, and I think this would be a worthwhile validation for this DyNetCP model. In particular, I think it would be valuable to understand how much the model overfits, and how accurately it can track known changes in coupling strength. If the only goal is "smoothing" time-varying CCGs, there are much easier statistical methods to do this (c.f. McKenzie et al. Neuron, 2021. Ren, Wei, Ghanbari, Stevenson. J Neurosci, 2022), and simulations could be useful to illustrate what the model adds beyond smoothing.

Stimulus vs noise correlations. For studying correlations between neurons in sensory systems that are strongly driven by stimuli, it's common to use shuffling over trials to distinguish between stimulus correlations and "noise" correlations or putative synaptic connections. This would be a valuable comparison for Figure 5 to show if these are dynamic stimulus correlations or noise correlations. I would also suggest just plotting the CCGs calculated with a moving window to better illustrate how (and if) the dynamic weights differ from the data.

Set design, costume, hair, color scheme, framing, composition,lighting are the aesthetic context to complete the film.

Movies are an illusion. It is just many frames combines into 1 with sound added.

in the 80s most media in the United States was ruled by about 50 companies. By 2019 it was down to 4.

(31) Marginal effects

*L6.(31) (Savannah) I am confused by the concept of marginal effects. I’m not sure I really understand what they are and why we need to do them on top of running the probit model.

Response: The marginal effect of X is the impact of X on the predicted probability; it’s different than the impact on the latent scale. The marginal effects are not constant.

*L3.(31) (Mia): When we talk about the marginal effects and we run the probitmfx and saw that an additional unit increase in x resulted in a 0.122 increase is this in like a Z unit or the actually probabilistic increase?

Response: The probability.

*L8.(31)(Rebecca) In lecture, you reference that the marginal effects are calculating the change in odds based on a one-unit increase in the independent variable – how does this translate if you have a dichotomous independent variable? Would you just end up using a completely different model in that case?

Response:It’s the same--i.e., the impact of going from X=0 to X=1.

(#16) *L4.(16)(Mia): Also I want to talk about why the variable Z is latent, in the example Z is not the explanatory variable (like how we use it in class)? So its saying we actually can’t observe likelihood or probability so its latent and measured through a dichotomous variable?

Response: Yes---we are predicting Z, not P. (Discuss more)

*L7.(16)(Syl): at this point i am bigger fan of logit models than probit models, but only because i do not see the point in the probit transformation when the logit one seems to work just fine? I guess would you mind explaining why we would prefer one over the other?

Response: In terms of results, they are the same. The reason to also understand the probit model is that it makes it easier to understand the impact of heteroskedasticity in the error term on the coefficients, which is the topic of Class S--it’s a big deal. In the case of categorical DV’s, heteroskedasticity affects the coefficients in logit/probit etc. models. That is why people often report the marginal effects, or elect to use LPM models.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Overall comments

We are pleased by the reviewers' comments and appreciate their suggestions for improvements. In addition to correcting small typos throughout the manuscript, we have made the following additions or changes in response to reviewer comments and suggestions:

1. New complementation experiments to verify the impacts of mgtA and PA4824 on bacterial fitness in fungal co-culture.
2. New experiments to measure intracellular Mg2+ levels in corA or mgtE mutants to strengthen our conclusion that neither of these constitutive Mg2+ transporters is required for maintaining intracellular Mg2+ levels in co-culture.
3. New experiments to confirm that the * cerevisiae mnr2D mutant does not have a fitness defect compared to WT in co-culture. This finding rules out the possibility that metabolic defects in the mnr2D mutant restore the fitness of bacterial mgtA* mutant in co-culture and strengthens our hypothesis that Mg2+ sequestration by fungal vacuole triggers Mg2+ nutritional competition with bacteria.
4. Clarification of bacterial species we tested in our study as suggested by Reviewer #3.
5. Revised discussion to highlight how our findings relate to any fungal-bacterial interaction both in ecological and infection contexts and any known role of mgtA in antibiotic susceptibility, as suggested by Reviewer #2. All changes in response to the reviewer's comments have been detailed in our point-by-point response (below).

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

This manuscript investigates polymicrobial interactions between two clinically relevant species, Pseudomonas aeruginosa and Candida albicans. The findings that C. albicans mediates P. aeruginosa tolerance to antibiotics through sequestration of magnesium provides insight into a specific interaction at play between these two organisms, and the underlying mechanism. The manuscript is well composed and generally the claims throughout are supported by the provided evidence. As a result, most comments are either for clarification, or minor in nature.

We thank the reviewer for their positive comments and their suggestions for improvement.

Major comments:

1) For their experiments, the authors frequently switch between 30C and 37C, but there is no rationale for why a specific temperature was used, or both were. E.g. some of the antibiotic survival assays, and fungal-bacterial co-culture assays were performed at both temperatures, while the colistin resistance, fitness competition and RNA sequencing were performed at 30C. Given the fact that the two organisms are both human pathogens and co-exist in human infections, it is not clear why 30C was used. The authors should provide clarity for why these two temperatures were used.

We thank the reviewer for raising this point. Fungal-bacterial interactions occur in a range of temperatures in ecological contexts (e.g., in soil or on plants) or during infection in animal hosts. Both 30oC and 37oC degree temperatures are used in C. albicans studies whereas 37oC is most preferred for P. aeruginosa studies. By providing data from both temperatures for critical experiments, we demonstrate that our findings are not dependent on temperature. Our studies also allow for an easy comparison to previously published studies performed at both temperatures. We chose to screen initial co-culture conditions showing fungal antagonism at 30oC, as C. albicans cells can reach higher CFUs than at 37oC due to growth in the single-celled yeast form.

We agree with the reviewer that 37oC is more physiologically relevant for conditions under which these two species coexist in animal hosts. Thus, we tested our findings of Mg2+ competition and antibiotic survival at 37oC.

We now clarify our reasoning in the revised Materials and Methods section as follows: "We chose 30oC for the initial co-culture assays for two reasons. First, C. albicans cells reached higher CFU at 30oC than 37oC, which would impose a stronger competition with bacteria. Second, C. albicans cells form hyphae at 37oC, which can have multiple cells in one filament and thus confound CFU measurements. We further confirmed that our findings of Mg2+ competition are independent of temperatures by setting up co-culture assays at both 30oC and 37oC."

2) Lines 184-191: It would be useful to measure intracellular Mg2+ (using the Mg sensor) in the corA and mgtB tn mutants in media as well as the fungal spent media, to provide stronger support for the claim that "MgtA is a key bacterial Mg2+ transporter that is highly induced under low Mg2+ conditions".

We thank the reviewer for this suggestion. Based on our experiments, neither CorA or MgtE are induced (in RNA-seq analyses) nor required in co-culture (in Tn-seq analyses), suggesting neither is involved in Mg2+ competition with C. albicans. In contrast, MgtA is highly induced in co-culture. Loss of mgtA significantly reduces bacterial fitness in co-culture and intracellular Mg2+ levels only in C. albicans-spent BHI, but not fresh BHI. These results suggest that MgtA is the key Mg2+ transporter required for bacterial Mg2+ uptake and fitness in co-culture.

Nevertheless, we agree with the reviewer that despite being constitutively expressed, CorA or MgtE might play an important role in importing Mg2+ in BHI and C. albicans-spent BHI. To test this possibility, we performed a new experiment suggested by the reviewer (now included in the revised manuscript) in which we measured intracellular Mg2+ levels in corA or mgtE loss-of-function mutants in BHI versus C. albicans-spent BHI, and compared them to intracellular Mg2+ levels in a mgtA loss-of-function mutant strain. We find that lack of either corA or mgtE does not significantly reduce bacterial Mg2+ levels in C. albicans-spent BHI compared to DmgtA mutant (Fig. S7C). Thus, our results strengthen our conclusion that MgtA is the key Mg2+ transporter that gram-negative bacteria use to overcome fungal-mediated Mg2+ sequestration.

3) Line no. 276. Does the mnr2∆ S. cerevisiae mutant have a growth defect compared to the WT? This would test whether the effect of the mnr2 mutant on P. aeruginosa fitness is strictly due to Mg2 and not due to reduced growth or metabolism of the mutant.

We agree with the possibility raised by the reviewer. In new experiments included with our revision as Figure S10, we find that the S. cerevisiae mnr2 deletion mutant exhibits similar CFU as WT in monoculture as well as co-culture. Thus, the rescuing effect of mnr2D is less likely due to reduced growth or metabolism.

4) The authors use the term 'antibiotic resistance' throughout the manuscript. However, the assays they perform do not directly test for antibiotic resistance which is defined as the ability to grow at higher concentrations of antibiotics (e.g. as measured by MIC tests). The authors should rephrase their phenotype as antibiotic survival or antibiotic tolerance.

We agree with the reviewer and thank them for raising this point. We replaced the phrase 'antibiotic resistance' with 'antibiotic survival' throughout the revised manuscript. We also accordingly changed our title to 'Widespread fungal-bacterial competition for magnesium lowers antibiotic susceptibility'

5) Also, the authors have two different assays, both measuring survival in antibiotic, but one is called a colistin resistance assay (line 508) and the other a colistin survival assay (line 523). It's not obvious what is the difference between what is being assayed in the two experiments, except perhaps the growth phase of the cells when they are exposed to the antibiotic? The authors should explain the difference, and the rationale for using two different assays.

We thank the reviewer for raising this point. In the revised manuscript, we explain the rationale of our two assays. The first assay measures the bacterial survival after colistin treatment in C. albicans-spent BHI, and the second measures the bacterial survival after colistin treatment in co-culture with C. albicans. We performed both assays because C. albicans-spent BHI mimics Mg2+-depleted conditions by C. albicans but might not represent all aspects of fungal presence in co-culture. To make sure our findings are consistent across these two experiments, we specify the difference in these two assays in the revised manuscript as the following: "Since fungal spent media cannot fully recapitulate fungal presence in co-culture conditions, we tested whether fungal co-culture also conferred increased colistin survival."

Minor comments:

• For almost all the figures, blue and orange dots are used for 'monoculture' and 'coculture' respectively, while orange and black dots are used for WT and the mgtA mutant. However, the black and blue dots are hard to tell apart, and for several figure sub-panels, the legends are not provided (e.g. figures 2D, 2F, S9H), making it a little confusing to figure out what is being shown. It would be best if the WT and mgtA symbols were in colors completely different from the monoculture/co-culture colors, making it easier to tell those apart.

We have updated these figures as the reviewer suggested.

Line no 122 and Figure 1A. The term "defense genes" in bacteria typically refers to genes conferring protection against phage infections. Perhaps the authors can use a different term (e.g. 'protective genes').

We agree with the reviewer. We have changed "defense genes" to "fungal-defense genes" to disambiguate the terms.

Line no 186. 'However, neither MgtA...' should be 'However, neither MgtE...'

We thank the reviewer for pointing out this typo. We have fixed this in our revision.

Line no 268. Does fungal-mediated Mg2+ competition extend to Gram positive bacteria?

We thank the reviewer for raising this interesting point. MgtA is prevalent in diverse gram-negative bacteria but rare in gram-positive bacteria. Using the fitness effect of mgtA mutants in co-culture vs monoculture allowed us to infer Mg2+ competition easily for diverse gram-negative bacteria. Currently, we do not have the experimental tools to extend this finding to gram-positive bacteria. Co-culture growth kinetics for gram-positive bacteria are also likely to be different from gram-negative bacteria in a way that makes direct comparisons challenging. We have clarified our writing in the revised manuscript: "This mode of competition might be highly specific between fungi and diverse gram-negative g-proteobacteria we have tested.... Whether fungi can suppress gram-positive bacteria through the same mechanism of Mg2+ competition remains an open question."

Line no 314. It is unclear whether the 'transient co-culture' is the same or a different assay as the colistin survival assay.

We apologize for the confusion and have removed the word 'transient' for clarity. The assays is the same as the 'colistin survival assay in fungal co-culture,' where we co-cultured log-phase P. aeruginosa cells with C. albicans for 5 hours and treated them with colistin.

Line no 316. For the bacterial survival assays shown in figures 3 and 4 (and other supplementary figures), please provide absolute numbers as cfu (as in figures 1 and 2), as opposed to a percentage, for cell counts. This will allow readers to appropriately interpret the data.

We thank the reviewer for this suggestion. We now include the raw CFU counts of colistin survival assays in Fig 3 and 4 and other supplementary figures in new supplementary figures (Fig. S11, S13, S14, S15, and S17) in our revision.

Line no 934-5: Italicize P. aeruginosa.

This typo has been fixed in our revision.

Reviewer #1 (Significance (Required)):

This study identifies a novel interaction between two the co-infecting human pathogens Pseudomonas aeruginosa and Candida albicans, where C. albicans causes Mg2+ limitation for P. aeruginosa. Further, the authors show that this interaction affects levels of antibiotic resistance, as well as the adaptive mutations seen during the evolution of antibiotic resistance. This advances the field by delineating how microbial interactions can affect clinically relevant phenotypes, and potentially clinical outcomes. The study should be of interest to a broad audience of researchers studying microbial ecology, evolutionary biology, microbiology, and infectious diseases.

We are grateful for the reviewer's positive appraisal.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This paper looks at the interaction between the fungus Candida albicans (Ca) and the bacterium Pseudomonas aeruginosa (Pa), which are found together in some environments. Co-culture experiments showed that Ca can inhibit the growth of Pa. The goal of this study is to determine the reason for this phenomenon and how widespread it is. This was performed by Tnseq analysis of Pa that identified 3 genes which showed significant decreases in the presence of Ca. Interestingly these were all in an operon that was recognized by the authors as being induced by RNAseq during co-culture. One of these genes, mgtA is a known Mg2+ transporter and therefore the remainder of the paper discusses the importance of competition for Mg2+.

The experiments seem to be well carried out and appropriately controlled.

We thank the reviewer's appreciation of our science and the rigor of our experiments.

The use of the Mg2+ genetic sensor reporter in Pa is an interesting approach to determine the intracellular Mg2+ concentrations, however how these levels relate to one another between different experiments is not clear. In Fig. S5, the levels are 5 AU for growth in minimal media +low (10uM) Mg2+ and 38 AU for growth in minimal media+high (10mM) Mg2+. But the levels seen in Figure 1E are all much lower. With such low levels, it is difficult to determine if the impact of ∆PA4824 and ∆mgtA (while perhaps additive) are relevant. Would differences be seen with these various strains grown under different conditions?

We thank the reviewer for this query. The reviewer is right in that we do not use absolute quantification of intracellular Mg2+ levels. While our Mg2+ genetic sensor assay does not facilitate comparison of absolute Mg2+ levels across experiments, it provides a robust comparative measurement of relative intracellular Mg2+ levels in mutants versus WT cells, or between two different media conditions.

Using this Mg2+ genetic sensor assay, we tested intracellular Mg2+ levels of WT P. aeruginosa under various media conditions. We found that lower intracellular Mg2+ levels in P. aeruginosa cells and the requirement of mgtA in these media are well-correlated at lower total Mg2+ levels in media (Fig. S9A-E). In contrast, there are no significant differences in intracellular Mg2+ levels between DmgtA (or DPA4824) and WT cells in BHI media, which has higher total Mg2+ levels than fungal-spent BHI media. Our experiments reveal that the lack of mgtA or PA4824 only affects intracellular Mg2+ levels when P. aeruginosa is cultured in media below a threshold level of Mg2+ concentration in media.

The experiments suggesting that the protein PA4824 is also a Mg2+ transporter seem to be related only to alpha fold predictions.

We clarify that our speculation that PA4824 encodes a potential novel Mg2+ transporter was first motivated by finding that it is induced in low Mg2+ conditions, its genetic importance in Tn-seq experiments independent of mgtA, and our finding that cells with loss-of-function mutations in PA4824 experience lower intracellular Mg2+ than WT cells. However, the reviewer is correct that this statement is speculative based on the Alphafold prediction. In the revised manuscript, we have clarified this point as the following: "Based on our co-culture RNA-seq and Tn-seq experiments, results from the Mg2+ genetic sensor assay, and the Alphafold prediction of PA4824 protein structure, we speculate that PA4824 potentially acts as a novel Mg2+ transporter."* * Is the statement in line 186 a typo? It is stated that "neither MgtA nor CorA was implicated in competition". Do the authors actually mean "MgtE"?

It is a typo. We thank the reviewer for pointing this out and have changed this to "MgtE".

Reviewer #2 (Significance (Required)):

Ca and Pa are known to inhabit the same niches and previous studies have shown both can have antagonist effects on one another. Nutritional competition is one mechanism of antagonism that has not been that well studied between these two genera. That makes the finding of some significance and relevant to those with an interest in either of these microbes and co-infections. The authors also found that it was not just Ca that had this effect, but other fungi as well. And this effect was not just reserved to effect Pa, but also other bacteria, suggesting a more global impact.

We thank the reviewer for an accurate summary of our findings.

However, diminishing the impact of this finding is the question as to whether this is simply a phenomena seen under the very specific laboratory conditions tested here. Furthermore how these findings exactly relate to any infection environment is not clear.

Fungal-bacterial interactions occur in a variety of broad biological contexts, including during infection in animal hosts or in environmental-associated microbial communities. Our study is the first to identify nutritional competition for Mg2+ as one of the most important axes of competition between fungi and bacteria. Our study also identifies MgtA as one of the key bacterial genes that mediates this interaction. MgtA is only induced upon experiencing low Mg2+ conditions; the fact that most gram-negative bacteria encode MgtA implies they must encounter low Mg2+ conditions and face fitness consequences in those conditions. To address the reviewer's concerns, we also highlight three additional points in our revised Discussion:

1. Fungal-bacterial competition for Mg2+ is not restricted only to BHI media alone. We also found the same phenomenon in TSB media medium. Indeed, we show (Fig. S9F) also that Mg2+ competition occurs whenever the environmental Mg2+ level is lower than 0.45mM, a critical threshold for fungi and bacteria to compete for this vital ion.
2. During infection in cystic fibrosis airways, proteomic experiments and Mg2+ measurement in CF sputum both suggest that * aeruginosa* experiences Mg2+ restriction.
3. Many previous studies have shown that many Gram-negative bacteria, including Salmonella Typhimurium, encounter reduced magnesium concentrations upon infection of hosts (PMID: 29118452). Our discovery that fungal co-culture may generally exacerbate fitness challenges associated with low magnesium levels is of high importance to all studies of gram-negative bacteria, not just to Pa.
4. In addition to infections in animal hosts, low Mg2+ is associated with worse outcomes of infections in plants. Our study suggests the importance of studying the role of Mg2+ competition in various infection contexts and the strategies of manipulating Mg2+ levels or fungal-bacterial interactions to constrain polymicrobial infectious diseases in diverse eukaryotic hosts and ecological conditions. The authors also seem to vastly overinterpret the significance of their findings; the impact on Pa is only to slow growth, not necessarily effect fitness, per se. The final number of bacteria appears to be the same, it just takes slightly longer to get there.

We are puzzled by this comment from the reviewer; slow growth IS a fitness effect! Although we agree with the reviewer's point that C. albicans is more likely to inhibit bacterial growth rate than viability (bacteriostatic, not bacteriocidal), there are many bacteriostatic antibiotic mechanisms.

In our co-culture assay, bacterial CFUs after 40 hours in co-culture are 10-100 times lower than in monoculture (this is not a subtle effect!). After 40 hours, bacterial cultures have already reached the stationary phase, which is why even slower growing bacterial cells in co-culture can 'catch up' (they are still lower by nearly 10-fold), despite fungal inhibition. Moreover, the co-culture condition provided enough of a fitness challenge to allow us to identify bacterial protective genes even in a pooled assay.

The authors speculate that that since Mg2+ supplementation did not totally restore growth to Pa during co-culture, that other Mg2+ independent "axes of antagonism" must exist. This also tends to diminish the significance of these finding.

Again, we are puzzled by this comment from the reviewer. Fungal-bacterial competition, like all microbial competition, is a multifactorial process, so we should not be surprised that Mg2+ isn't the only axis of competition. Indeed, our study reinforces the importance of investigating all potential axes of competition to get a complete understanding of the mechanisms of fungal-bacterial competition.

The importance of mgtA on antibiotic susceptibility has been well studied in a number of bacteria including Pa making these findings generally confirmatory.

We would like to clarify this comment. To the best of our knowledge, mgtA in P. aeruginosa has not been reported in antibiotic susceptibility studies. Instead, P. aeruginosa mgtE is induced upon treatment with aminoglycoside antibiotics, but its expression does not change antibiotic resistance (PMID: 24162608).

The reviewer may be referring to studies in S. Typhimurium, where the DmgtA mutant shows increased susceptibility to nitrooxidative stress (PMID: 29118452) and to cyclohexane (PMID: 18487336), suggesting Mg2+ homeostasis might be generally important for bacterial survival to antimicrobial treatments. Although this is not the main focus of our study, we now include these references in our revised discussion to provide readers with more background on the relevance of our work: "Mg2+ has been implicated in altering the susceptibility of gram-negative bacteria to antibiotics other than colistin. For instance, in S. Typhimurium, impaired mgtA or Mg2+ homeostasis increases susceptibility to cyclohexane or nitrooxidative stress. In line with these observations, our study also highlights the importance of studying how Mg2+ homeostasis broadly impacts antimicrobial resistance in gram-negative bacteria."

The importance of different mutations that emerge in Pa during mono vs. co-culture in the presence of colistin is not clearly explained. Why should co-culture inhibit the emergence of hypermutator Pa strains?

We thank the reviewer for the opportunity to clarify this important point. Previous studies have shown, both in Pa as well as other bacteria, that hypermutator strains often arise when bacteria adapt to strong and continuous antibiotic stress (PMID: 28630206) to maximize exploration of mutation space necessary to acquire beneficial resistance mutations even though hypermutation itself is inherently deleterious to bacterial fitness. We show that fungal co-culture protects P. aeruginosa from high concentrations of colistin by sequestering the Mg2+ co-factor required for colistin action (Fig. 4C). Thus, under co-culture conditions, bacteria experience lower levels of colistin than the levels administered and are subject to less severe fitness challenges, allowing them to eschew the deleterious route of acquiring adaptive mutations with hypermutation.

Our discovery that bacteria have an entirely different means of enhancing colistin resistance under fungal co-culture (or low Mg2+) conditions is one of the highlights of our study. Understanding the biological basis of this novel model of colistin resistance will be an active area of investigation to pursue in the future.

No additional experiments are likely needed but the authors should be encouraged to place their findings more clearly in what is already known in the field as well as articulate the limitations of their study.

We thank the reviewer for their detailed comments and suggestions. We hope our revisions have both clarified the importance and limitations of our study and provided the right context sought by the reviewer.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this study, Hsieh et al. find a critical axis of competition between Pseudomonas aeruginosa and Candida albicans is Mg2+ sequestered by Candida. The authors find that use of BHI, which is has lower Mg2+ levels compared to other media, allowed this discovery. The authors further demonstrate critical genes for this axis in multiple gammaproteobacteria and fungal species. The authors further show that fungal Mg2+ sequestration promotes polymyxin resistance in multiple gammaproteobacteria and show that it alters the course of Pseudomonas aeruginosa evolution of polymyxin resistance. Finally, they show that for populations evolved polymyxin resistance in the presence of Candida, removal of Candida by antifungal treatment re-induces sensitivity to polymyxins.

We thank the reviewer for a concise and accurate summary of our study.

Major comments: -The claims and conclusions are generally supported; however, a key phenotype of the ∆mgtA and ∆PA4824 mutants should be complemented in trans or in a second site of the chromosome.

We thank the reviewer for this comment and agree with the suggestion. In our revised manuscript, we now provide results of new complementation experiments recommended by the reviewer, which find that expression of PA4824 or mgtA in trans restore the fitness cost of either deletion mutant (Fig. S4C and S4D).

-The authors note that "This mode of competition appears to be highly specific between fungi and gram-negative bacteria." However, it does not appear that gram-positive bacteria were tested in competition with fungi. Additionally, the only gram-negative tested were gammaproteobacteria (although do represent diverse gammaproteobacteria). This could be addressed by clarifying the text or OPTIONAL additional experimentation.

We agree with the reviewer. We had intended to highlight that we had only tested this mode of competition between fungi and gram-negative bacteria, but inadvertently phrased this to suggest that gram-positive bacteria are not subject to this competition. As we highlight in our response to Reviewer 1, we are unable to test this (so far) for gram-positive bacteria. We clarify this in our revision: ""This mode of competition might be highly specific between fungi and diverse gram-negative g-proteobacteria we have tested.... Whether fungi can suppress gram-positive bacteria through the same mechanism of Mg2+ competition remains an open question."

-Figure 3A: is this depiction of modifications on the O-antigen correct? PhoQ- and PmrB-activated enzymes seem to modify the lipid A portion of LPS (eg PMID: 31142822)

We thank the reviewer for noting this error, which we have now fixed in the revision.

• For many of the figures, multiple t-tests are used and it seems like perhaps an ANOVA with multiple comparisons would be more appropriate

We thank the reviewer for this feedback. In our revision, we now use Dunnett's one-way ANOVA test for figures with multiple comparisons; our conclusions are unchanged.

Minor comments: - The text and figures are clear and accurate

We thank the reviewer for this feedback.

-the cited nutritional immunity reviews are out of date (e.g. reference 37) and there are more recent reviews on the topic (e.g. PMID: 35641670)

We have added the suggested reference in our revision.

-Line 293: Unclear why polymyxin resistance would be "unexpected" following the explanation of why Mg2+ depletion might confer it

We agree and have removed 'unexpected.'

-Line 318: "antibitoics" typo

We thank the reviewer for pointing out this typo, which we have now corrected.

Reviewer #3 (Significance (Required)):

The following aspects are important:

• General assessment: This study is very mechanistic, identifying the role of Mg2+ sequestration by fungi that limit gram-negative bacterial growth in Mg2+ deplete environments. The strengths are that relevant Mg2+ acquisition genes are identified or tested in Pseudomonas aeruginosa, the main test organism, as well as Salmonella enterica and Escherichia coli. Additionally, the authors identify a relevant Mg2+ mechanism in fungal species tested, including showing the importance with a genetic knockout. The limitations are relatively minor, and include lack of complementation, potential issues in model figure depiction of LPS modifications, and potential minor issues in statistical tests used. Future directions discussed include expanding analysis to clinical isolates, which is outside the scope of this manuscript which already showed the same mechanism in diverse gammaproteobacterial.

We thank the reviewer for their positive appraisal.

• Advance: This study has two major advances: The first is uncovering this critical Mg2+ sequestration axis in competition between fungal species and gammaproteobacteria. The second is the finding that the Mg+ sequestration induces polymyxin resistance and alters the evolutionary path to further polymyxin resistance. While nutrient metals as an axis of competition is not a conceptual advance, the specific role of Mg2+ and its affect on evolution of polymyxin antibiotic resistance is a conceptual advance.
• Audience: I think this study would be of interest to a relatively broad audience. The study itself touches on multiple fields including intermicrobial competition, nutritional immunity, antimicrobial resistance, and microbial evolution. Additionally, there are clinical implications for the potential to use antifungals to resensitize polymyxin-resistant P. aeruginosa to polymyxins.
• My field of expertise is bacterial genetics and physiology, nutritional immunity, and bacterial cell envelope. I do not have expertise in fungus.

We appreciate the reviewer's positive and constructive feedback on our study and for highlighting the relevance of our research to a broader audience in microbiology and evolution. We do hope our mechanistic understanding of fungal-bacterial competition will spark further conversation or collaboration between evolutionary microbiologists and physician-scientists.

Author Response

The following is the authors’ response to the original reviews.

We greatly thank you and the reviewers for your expert comments and valuable suggestions on our manuscript. After reading these comments, we realized that the previous version of the manuscript contained some weak points. Surely, the issues raised by the six reviewers were of great help in the revision of our manuscript.

According to the comments, we have now fully revised the manuscript to address most of the questions and suggestions. In addition, we reworded some parts of the Introduction, Results and Discussion, Figures, Figure legends and Experimental Methods to increase the rigor of our conclusions.

Overall, you will see that we have paid serious attention to all the concerns and criticisms expressed by reviewers. Addressing these various issues has most certainly allowed us to prepare a much-improved manuscript and for this we offer our hearty thanks.

Reviewer #1 (Public Review):

Summary:

The organization of cell surface receptors in membrane nanodomains is important for signaling, but how this is regulated is poorly understood. In this study, the authors employ TIRFM single-molecule tracking combined with multiple analyses to show that ligand exposure increases the diffusion of the immune receptor FLS2 in the plasma membrane and its co-localization with remorin REM1.3 in a manner dependent on the phosphosite S938. They additionally show that ligand increases the dwell time of FLS2, and this is linked to FLS2 endocytosis, also in a manner dependent on S938 phosphorylation. The study uncovers a regulatory mechanism of FLS2 localization in the nanodomain crucial for signaling.

Strengths:

TIRFM single-molecule tracking, FRAP, FRET, and endocytosis experiments were nicely done. The role of S938 phosphorylation is convincing.

Weaknesses:

Question 1: The model suggests that S938 is phosphorylated upon flg22 treatment. This is actually not known.

Reply: Thank you for your expert comments. Although the phosphorylation of Ser-938 upon flg22 treatment is not known, the model presented in the manuscript is based on previous studies that have shown the importance of Ser-938 phosphorylation for the function of FLS2 (Cao et al, 2013). When it is mutated to the phosphorylation-mimicking residues aspartate or glutamate, immune responses remain normal. These findings suggest that the phosphorylation of Ser-938 plays a critical role in activating defense mechanisms upon flagellin detection (Cao et al, 2013). Now we added the results of Cao et al. (2013) to the introduction to strengthen in the revised manuscript.

Question 2: In addition, the S938D mutant does not show constitutively increased diffusion and co-localization with remorin. It is necessary to soften the tone in the conclusion.

Reply: We appreciate the valuable suggestions from the reviewer. Based on our findings, we observed that the phosphorylation of Ser-938 significantly impacts the dynamics of flg22-induced FLS2. However, it does not alter the diffusion coefficient of FLS2 itself. In the revised manuscript, we have carefully adjusted the conclusion by softening the tone to reflect these findings.

Question 3: The introduction (only two paragraphs) and discussion are not properly written in the context of the current understanding of plant receptors in nanodomains. The authors basically just cited a few publications of their own, and this is not acceptable.

Reply: We accepted the criticisms here. Now, we have reworded the introduction and discussion sections to improve clarity. Furthermore, we have incorporated several new reports on plant receptors in nanodomains into the revised manuscript. Besides, we deleted some publications from our own group, while citing the latest references on plant receptors and nanodomains.

Reviewer #2 (Public Review):

Summary:

The research conducted by Yaning Cui and colleagues delves into understanding FLS2-mediated immunity. This is achieved by comparing the spatiotemporal dynamics of an FLS2-S938A mutant and FLS2-WT, especially in relation to their association with the remorin protein. To delineate the differences between the FLS2-S938A mutant and FLS2-WT, they utilized a plethora of advanced fluorescent imaging techniques. By analyzing surface dynamics and interactions involving the receptor signal co-receptor BAK1 and remorin proteins, the authors propose a model of how FLS2 and BAK1 are assembled and positioned within a remorin-specific nano-environment during FLS2 ligand-induced immune responses.

Strengths:

These techniques offer direct visualizations of molecular dynamics and interactions, helping us understand their spatial relationships and interactions during innate immune responses. Advanced cell biology imaging techniques are crucial for obtaining high-resolution insights into the intracellular dynamics of biomolecules. The demonstrated imaging systems are excellent examples to be used in studying plant immunity by integrating other functional assays. Weaknesses:

It's essential to acknowledge that every fluorescence-based method, just like biochemical assays, comes with its unique limitations. These often pertain to spatial and temporal resolutions, as well as the sensitivity of the cameras employed in each setup. Meticulous interpretation is pivotal to guarantee an accurate depiction and to steer clear of potential misunderstandings when employing specific imaging systems to analyze molecular attributes. Moreover, a discerning interpretation and accurate image analysis can offer invaluable guidance for future studies on plant signaling molecules using these nice cell imaging techniques. For instance, although single-particle analysis couldn't conclusively link FLS2 and remorin, FLIM-FRET effectively highlighted their ligand-triggered association and the disengagement brought on by mutations. While these methodologies seemed to present differing outcomes, they were described in the manuscript as harmonious. In reality, these differences could highlight distinct protein populations active in immune responses, each accentuated differently by the respective imaging techniques due to their individual spatial and temporal limitations. Addressing these variations is imperative, especially when designing future imaging explorations of immune complexes.

Reply: Thank you for your insightful comments and suggestions. We appreciate your expertise in fluorescence-based methods and the importance of careful interpretation and accurate image analysis. We agree with you that different imaging techniques may have their limitations and can highlight distinct aspects of protein dynamics and interactions.

In our study, we used single-particle analysis and FLIM-FRET to investigate the spatiotemporal dynamics of FLS2 and its association with remorin. While single-particle analysis did not conclusively link FLS2 and remorin, FLIM-FRET effectively highlighted their ligand-triggered association and the disengagement caused by mutations. We acknowledge that these techniques may have different spatial and temporal resolutions, leading to the discrepancy in their results. However, after the normalized treatment, we can provide very similar conclusions. Accordingly, we have revised the manuscript.

Reviewer #3 (Public Review):

Summary:

Receptor kinases (RKs) perceive extracellular signals to regulate many processes in plants. FLS2 is an RK that acts as a pattern-recognition receptor (PRR) to recognize bacterial flagellin and activate pattern-triggered immunity (PTI). PRRs such as FLS2 have been previously shown to reside within PM nanodomains, which can regulate downstream PTI signaling. In the current manuscript, Cui et al use single particle tracking to characterize the effect of previously-described phosposite mutants (FLS2-S938A/D) on the PM organization, endocytosis, and signaling functions of FLS2. The authors confirm that FLS2-S938D but not -S938A is functional for flg22-induced responses, while also demonstrating that phopshodead mutation at this site (S938A) prevents flg22-induced sorting into nanodomains and endocytosis. These results are consistent with S938 being an important phosphorylation site for FLS2 function, however, they fall short of demonstrating that membrane disorganization of FLS2-938A is responsible for downstream signaling defects.

Strengths:

The authors' experiments (single particle tracking, co-localization, etc) do a good job of demonstrating how a non-functional version of FLS2 (S938A) does not alter its spatio-temporal dynamics, nanodomain organization, and endocytosis in response to flg22, suggesting that these require a functional receptor and are regulated by intracellular signaling components.

Weaknesses:

Question 1: The authors do not provide direct evidence that S938 phosphorylation specifically affects membrane organization, rather than FLS2 signaling more generally. All evidence is consistent with S938A being a non-functional version of FLS2, wherein an activated/functional receptor is required for all downstream events including membrane re-organization, downstream signalling, internalization, etc. Furthermore, the authors never demonstrate that this site is phosphorylated in planta in the basal or flg22-elicited state.

Reply: Sorry that we did not describe clearly in the original manuscript. In fact, we found in our study that the phosphorylation of the Ser-938 site influences the efficient sorting of FLS2 into AtRem1.3-associated microdomains rather than membrane organization, as depicted in Figure 2. Furthermore, we found that the immune responses are disrupted when Ser-938 is mutated to alanine, which is consistent with previously reported results (Cao et al, 2013). However, they remain normal when mutated to the phosphorylation-mimicking residues aspartate or glutamate. These results suggest that the phosphorylation of Ser-938 is crucial for activating defense mechanisms upon flagellin detection. Although the phosphorylation of Ser-938 in plant at the basal or flg22-elicited state is not known, the model presented in the manuscript is based on the results of our current investigation together with those in the previous study that have shown the importance of Ser-938 phosphorylation for FLS2 function (Cao et al, 2013).

Question 2: As written, the manuscript also has numerous scientific issues, including a misleading/incomplete description of plant immune signaling, lack of context from previous work, and extensive use of inappropriate references.

Reply: We accept the criticism here. After reading the comments, we realized the problem. Now we have revised the misleading or incomplete description of plant immune signaling, added the context of previous works and deleted inappropriate references in the revised manuscript.

Reviewer #1 (Recommendations For The Authors):

Question 1: The description of the data has no details. How many biological repeats were done? How were statistical analyses done? What is the concentration of flg22? How was the calcium flux done (Fig. 4A)? The method also lacks details and relevant references.

Reply: We apologize for the lack of detail in presenting the data. Following your suggestion, we added comprehensive figure legends that provide clear explanations for each figure. Additionally, we included supplementary information on the measurement methods and references pertaining to calcium flux in the revised manuscript.

Question 2: Data in Fig. 4 basically repeated the 2013 PLoS Pathog paper. Why were these experiments even performed? Were GFP-tagged FLS2 lines used in these experiments? If this is the case, the data just verified that the GFP-tagged FLS2 functions as expected and should be moved to supporting data.

Reply: Thanks for the expert suggestions. In our study, we utilized GFP-tagged FLS2 lines to generate FLS2-S938 mutants and conducted experiments to investigate the flg22-induced immune response. Although some experiments in Figure 4 are similar to those reported (Cao et al, 2013), we provided a more detailed analysis of the immune response. The comprehensive analysis included early immune responses and late immune responses, e.g., the activation of a calcium burst, mitogen-activated protein kinases (MAPKs), the induction of immune-responsive genes and callose deposition, ultimately resulting in the inhibition of plant growth. As some results are analogous to the previous paper, we transfer some of the experiments as suggested, including the analysis of MAPKs and callose deposition, to the supporting data section of the revised manuscript.

Question 3: Flg22-induced FLS2-BAK1 association does not require S938, this is consistent with prior study that flg22 acts as a molecular glue for the ectodomains of FLS2 and BAK1 (Sun et al., 2013 Science). This needs to be cited.

Reply: Yes, we agree with the comment. Now we added an additional sentence in the revised manuscript: “ This aligns with the previous finding that flg22 acts as a molecular glue for FLS2 and BAK1 ectodomains (Sun et al., 2013).”

Question 4: Line 50, the references cited do not match what they say here.

Reply: We are sorry for the mistake in citing inappropriate references. In the revised manuscript, we deleted this sentence as well as the incorrect reference.

Question 5: Line 105, "flg22 can act as a ligand-like factor". It is a ligand!

Reply: Sorry for the mistake. Now, the sentence was corrected in the revised manuscript by deleting the word “like”.

Question 6: Line 107, FLS2/BAK1 heterodimerization, not heteroologomerization.

Reply: Now we used “heterodimerization” to replace “heteroologomerization” in the revised manuscript.

Question 7: Line 114, are these really the best references to cite here?

Reply: After reading the comment, we found the references were not suitable here. Now we changed references by citing “(Martinière et al., 2021)” in the revised manuscript.

Question 8: Lines 123-124, the sentence is incomplete.

Reply: In the revised manuscript, we reworded the sentence to make it complete now. We changed “In a previous investigation, we demonstrated that flg22 induces FLS2 translocation from AtFlot1-negative to AtFlot1-positive nanodomains in the plasma membrane, implying a connection between FLS2 phosphorylation and membrane nanodomain distribution (Cui et al., 2018). To validate this, we assessed the association of FLS2/FLS2S938D/FLS2S938A with membrane microdomains, using AtRem1.3-associated microdomains as representatives (Huang et al., 2019).” in the revised manuscript.

Question 9: Lines 169-170, Why is this "most important"?

Reply: Sorry for the unsuitable description. As we have dramatically changed the manuscript, this sentence was deleted from the new version.

Reviewer #2 (Recommendations For The Authors):

Here are some specific areas of ambiguity in the study to be improved.

Question 1: Clarity in statistical analysis is necessary. Many figure legends omit details such as the sample size "n", and the nature of the measurements, like ROIs, images, and dots, the size of the seedlings, etc.

Reply: We appreciated this suggestion, which was raised by the reviewer I as well. Now, we provided the details for each figure, including the sample size, the nature of the measurements in the revised manuscript.

Question 2: Additional background about the choice of FLS2-S938 mutant would be beneficial, given that this mutant doesn't affect the BAK1 interaction but nullifies several PTI responses.

Reply: Yes, we agreed that some additional background is required for the FLS2-S938 mutant. Therefore, we added a sentence here: “FLS2 Ser-938 mutations impact flg22-induced signaling, while BAK1 binding remains unaffected, thereby suggesting Ser-938 regulates other aspects of FLS2 activity (Cao et al., 2013).” in the revised manuscript.

Question 3: A specific segment "... Using CLSM, Fluorescence Correlation Spectroscopy (FCS) and Western blotting, we found that the endocytic vesicles of FLS2S938D increased significantly after flg22 treatment (Figure 3B-3E)..." is not easy to follow. The author may want to differentiate these methods and highlight them by indicting them as endocytic vesicle counting, receptor density on PM measurement by FCS, and WB-based protein degradation characterization to understand such mixed descriptions better. By the way, "Number of Endocytosis" should be "number of endocytic vesicles". Endocytosis is a process and uncountable.

Reply: We thank the reviewer for kindly reminding us to differentiate experimental methods. Therefore, we changed the sentences in the revised manuscript: “Employing confocal laser-scanning microscopy (CLSM) during 10μM flg22 treatment, we tracked FLS2 endocytosis and quantified vesicle numbers over time (Figure 3B). It is evident that both FLS2 and FLS2S938D vesicles appeared 15 min after-flg22 treatment, significantly increasing thereafter (Figure 3C). Notably, only a few vesicles were detected in FLS2S938A-GFP, indicating Ser-938 phosphorylation's impact on flg22-induced FLS2 endocytosis. Additionally, fluorescence correlation spectroscopy (FCS) (Chen et al., 2009) monitored molecular density changes at the PM before and after flg22 treatment (Figure S3F). Figure 3D shows that both FLS2-GFP and FLS2S938D-GFP densities significantly decreased after flg22 treatment, while FLS2S938A-GFP exhibited minimal changes, indicating Ser-938 phosphorylation affects FLS2 internalization. Western blotting confirmed that Ser-938 phosphorylation influences FLS2 degradation after flg22 treatment (Figure 3E), consistent with single-molecule analysis findings.” Besides, we also changed “number of endocytosis” to “the number of endocytic vesicles” in Figure 3C as suggested.

Question 4: In Figure 1 E, a discrepancy exists where the total percentages in the red and black columns don't sum up to 100%, while other groups look right. This needs clarification.

Reply: We are sorry for our carelessness in making the data incomplete. Now we thoroughly supplemented, collated, and rechecked the data in Figure 1E. Due to an oversight during the production of the figure, some data was inadvertently omitted, resulting in the red column not reaching 100%. Besides, we checked the data in the black column again, and the total percentage indeed added up to 100%.

Question 5: Although Figure 1F uses UMAP analysis to differentiate between FLS2WT and A mutants, only data pertaining to the "D" mutant is shown.

Reply: Thank you for the expert comments. Because there are several images in Figure 1, we only selected the data related to the “D” mutant as a representative for display. As suggested, we have added all the UMAP images in the revised supplement figure S1F.

Question 6: There are apparent inconsistencies in the FRAP results, particularly regarding the initial recovery points post-bleaching. A detailed statistical analysis, supplemented with FRAP images over time, should be included for clarity. Were they bleached to a similar ground level before monitoring their recovery? The data points from "before" and "after "bleaching were not shown. I found the red and blue curves showed similar recovery slop, which suggests no long-distance movement changes for all three FLS2 versions, with or without flg22. This is opposite from the conclusions made by the author.

Reply: Thank you for the expert comments. After reading the comments, we recognized this terrible problem. Therefore, we carried out a new FRAP experiment. The new results showed that, following complete bleaching of three samples of FLS2 to ground level, the recovery rates of FLS2 and FLS2S938D under flg22 treatment were significantly higher compared to the control group (Fig. 1G). In contrast, the recovery rates of the FLS2S938A-GFP after flg22 treatment remain similar to that before treatment (Fig. 1G), indicating that the Ser-938 phosphorylation site indeed affects the flg22-induced lateral diffusion of FLS2 at the PM. The new results are basically consistent with the motion range of single-molecule results, which is not contradictory to long-distance movement changes. Accordingly, we incorporated the new time-lapse FRAP images into Figure 1G and S1B.

Question 7: There's a potential typo in Figure 1B regarding the bar size. It could neither possibly be 200 um nor 200 nm. Figure 1A also needs a scale bar.

Reply: Apologies for the mistake. We now corrected “200 μm” to “2 μm”. Besides, we also included a scale bar in Figure 1A in the revised manuscript.

Question 8: Due to the unreliable tracking for a long-time by Imaris, the authors analyzed the tracks within 10s and quantified very short live particles under 4s. Such 4S surface retention for a receptor does not seem to match functional endocytic internalization time for cargo. Even after the endocytic adaptor module recruitment, it would take at least more than 10s to finish the internalization. In the field of endocytosis, these events are often described as abortive endocytic events. However, the disappearance of cargoes, FLS2 in this case, indicates internalization into the cytoplasm, which is interesting. May the author discuss more on how these short events analyzed enhance our understanding of the functional behavior of FLS2?

Reply: We greatly appreciated the valuable comments provided by the reviewer. After thorough consideration, we acknowledged that in our original manuscript, we failed to distinguish the short-lived from the long-lived particles and vaguely put them collectively into the internalized particles. We realized that and it is inappropriate to ambiguously categorize all particles as internalized. Therefore, we added the sentence “Additionally, numerous FLS2 exhibited short-lived dwell times, indicating abortive endocytic events associated with the endocytic pathway and signal transduction (Bertot et al., 2018)” in the revised manuscript.

Question 9: Figure 2D should be comprehensive, presenting data for the WT, A, and D versions.

Reply: Yes, we agreed with the suggestions. Now, we added several representative images for the WT, A, and D versions in the revised manuscript.

Question 10: In Figure 2D, TIRM-SIM should be a typo and rectified to TIRF-SIM. Also, a detailed explanation of the TIRF-SIM setup and its specifics would be important. The imaging approach of SIM, especially the time duration for finishing all frames before reconstruction, is essential to rationalize its use in capturing and measuring an appropriate speed range of particle movement. May the author elaborate on the technique details and the use of TIRF-SIM for colocalization analysis? To clarify these, the author may provide additional TIRF-only movies of FLS2 (WT, A, D) and AtRem1.3 for comparison with TIRF-SIM still images.

Reply: Sorry for the mistake. In the revised manuscript, we have corrected “TIRM-SIM” to “TIRF-SIM”. In order to rationalize its use in capturing and measuring an appropriate speed range of particle movement, we included a more detailed description of the imaging approach and the colocalization analysis of TIRF-SIM in the Materials and Methods section as follows: “The SIM images were taken by a 60 × NA 1.49 objective on a structured illumination microscopy (SIM) platform (DeltaVision OMX SR) with a sCMOS camera (Camera pixel size, 6.5 μm). The light source for TIRF-SIM included diode laser at 488 nm and 568 nm with pixel sizes (μm) of 0.0794 and 0.0794 (Barbieri et al., 2021). For the dual-color imaging, FLS2/FLS2S938A/FLS2S938D-GFP (488 nm/30.0%) and AtRem1.3-mCherry (561 nm/30.0%) were excited sequentially. The exposure time of the camera was set at 50 ms throughout single-particle imaging. The time interval for time-lapse imaging was 100 ms, the total time was 2s, and the total time points were 21s. The Imaris intensity correlation analysis plugin was used to calculate the co-localization ratio.” in the revised manuscript. Furthermore, we provided additional TIRF-SIM movies of FLS2 (WT, A, D) and AtRem1.3.

Question 11: The colocalization displayed in Figure 2D is hard to tell. A colocalization ratio of FLS2-AtRem1.3 is shown as ~0.8%, which has only ~0.2% difference from the flg22-treated condition. "n" of Figure 2F should be specified in the legend, such as a line with a specific length, or an ROI with a specific area size.

Reply: Thank you for the expert comments. Although the increased colocalization after flg22 treatment is not high, the change is statistically significant as compared with the wild type. We agreed that every fluorescence-based method, like biochemical analysis, has its own unique limitations, which were raised by the Reviewer #2 (Public Review) as well. In order to provide strong evidence, we also carried out the FLIM-FRET experiment as a supplement, which can effectively detect their ligand-triggered association or disassociation. From figure 2G and H, we clearly found that the co-localization of FLS2/FLS2S938D-GFP with AtRem1.3-mCherry significantly increase in response to flg22 treatment (FLS2-GFP control: 2.45 ± 0.019 s; FLS2-GFP flg22-treated: 2.39 ± 0.016 s; FLS2S938D-GFP control: 2.42 ± 0.010 ns; FLS2S938D-GFP flg22-treated: 2.35 ± 0.028 ns). In contrast, FLS2S938A-GFP shows no significant changes (control: 2.53 ± 0.011 ns; flg22-treated: 2.56 ± 0.013 ns), indicating that Ser-938 phosphorylation influences efficient sorting of FLS2 into AtRem1.3-associated microdomains. Following the suggestion of the reviewer, we now rearranged the order of 2E and 2F, in which N represents the entire image region used for analysis rather than a specific region of interest.

Question 12: I appreciate the nice results of the FLIM-FRET results for FLS2-Rem1.3. Figure 2H should be supplemented with additional representative images of all FLS2 variants including WT and mutants.

Reply: Thanks for your warm encouragement. As suggested, we added all the representative images in the revised manuscript.

Question 13: The unit of the X-axis of Figure 2E can not be pixel. Should it be, um? In the method, the author could specify the camera model and magnification for TIRF-SIM to understand pixel size of the image better.

Reply: Sorry for the mistake here. Indeed, the unit of the X-axis in Figure 2E should be μm. Now we correct this mistake in Figure 2E in the revised manuscript. Besides, we included a detailed description of the imaging approach of TIRF-SIM in the Materials and Methods section as follows: “The SIM images were taken by a 60 × NA 1.49 objective on a structured illumination microscopy (SIM) platform (DeltaVision OMX SR) with a sCMOS camera (Camera pixel size, 6.5 μm)”.

Question 14: "... as shown in A..." in Figure Legend 2E should be "... as shown in D..."

Reply: Thanks for pointing out this mistake. In the revised manuscript, we used “as shown in D” to replace “as shown in A”.

Question 15: I recommend that the authors exercise caution when drawing conclusions based on the Rem1.3 data and when representing the "microdomain" concept in their final model. While Rem1.3 punctate is a nanometer-sized protein cluster specific to its identity, its shape can be categorized as a nanodomain. Conceptually, however, it neither universally represents all nanodomains nor microdomains, as depicted in Figure 4. We should exercise caution to prevent providing misleading information to the field.

Reply: We thank the reviewer for expert comments. To avoid misleading conclusions, we changed “nanodomains” to “AtRem1.3-associated microdomains” in the revised manuscript. Besides, we have also made modifications to Figure 4.

Reviewer #3 (Recommendations For The Authors):

Question 1: The manuscript needs to be extensively re-written and has severe issues as-is. Many references are either not quite appropriate or are completely unrelated to the use in the text. In general, the current state-of-the-art of PTI and RK signaling is not correctly described or incorporated.

Reply: We accepted the criticisms here. As suggested, we thoroughly rewrote the manuscript to address the concerns raised. Furthermore, we have thoroughly checked and revised the manuscript by removing 21 irrelevant references and adding 30 relevant references. We also incorporated the most up-to-date descriptions of the PTI and RK signaling pathways.

Question 2: Receptor-like kinase (RLK) should generally be receptor kinase (RK) as receptor functions are now well established.

Reply: Yes, we agreed with your expert comment here. Now, we changed “Receptor-like kinase (RLK)” into “receptor kinase (RK)” in the revised manuscript.

Question 3: Line 20 - is this really true?

Reply: Sorry for the mistake. In the revised manuscript, we changed “However, the mechanisms underlying the regulation of FLS2 phosphorylation activity at the plasma membrane in response to flg22 remain largely enigmatic.” to “However, the dynamic FLS2 phosphorylation regulation at the plasma membrane in response to flg22 needs further elucidation.”

Question 4: S938D sorts better in response to Flg22; S938A is unaffected - suggests phosphorylation of S938 is not dynamic in response to Fig 22 but is required for pre-elicitation sorting. Overall, there is a chicken-and-egg problem in this paper: which comes first, immune/signalling functionality or nanodomain sorting? And which is explaining the defects of S938A?

Reply: We thank the reviewer for expert suggestions. In fact, the previous studies showed that membrane microdomains serve as signaling platforms that mediate cargo protein sorting and protein-protein interactions in a variety of contexts (Goldfinger et al. 2017). Since our previous research showed that the disruption of membrane microdomains affected flg22-induced immune signaling (Cui et al. 2018), we speculate that the immune signal occurred after entering the membrane microdomains.

As shown in Figure 1 and 2, ligand exposure leads to an increase in diffusion coefficient and enhanced co-localization with REM1.3, both of which are dependent on the phosphorylation of the Ser-938 site. Deducing from these results, we inferred that the defects in S938A resulted largely from its failure to sort into membrane microdomains. The phosphorylation of the Ser-938 site can regulate FLS2 into functional AtRem1.3-associated microdomains, thereby affecting flg22-induced plant immunity.

Question 5: Line 37 conserved, not conservative (though not technically true - the domain organization is conserved but the ECDs are not conserved).

Reply: Thank you for pointing this mistake out. In the revised manuscript, we used “conserved” to replace “conservative”.

Question 6: Lines 40-42 - not all phosphorylation sites are within the kinase domain, for example, sites are well-described on the JM and/or C-tail regions outside of the kinase domain.

Reply: We accepted the criticisms here. We have corrected the sentence to “with phosphorylation sites mainly located in PKC” in the revised manuscript.

Question 7: Line 42 - what is BIK1? Intro to relevant topics is severely lacking.

Reply: Sorry for the incomplete introduction here. We added the relevant introduction of BIK1 by adding that “Upon recognizing flg22, FLS2 interacts with the co-receptor Brassinosteroid-Insensitive 1-associated Kinase 1 (BAK1), initiating phosphorylation events through the activation of receptor-like cytoplasmic kinases (RLCKs) such as BOTRYTIS-INDUCED KINASE 1 (BIK1) to elicit downstream immune responses (Chinchilla et al., 2006; Li et al., 2016b; Majhi et al., 2021). ” in the revised manuscript.

Question 8: Lines 42-44 - not sure this sequence of events is being properly described (e.g. BIK1 release is unlikely to precede activation by BAK1/SERKs).

Reply: We apologize for not expressing this sentence clearly. Now, we reworded the sentence: “Upon recognizing flg22, FLS2 interacts with the co-receptor Brassinosteroid-Insensitive 1-associated Kinase 1 (BAK1), initiating phosphorylation events through the activation of receptor-like cytoplasmic kinases (RLCKs) such as BOTRYTIS-INDUCED KINASE 1 (BIK1) to elicit downstream immune responses (Chinchilla et al., 2006; Li et al., 2016b; Majhi et al., 2021).” in the revised manuscript.

Question 9: Line 61 - S938 was identified by Cao et al (2013) based on in vitro MS, but was functionally validated using genetic assays, not based on MS.

Reply: Thank you for your comments. Now, we changed the sentence: “In vitro mass spectrometry (MS) identified multiple phosphorylation sites in FLS2. Genetic analysis further identified Ser-938 as a functionally important site for FLS2 in vivo (Cao et al., 2013).” in the revised manuscript.

Question 10: Line 68-69 - phospho-dead and phospho-mimic, not phosphorylated/non-phosphorylated.

Reply: We thank the reviewer for expert suggestions. In the revised manuscript, we changed the sentence by replacing “phosphorylated/non-phosphorylated” with “phospho-mimic” and “phospho-dead”.

Question 11: Lines 104-106 - this is wildly misleading. Flg22 is more than a ligand-like factor, as it is a bona fide ligand, and the heterodimerization with BAK1/SERKs is extremely well-established (and relevant foundational papers should be cited here in place of the authors' previous work).

Reply: We apologize for the incorrect expression here. After reading the comments, we realized the problem which was raised by the reviewer I as well. Now, we changed “ligand-like factor” to “ligand”. Besides, we cited the new references “(Orosa et al., 2018)” to replace the references of our group in the revised manuscript.

Question 12: Lines 107-112 - again, this is confusing. There is a decade of (uncited, undiscussed) work previously establishing that heterodimerization of RK-co-receptor complexes is mediated by extracellular ligand binding and independent of intracellular phosphorylation.

Reply: We thank the reviewer for expert suggestions. Now, we added several sentences in the revised manuscript: “Therefore, we further investigated if Ser-938 phosphorylation affects FLS2/BAK1 heterodimerization. Tesseler segmentation, FRET-FLIM, and smPPI analyses revealed no impact of Ser-938 phosphorylation on FLS2/BAK1 heterodimerization (Figure 2A-C and S2). This aligns with the previous finding that flg22 acts as a molecular glue for FLS2 and BAK1 ectodomains (Sun et al., 2013), confirming the independence of FLS2/BAK1 heterodimerization from phosphorylation, with these events occurring sequentially.”

Question 13: Line 119 - this is the wrong citation - Yu et al 2020 is a review and does not cover RALFs; correct citation is Gronnier et al 2022 eLife.

Reply: In the revised manuscript, we updated the reference from “ (Yu et al., 2020)” to “(Gronnier et al., 2022)”.

Question 14: Lines 123-124 - this sentence is incomplete.

Reply: Sorry for the incomplete sentence. Now we reworded the sentence to “In a previous investigation, we demonstrated that flg22 induces FLS2 translocation from AtFlot1-negative to AtFlot1-positive nanodomains in the plasma membrane, implying a connection between FLS2 phosphorylation and membrane nanodomain distribution (Cui et al., 2018). To validate this, we assessed the association of FLS2/FLS2S938D/FLS2S938A with membrane microdomains, using AtRem1.3-associated microdomains as representatives (Huang et al., 2019).” in the revised manuscript.

Question 15: Line 126 - this requires a reference.

Reply: Yes, we added a new reference: “(Huang et al., 2019)” in the revised manuscript.

Question 16: Lines 125-128 - should clarify that the authors are not looking at direct interaction between FLS2 and REM1.3.

Reply: Sorry for the inappropriate expressions here. In the revised manuscript, we reworded the sentence as follows: “To validate this, we assessed the association of FLS2/FLS2S938D/FLS2S938A with membrane microdomains, using AtRem1.3-associated microdomains as representatives (Huang et al., 2019)” .

Question 17: Line 138 - these are odd references to use for such a broad statement.

Reply: Now the inappropriate references cited here have been deleted.

Question 18: Line 161 - incorrect reference, again.

Reply: Sorry for this mistake. In the revised manuscript, we reworded the sentence and changed the reference.

Question 19: Lines 160-165 - this is very confusing and misleading. I would suggest just having a short section introducing PTI earlier on (with appropriate references).

Reply: As suggestion, we reworded and added a section in the revised manuscript as follows: “PTI plays a pivotal role in host defense against pathogenic infections (Lorrai et al., 2021; Ma et al., 2022). Previous studies demonstrated that FLS2 perception of flg22 initiates a complex signaling network with multiple parallel branches, including calcium burst, mitogen-activated protein kinases (MAPKs) activation, callose deposition, and seedling growth inhibition (Baral et al., 2015; Marcec et al., 2021; Huang et al., 2023). Our focus was to investigate the significance of Ser-938 phosphorylation in flg22-induced plant immunity. Figure 4A-F illustrates diverse immune responses in FLS2 and FLS2S938D plants following flg22 treatment. These responses encompass calcium burst activation, MAPKs cascade reaction, callose deposition, hypocotyl growth inhibition, and activation of immune-responsive genes. In contrast, FLS2S938A (Figure S4A-D) exhibited limited immune responses, underscoring the importance of Ser-938 phosphorylation for FLS2-mediated PTI responses”.

Question 20: Line 166 - these are not appropriate references, again.

Reply: Thank you for the suggestion. In the revised manuscript, we removed the inappropriate references. Besides, we added new references by citing: “(Baral et al., 2015; Marcec et al., 2021)”.

Question 21: Lines 169-173 - this is not relevant, the inhibition of growth by elicitors is extremely well-documented (though not by the refs cited here).

Reply: We reworded the sentence and deleted the inappropriate reference in the revised manuscript.

Question 22: Lines 174-175 - I don't see why this is unexpected, as nanodomain organization of PRRs has been previously described.

Reply: Sorry for the inappropriate expressions here. As we have dramatically changed the manuscript, this sentence was deleted from the new version.

References we added into the revised manuscript

Baral A, Irani NG, Fujimoto M, Nakano A, Mayor S, Mathew MK. 2015. Salt-induced remodeling of spatially restricted clathrin-independent endocytic pathways in Arabidopsis root. Plant Cell 27:1297-315. DOI: 10.1105/tpc.15.00154, PMID: 25901088

Barbieri L, Colin-York H, Korobchevskaya K, Li D, Wolfson DL, Karedla N, Schneider F, Ahluwalia BS, Seternes T, Dalmo RA, Dustin ML, Li D, Fritzsche M. 2021. Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM). Nature Communications 12:2169. DOI: 10.1038/s41467-021-22377-9, PMID: 33846317

Bertot L, Grassart A, Lagache T, Nardi G, Basquin C, Olivo-Marin J, Sauvonnet N. 2018. Quantitative and statistical study of the dynamics of clathrin-dependent and -independent endocytosis reveal a differential role of endophilinA2. Cell Reports 22: 1574–1588. DOI:org/10.1016/j.celrep.2018.01.039, PMID: 29425511

Bücherl CA, Jarsch IK, Schudoma C, Segonzac C, Mbengue M, Robatzek S, MacLean D, Ott T, Zipfel C. 2017. Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains. eLife 6:e25114. DOI: https://doi.org/10.7554/eLife.25114, PMID: 28262094

Chen Y, Munteanu AC, Huang YF, Phillips J, Zhu Z, Mavros M, Tan W. 2009. Mapping receptor density on live cells by using fluorescence correlation spectroscopy. Chemistry 15:5327-36. DOI: https://doi.org/10.1002/chem.200802305, PMID: 19360825

Chinchilla, D., Bauer, Z., Regenass, M., Boller, T., and Felix, G. 2006. The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell 18:465-476. doi:10.1105/tpc.105.036574, PMID: 16377758

Gada KD, Kawano T, Plant LD, Logothetis DE. 2022. An optogenetic tool to recruit individual PKC isozymes to the cell surface and promote specific phosphorylation of membrane proteins. The Journal of Biological Chemistry 298:101893. DOI: https://doi.org/10.1016/j.jbc.2022.101893, PMID: 35367414

Gronnier J, Franck CM, Stegmann M, DeFalco TA, Abarca A, von Arx M, Dünser K, Lin W, Yang Z, Kleine-Vehn J, Ringli C, Zipfel C. 2022. Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors. eLife 11:e74162. DOI: https://doi.org/10.7554/eLife.74162, PMID: 34989334

Hohmann U, Lau K, Hothorn M. 2017. The structural basis of ligand perception and signal activation by receptor kinases. Annual Review of Plant Biology 68:109–137. DOI: https://doi.org/10.1146/annurev-arplant-042916-040957, PMID: 28125280.

Huang D, Sun Y, Ma Z, Ke M, Cui Y, Chen Z, Chen C, Ji C, Tran TM, Yang L, Lam SM, Han Y, Shu G, Friml J, Miao Y, Jiang L, Chen X. 2019. Salicylic acid-mediated plasmodesmal closure via Remorin-dependent lipid organization. Proceedings of the National Academy of Sciences 116:21274–21284. DOI: https://doi.org/10.1073/pnas.1911892116, PMID: 31575745

Huang Y, Cui J, Li M, Yang R, Hu Y, Yu X, Chen Y, Wu Q, Yao H, Yu G, Guo J, Zhang H, Wu S, Cai Y. 2023. Conservation and divergence of flg22, pep1 and nlp20 in activation of immune response and inhibition of root development. Plant Science 331:111686. DOI: https://doi.org/10.1016/j.plantsci.2023.111686, PMID: 36963637

Jiao C, Gong J, Guo Z, Li S, Zuo Y, Shen Y. 2022. Linalool activates oxidative and calciμm burst and CAM3-ACA8 participates in calciμm recovery in Arabidopsis leaves. International Journal of Molecular Sciences, 23:5357. DOI: https://doi.org/10.3390/ijms23105357, PMID: 35628166

Kim TJ, Lei L, Seong J, Suh JS, Jang YK, Jung SH, Sun J, Kim DH, Wang Y. 2018. Matrix rigidity-dependent regulation of Ca2+ at plasma membrane microdomains by FAK visualized by fluorescence resonance energy transfer. Advanced science, 6:1801290. DOI: https://doi.org/10.1002/advs.201801290, PMID: 30828523

Kontaxi C, Kim N, Cousin MA. 2023. The phospho-regulated amphiphysin/endophilin interaction is required for synaptic vesicle endocytosis. Journal of Neurochemistry 166:248–264. DOI: https://doi.org/10.1111/jnc.15848, PMID: 37243578

Lee Y, Phelps C, Huang T, Mostofian B, Wu L, Zhang Y, Tao K, Chang YH, Stork PJ, Gray JW, Zuckerman DM, Nan X. 2019. High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking. eLife 8:e46393. DOI: https://doi.org/10.7554/eLife.46393, PMID: 31674905

Li B, Meng X, Shan L, He P. 2016a. Transcriptional regulation of pattern-triggered immunity in plants. Cell Host Microbe 19:641-50. DOI: 10.1016/j.chom.2016.04.011, PMID: 27173932

Li L, Kim P, Yu L, Cai G, Chen S, Alfano JR, Zhou JM. 2016b. Activation-dependent destruction of a co-receptor by a pseudomonas syringae effector dampens plant immunity. Cell Host Microbe 20:504-514. DOI: https://doi.org/10.1016/j.chom.2016.09.007, PMID: 27736646.b

Lorrai R, Ferrari S. 2021. Host cell wall damage during pathogen infection: mechanisms of perception and role in plant-pathogen interactions. Plants (Basel) 10:399. DOI: https://doi.org/10.3390/plants10020399, PMID: 33669710

Marcec MJ, Tanaka K. 2021. Crosstalk between Calcium and ROS signaling during flg22-triggered immune response in Arabidopsis leaves. Plants 11:14. DOI: 10.3390/plants11010014. PMID: 35009017

Ma M, Wang W, Fei Y, Cheng HY, Song B, Zhou Z, Zhao Y, Zhang X, Li L, Chen S, Wang J, Liang X, Zhou JM. A surface-receptor-coupled G protein regulates plant immunity through nuclear protein kinases. 2022. Cell Host Microbe 30:1602-1614. DOI: 10.1016/j.chom.2022.09.012. Epub 2022 Oct 13. PMID: 36240763.

Martinière A, Zelazny E. 2021. Membrane nanodomains and transport functions in plant. Plant Physiology 187:1839–1855. DOI: https://doi.org/10.1093/plphys/kiab312, PMID: 35235669

Majhi, B.B., Sobol, G., Gachie, S., Sreeramulu, S., and Sessa, G. 2021. BRASSINOSTEROID-SIGNALLING KINASES 7 and 8 associate with the FLS2 immune receptor and are required for flg22-induced PTI responses. Molecular Plant Pathology 22:786-799. DOI:https://doi.org/10.1111/mpp.13062, PMID: 33955635

Mitra SK, Chen R, Dhandaydham M, Wang X, Blackburn RK, Kota U, Goshe MB, Schwartz D, Huber SC, Clouse SD. 2015. An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. The Plant Journal 82:1042–1060. DOI: https://doi.org/10.1111/tpj.12863, PMID: 25912465

Orosa B, Yates G, Verma V, Srivastava AK, Srivastava M, Campanaro A, De Vega D, Fernandes A, Zhang C, Lee J, Bennett MJ, Sadanandom A. 2018. SμmO conjugation to the pattern recognition receptor FLS2 triggers intracellular signalling in plant innate immunity. Nature Communications 9:5185. DOI: https://doi.org/10.1038/s41467-018-07696-8, PMID: 30518761

Sun Y, Li L, Macho AP, Han Z, Hu Z, Zipfel C, Zhou JM, Chai J. 2013. Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex. Science 342:624-628. DOI: https://doi.org/10.1126/science.1243825, PMID: 24114786

Vitrac H, Mallampalli VKPS, Dowhan W. 2019. Importance of phosphorylation/dephosphorylation cycles on lipid-dependent modulation of membrane protein topology by posttranslational phosphorylation. The Journal of Biological Chemistry 294:18853–18862. DOI: https://doi.org/10.1074/jbc.RA119.010785, PMID: 31645436

Xue Y, Xing J, Wan Y, Lv X, Fan L, Zhang Y, Song K, Wang L, Wang X, Deng X, Baluška F, Christie JM, Lin J. 2018. Arabidopsis blue light receptor phototropin 1 undergoes blue light-induced activation in membrane microdomains. Molecular Plant 11:846-859. DOI: 10.1016/j.molp.2018.04.003, PMID: 29689384

Xing J, Ji D, Duan Z, Chen T, Luo X. 2022. Spatiotemporal dynamics of FERONIA reveal alternative endocytic pathways in response to flg22 elicitor stimuli. New Phytologist 235: 518-532. DOI: 10.1111/nph.18127, PMID: 35358335

Zhai K, Liang D, Li H, Jiao F, Yan B, Liu J, Lei Z, Huang L, Gong X, Wang X, Miao J, Wang Y, Liu JY, Zhang L, Wang E, Deng Y, Wen CK, Guo H, Han B, He Z. 2021. NLRs guard metabolism to coordinate pattern- and effector-triggered immunity. Nature 601:245-251. DOI: https://doi.org/10.1038/s41586-021-04219-2, PMID: 34912119

Zhong YH, Guo ZJ, Wei MY, Wang JC, Song SW, Chi BJ, Zhang YC, Liu JW, Li J, Zhu XY, Tang HC, Song LY, Xu CQ, Zheng HL. 2023. Hydrogen sulfide upregulates the alternative respiratory pathway in mangrove plant Avicennia marina to attenuate waterlogging-induced oxidative stress and mitochondrial damage in a calciμm-dependent manner. Plant Cell and Environment 46:1521-1539. DOI: https://doi.org/10.1111/pce.14546, PMID: 36658747

Inappropriate references we deleted from the revised manuscript

Schulze S, Yu L, Hua C, Zhang L, Kolb D, Weber H, Ehinger A, Saile SC, Stahl M, Franz-Wachtel M, Li L, El Kasmi F, Nürnberger T, Cevik V, Kemmerling B. 2022. The Arabidopsis TIR-NBS-LRR protein CSA1 guards BAK1-BIR3 homeostasis and mediates convergence of pattern- and effector-induced immune responses. Cell Host Microbe 30:1717-1731.e6. DOI: 10.1016/j.chom.2022.11.001, PMID: 36446350

Wang Q, Zhao Y, Luo W, Li R, He Q, Fang X, Michele RD, Ast C, von Wirén N, Lin J. 2013. Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization. Proceedings of the National Academy of Sciences of the United States of America 110:13204-9. DOI: 10.1073/pnas.1301160110, PMID: 23882074

Eichel K, Jullié D, von Zastrow M. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nature Cell Biology 18:303-10. DOI: 10.1038/ncb3307, PMID: 26829388

Van Itallie CM, Anderson JM. Phosphorylation of tight junction transmembrane proteins: Many sites, much to do. Tissue Barriers 6:e1382671. DOI: 10.1080/21688370.2017.1382671, PMID: 29083946

Monje-Galvan V, Warburton L, Klauda JB. Setting up all-atom molecular dynamics simulations to study the interactions of peripheral membrane proteins with model lipid bilayers. Methods in Molecular Biology 1949:325-339. DOI: 10.1007/978-1-4939-9136-5_22, PMID: 30790265.

Trotta A, Bajwa AA, Mancini I, Paakkarinen V, Pribil M, Aro EM. The role of phosphorylation dynamics of CURVATURE THYLAKOID 1B in plant thylakoid membranes. Plant Physiology 181:1615-1631. DOI: 10.1104/pp.19.00942, PMID: 31615849

Dorrity MW, Saunders LM, Queitsch C, Fields S, Trapnell C. Dimensionality reduction by UMAP to visualize physical and genetic interactions. Nature Communications 11:1537. DOI: 10.1038/s41467-020-15351-4, PMID: 32210240

Sato KI, Tokmakov AA. Membrane microdomains as platform to study membrane-associated events during Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis. Methods in Molecular Biology 920:59-73. DOI: 10.1007/978-1-4939-9009-2_5, PMID: 30737686.

Ozolina NV, Kapustina IS, Gurina VV, Bobkova VA, Nurminsky VN. Role of plasmalemma microdomains (Rafts) in protection of the plant cell under Osmotic stress. Journal of Membrane Biology 254:429-439. DOI: 10.1007/s00232-021-00194-x, PMID: 34302495

Boutté Y, Moreau P. Plasma membrane partitioning: from macro-domains to new views on plasmodesmata. Frontiers in Plant Science 5:128. DOI: 10.3389/fpls.2014.00128. PMID: 24772114

Yu M, Cui Y, Zhang X, Li R, Lin J. Organization and dynamics of functional plant membrane microdomains. Cellular and Molecular Life Sciences 77:275-287. DOI: 10.1007/s00018-019-03270-7, PMID: 31422442

Zhao Z, Li M, Zhang H, Yu Y, Ma L, Wang W, Fan Y, Huang N, Wang X, Liu K, Dong S, Tang H, Wang J, Zhang H, Bao Y. Comparative proteomic analysis of plasma membrane proteins in rice leaves reveals a vesicle trafficking network in plant immunity that is provoked by Blast Fungi. Frontiers in Plant Science 13:853195. DOI: 10.3389/fpls.2022.853195, PMID: 35548300

Hilgemann DW, Dai G, Collins A, Lariccia V, Magi S, Deisl C, Fine M. Lipid signaling to membrane proteins: From second messengers to membrane domains and adapter-free endocytosis. Journal of General Physiology 150:211-224. DOI: 10.1085/jgp.201711875, PMID: 29326133

Joshi R, Paul M, Kumar A, Pandey D. Role of calreticulin in biotic and abiotic stress signalling and tolerance mechanisms in plants. Gene 714:144004. DOI: 10.1016/j.gene.2019.144004, PMID: 31351124

Chen Y, Cao C, Guo Z, Zhang Q, Li S, Zhang X, Gong J, Shen Y. Herbivore exposure alters ion fluxes and improves salt tolerance in a desert shrub. Plant Cell and Environment 43:400-419. DOI: 10.1111/pce.13662, PMID: 31674033

Chi Y, Wang C, Wang M, Wan D, Huang F, Jiang Z, Crawford BM, Vo-Dinh T, Yuan F, Wu F, Pei ZM. Flg22-induced Ca2+ increases undergo desensitization and resensitization. Plant Cell and Environment 44:3563-3575. DOI: 10.1111/pce.14186, PMID: 34536020

Zhang M, Su J, Zhang Y, Xu J, Zhang S. Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Current Opinion in Plant Biology 45:1-10. DOI: 10.1016/j.pbi.2018.04.012, PMID: 29753266

Arnaud D, Deeks MJ, Smirnoff N. RBOHF activates stomatal immunity by modulating both reactive oxygen species and apoplastic pH dynamics in Arabidopsis. Plant Journal 116:404-415. DOI: 10.1111/tpj.16380, PMID: 37421599

Zou Y, Wang S, Zhou Y, Bai J, Huang G, Liu X, Zhang Y, Tang D, Lu D. Transcriptional regulation of the immune receptor FLS2 controls the ontogeny of plant innate immunity. Plant Cell.30:2779-2794. DOI: 10.1105/tpc.18.00297, PMID: 30337428

Ngou BPM, Jones JDG, Ding P. Plant immune networks. Trends in Plant Science 27:255-273. DOI: 10.1016/j.tplants.2021.08.012, PMID: 34548213.

Yu M, Liu H, Dong Z, Xiao J, Su B, Fan L, Komis G, Šamaj J, Lin J, Li R. 2017. The dynamics and endocytosis of Flot1 protein in response to flg22 in Arabidopsis. Journal of Plant Physiology 215:73–84. DOI: https://doi.org/10.1016/j.jplph.2017.05.010, PMID: 28582732

Reviewer #2 (Public Review):

Summary:

The research conducted by Yaning Cui and colleagues delves into understanding FLS2-mediated immunity. This is achieved by comparing the spatiotemporal dynamics of a FLS2-S938A mutant and FLS2-WT, especially in relation to their association with the remorin protein. To delineate the differences between the FLS2-S938A mutant and FLS2-WT, they utilized a plethora of advanced fluorescent imaging techniques. By analyzing surface dynamics and interactions involving the receptor signal co-receptor BAK1 and remorin proteins, the authors propose a model of how FLS2 and BAK1 are assembled and positioned within a remorin-specific nano-enviroment during FLS2 ligand-induced immune responses.

Strengths:

These techniques offer direct visualizations of molecular dynamics and interactions, helping us understand their spatial relationships and interactions during innate immune responses.

Advanced cell biology imaging techniques are crucial for obtaining high-resolution insights into the intracellular dynamics of biomolecules. The demonstrated imaging systems are excellent examples to be used in studying plant immunity by integrating other functional assays.

Weaknesses:

It's essential to acknowledge that every fluorescence-based method, just like biochemical assays, comes with its unique limitations. These often pertain to spatial and temporal resolutions, as well as the sensitivity of the cameras employed in each setup. Meticulous interpretation is pivotal to guarantee an accurate depiction and to steer clear of potential misunderstandings when employing specific imaging systems to analyze molecular attributes. Moreover, a discerning interpretation and accurate image analysis can offer invaluable guidance for future studies on plant signaling molecules using these nice cell imaging techniques.

For instance, although single-particle analysis couldn't conclusively link FLS2 and remorin, FLIM-FRET effectively highlighted their ligand-triggered association and the disengagement brought on by mutations. While these methodologies seemed to present differing outcomes, they were described in the manuscript as harmonious. In reality, these differences could highlight distinct protein populations active in immune responses, each accentuated differently by the respective imaging techniques due to their individual spatial and temporal limitations. Addressing these variations is imperative, especially when designing future imaging explorations of immune complexes.

What are some good methods and practices someone can use to fully grasp the experience that comes with the process of doing something? It's so easy to get caught up in life and hyperfocus about the outcome for the things we want to do, rather than the process of getting there which can lead to not even getting there. So, how often should we check ourselves to make sure we are being and living in the full moment rather than just doing.

I think Brewer's opinion on this is very valid especially when it comes to education. I think even with being in college I find myself thinking more about what assignments I have and when I have to do them by to receive full credit rather than the assignment itself. It's in this that I lose sight of the knowledge I'm meant to gain, and it takes away from process of actually completing the assignment; setting aside intentional time, understanding what makes sense, what doesn't, asking all the questions, etc.

I find this point to be incredibly engaging because it shows how people are so easily manipulated into engaging with, and trying to attain, a number that is given intrinsic value. In the same way that people are pit against one another to get the highest number of capital in their bank account, attain the highest credit score, and kill the most zombies in a video game. Putting value onto numbers creates a trap for the people who engage with them. Our society of capitalism invites competition, and does not function without it. The easiest way to demonstrate competition in a tangible way is by associating a number with it, so, within schools, deans and administrations are prompted to offer an exact score to prove that you have won the competition. It's a game that we all compete in whether or not we actively work to participate in it.

This is absolutely an important aspect to consider for learners. When conducting a learner analysis, it's important to not just look at the surface level conditions of access (whether a student can or cannot access technology), but at the deeper dimensions of why. As the article points out, it is not as simple as "Student A does not have a computer." Student A may have a computer that she shares with her entire family, and Student B may have a computer that is old or faulty. Access issues are oftentimes more complex than the surface level, which this article does a wonderful job at pointing out.

Within this entire opening, Rabelais is basically saying that the reader should enjoy themselves and not be so serious and judgmental while reading. He is prefacing that he knows that these stories are filled with crude jokes, but that there is a deeper meaning behind them that people are too quick to judge. While reading other sources, I was able to understand more about how Rabelais was relaying deeper meanings in his work. He compares it to a monk, that on the outside they look like they should be respected, but on the inside it's the opposite. He is essentially telling the readers not to judge a book by its cover, or in this case to think beyond just the words he uses. https://itech.fgcu.edu/&/issues/vol2/issue2/rabelais.htm

My second language is Spanish, and I understand quite well what 'Libre' means. In my view, there's no real difference between FLOSS and FOSS, since 'Free' translates to 'Libre'. However, when 'Free' pertains solely to 'price', it's more accurate to use 'Gratis', which means 'No Cost'.

Additionally, considering that words can have multiple meanings in English as well, I question the necessity of incorporating an 'L' from another language into an English acronym. Instead of complicating the acronym, I believe in giving a clear explanation.

I found the explanation of variables in programming to be really helpful. The analogy of bowls in cooking made it easier to understand how information is stored and manipulated in a computer program. Just like you use different bowls for different mixtures in cooking, variables in programming hold different types of information that can be used later in the program. This comparison helped me grasp the concept of variables and their importance in programming. It's fascinating to see how programming concepts can be explained using everyday examples like cooking.

After a quick google search, I'm a little disappointed that so many Instagram bots are used for growth. I suppose there's nothing inherently wrong with that, but it's a bit sad that platforms that purport to be used for connection are just another place for business.

My favorite bots by far are the ones that post themed images regularly, whether it be cats, red pandas, or old videogame posters. The gender pay gap bot also looked pretty cool.

I can appreciate this because there are times that I just jot down information and then when I try to piece together my paper I get so lost. It's actually a very bad habit that I have. Being aware of where and how you organize your ideas is very important.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Dear Editor,

We have addressed the points and concerns raised by the reviewers and wish to thank them for their effort and time. We agree with all the comments and suggestions, which resulted in a significant improvement of the manuscript. Below, we provide a point-by-point response to all comments.

Sincerely,

Anders Hofer, corresponding author

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). The authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this work will be paramount aid in better understanding of the reaction mechanism, especially in the context of molecules which can be used as inhibitors of such a crucial enzyme (metabolic vulnerability for this parasite).

This manuscript, in its current form does not require additional experiments but I would like to have a few aspects corrected/clarified, before it can be accepted for publication:

Line 30: "whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower." Better not to use term "affinity" when KM or kcat/KM are implied (unless ITC was used to measure true Kds).

-This is a good point, and we are now using KM values in all instances were actual numbers are implied and only kept the word affinity in cases where it is discussed in more general terms.

Line 31: "Deoxyadenosine analogues halogenated at the 2- and/or 2´-positions were also potent substrates, with comparable EC50 values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites." Not sure this sentence is clear as written.

-We have now rewritten the sentence as follows: "Deoxyadenosine analogues halogenated at the 2- and/or 2´-positions were also potent substrates with comparable EC50 values on cultured G. intestinalis cells as metronidazole, the first line treatment today, with the additional advantage of being effective against metronidazole-resistant parasites."

Line 55: "..G. intestinalis (synonymous to G. lamblia and G. duodenalis)..". Very nice that authors provide this information as it is usually a point of confusion i.e. multiple names for the same organism.

-Thanks a lot, we are happy that you liked it.

Line 61 and above as well: "Treatment regimes are mainly based on metronidazole and to a lesser extent other 5-nitroimidazoles...". MT is introduced a bit sporadically, and not completely clear which enzyme it inhibits and its mode of action." Common knowledge is that MT is known for its action in aerobic parasites/bacteria and known as Flagyl, where it is mode of action was linked to "activation" due to microaerophilic conditions. Maybe MT can be introduced after text starting from Line 71?

-The description of metronidazole is adjusted as following: "Metronidazole (Flagyl) is the most commonly used drug to treat giardiasis and selectively kills the parasite and other anaerobic organisms by forming free radicals under oxygen-limited conditions, but it has side effects such as nausea, abdominal pain, diarrhea, and in some cases neurotoxicity reactions."

Line 70: what is "cyst-wall"?

-It is a cell wall consisting of three major cyst wall proteins and N-acetylgalactosamine. We have adjusted the sentence to the following to make the term clearer: The trophozoites can also secrete material to form a cyst wall and go through two rounds of DNA replication to form cysts, which contain four nuclei and a 16N genome per cell (4N in each nucleus).

Line 90: "The reaction is catalyzed by deoxyribonucleoside kinases (dNKs), which are.." I really do not like when in order to find a reaction which is catalyzed by an enzyme in a particular study one needs to dive into the literature, sometimes it requires a lot of time as in most of recent papers on the subject reactions catalyzed are not listed. Please add a Figure or a panel with reactions catalyzed by both dNKs families.

-It is a good idea and we have now added a figure (Fig. 1), which compares the deoxyribonucleotide metabolism of G. intestinalis with mammalian cells. The different deoxyribonucleoside kinases in the parasite and mammalian cells are included in the figure.

Line 96: "..was found to have a ~10-fold higher affinity to thymidine.." as I mentioned above I really do not like the usage of "affinity", when actually low KM is implied.

-It is corrected now (see above).

Line 113: "This does not match the current knowledge that there are three dNKs in total whereof one completely specific for thymidine. The lack of knowledge about these essential enzymes in the parasite has hampered the understanding of Giardia deoxyribonucleoside metabolism and hence its exploitation as a target for antiparasitic drugs." Very good rationale, as I mentioned above, I think a Figure needs to be introduced that depicts different enzymes involved in deoxyribonucleoside metabolism (both TK1 and non- TK1 members) in Giardia with clearly labeled all known paralogs and corresponding enzymatic reactions.

-Thanks a lot for the suggestion. Information about the different dNKs in G. intestinalis with mammalian cells for comparison is included in the new figure (Fig. 1).

Line 132: Odd designation of supplementary figures, usually it is "Fig. S1" etc. The legend for Fig. S1 is not adequate, please add description of species and name of enzymes for all sequences shown. Also each sequences in alignment should start with number (a.a. number) as it is not clear if a full sequence is shown or not. Overall comment about the multiple sequence alignment (relevant to Fig. S1): with such a small number of sequences it is very hard to make any substantial predictions about conserved regions etc.

-Thanks for the suggestions. We have now included more sequences, sequence numbering, and description of species as well as enzyme names. Some other changes are also that we have now used the same G. intestinalis dAK sequence in the alignment as in the experiments (same strain and accession number), and that we have made a realignment using Clustal W instead of Clustal Omega (gives better alignment of the termini). The designation of supplementary figures is according to the style of PLoS journals.

Fig. 1 and elsewhere: I will prefer that all bar graphs show individual values + the error bar (if possible);

-We have now added individual values to the bar graphs.

I do not have any issues with X-ray data and cryo-EM studies (refinement statistics, particles classification etc).

**Referees cross-commenting**

I also agree with all the comments provided by Reviewer 2 and very pleased to see that we were very similar in our evaluations.

Reviewer #1 (Significance (Required)):

In their paper Ranjbarian and colleagues provide a tour de force at characterizing dAK from G. intestinalis using both enzymology and structural biology. G. intestinalis does not have RNR and therefore this organism relies on dAK which catalyzes formation of dAMP and ADP from deoxyadenosine and ATP (among other substrate pairs). The authors performed a terrific job at testing this reaction in depth using a recombinant dAK and a battery of various co-substrates (both natural as well as synthetic ones, Table 1). Extensive structural information on dAK was obtained using a combination of X-ray crystallography and cryo-EM. Overall, this work will be paramount aid in better understanding of the reaction mechanism, especially in the context of molecules which can be used as inhibitors of such a crucial enzyme (metabolic vulnerability for this parasite).

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary:

Ranjbarian et al. investigated a non-TK1-Like deoxyribonucleoside kinase (dNK) found in the protozoan parasite Giardia intestinalis. They used enzyme kinetic assays on heterologously expressed Gi dNK in E. coli to determine which deoxyribonucleotides were most likely physiological substrates for the enzyme. Their characterization revealed that this Gi dNK has a strong affinity to deoxyadenosine. They further investigated the affinity and activity of the dNK on deoxyadenosine analogues, some of which have known pharmaceutical utility. Finally, using a combination of crystallography, cryo-EM, chromatography, and mass photometry, they reveal that unlike other dNKs, Gi dNK forms a tetramer. They characterize important regions required for tetramerization and postulate that this tetramerization evolved to provide Gi dNK with a heightened affinity for deoxyadenosine.

Major comments and questions:

• The claims in this manuscript are well-supported, and I found no major issues with experimental methods. • The authors provide a structure of tetrameric dNK and suggest that this tetramer leads to the increased affinity to substrate compared to non-giardia dNKs. They also show through mutations that removing the novel dimerization regions decreases substrate affinity by 100-fold. However, I was left unclear about why the tetramer would lead to such high affinity for substrate compared to two dimers. This is especially notable, since the authors state that there are no signs of cooperativity, which is a common way that oligomerization may lead to heightened affinity. If the authors have no current evidence explaining this, they can consider adding a short amount of discussion speculating on the mechanism and future directions of study. -Thanks for this suggestion. We have now added a section in the last paragraph of the discussion where we speculate on the subject.

Minor comments and questions:

• The authors state that dATP acts as a mixed inhibitor and not a simple competitive inhibitor, and that previous studies have shown that this is because the dNTP competes in two locations (line 163). Is it also possible that competitive inhibition + allosteric regulation could be causing this behavior instead? -It is true that this can be theoretically explained in many ways. In fact, many allosteric regulators affect both the Vmax and Km values. However, in all studied dNKs, the dNTP acts as a dual competitor and no proper allosteric regulation with a separate allosteric site has ever been observed so far. We have rephrased this part as following to make it clear: "Mixed inhibition is often the result of allosteric regulation but studies of other dNKs have shown that this is not the case [17]. Instead, the far-end dNTP product gives a dual inhibition where the deoxyribonucleoside moiety competes with the substrate and the phosphate groups mimic those of ATP but coming from the opposite direction."

• In the introduction (line 93), non-TK1-like dNKs are described as "not structurally related to TK1-like". This left me unclear, are they still interrelated among themselves? -We have added the following sentence for clarification: "The non-TK1-like dNKs are further subdivided into a monophyletic group of canonical non-TK1-like dNKs and a second group with thymidine kinases from Herpesviridae, which are structurally related to the canonical group but share very little amino acid sequence homology."

• I was left confused by lines 106-116 in the introduction, where the specificities of dNKs in giardia are discussed. This is touched upon again in the discussion, but it was not clear here that there are several deoxyribonucleotides unaccounted for. -We think this should be clear now with the added Fig. 1 where the dNKs are shown.

• When describing enzyme assays (Line 145), the authors say there is no salt dependence, but there looks to be MgCl2 always included in the assays (presumably for the ATP). -This is a good point and something we have overlooked when the sentence was written (Mg2+ is required). We have now corrected the sentence as follows: "Based on initial enzyme activity studies, it was confirmed that the assay did not have any specific requirements regarding K+, Na+, NH4+, acetate or reducing agents, and that it was linear with respect to time (S2 Fig)."

• I was confused by the y-axis of Fig 2. How is enzyme activity lower when dAdo is added? I think I read "enzyme activity" as total substrate depleted, when it is actually referring exclusively to the given non-dAdo substrate in each column. -This is a very good point that we seem to have overlooked. We have now adjusted the y-axis title to "Indicated enzyme activity" and added the following sentence to the figure legend: "The recorded enzyme activities are for the substrates indicated on the x-axis (excluding the activity with the competing substrate)."

• Lines 239 - 255 and Figure 3 were a little unclear to me. Specifically, I was having trouble following in the text which dimer is in the ASU, which is symmetry related, and matching those terms with which are canonical and non-canonical. -We agree with the reviewer and thank them for their comment. In order to improve the presentation of these results, we chose to extensively rearrange the figures and accompanying text. We now present the initial X-ray data together with the cryo-EM data in a new Figure 4 that focuses on the overall architecture of the tetramer. We realize that some of the nomenclature previously used in that figure was, as the reviewer pointed out, confusing and superfluous, and we have now simplified and unified it. The structural details of how the extended N- and C-termini interact with the neighboring subunit have been moved to the new figure 6 in order to present them just before the functional analyses of the consequences of truncating the termini. As a consequence of these changes to the figure layout, we made substantial changes in the organization of the text surrounding these figures, which also led to a clearer presentation. Since the changes to the figures and text are quite substantial, we would like to point out that they are only changes to the presentation, not to the data shown.

• The authors suggest that in the experiment shown in Figure S9 (Line 285), low activity may be caused by minor impurities. I'm not sure why impurities would lower activity significantly. Could there be other differences in experimental conditions that are at play instead? -The sentence refers to a side activity (dATP dephosphorylation) which is not the normal reaction of deoxyadenosine kinase. We have rephrased the sentence to make it clearer: "The dATP-dephosphorylating activity was several orders of magnitude lower than the regular dAK activity (to phosphorylate deoxyadenosine) and was possibly catalyzed by other enzymes present as minor impurities in the protein preparation."

• (Optional) From looking at the crystallography stats, I think the authors can potentially push the resolution more. At higher resolutions, Rmerge may become high, but depending on the data collection strategy, Eiger detectors can lead to high Rmerge just out of sheer data redundancy. Cc 1/2 can be a more useful metric in these contexts. -This is a good point and well spotted by the reviewer. Indeed, a CC1/2 of 0.802 suggests that the resolution can be pushed further. However, due to contaminating spots at higher resolutions the statistics significantly worsen when trying to push the resolution beyond 2.1 A, which is why we did not process the data to a higher resolution.

• For Figure S8, the Polder map feature in Phenix is another option for showing ligand occupancy in an unbiased way. Did the authors try this? -We want to thank the reviewer for suggesting this. We have calculated a polder map using the Polder map feature in Phenix and both the resulting map and correlation coefficients support the presence of a dADP in the active site of monomer I. We added a section to the relative paragraph to include these new findings: "To increase our confidence that dADP was correctly placed within active site I, we calculated a polder map for dADP to test whether the b-phosphate density is correctly attributed or if it rather belongs to the bulk solvent. The resulting polder map and statistics support the placement of dADP in active site I with correlation coefficients of CC1,2=0.7627, CC1,3=0.9424, and CC2,3=0.7423 suggesting that the density does belong to dADP as CC1,3 > CC1,2 and CC1,3 > CC2,3 (S8 Fig.)."

• It's disappointing that the tetramers show so much preferred orientation in the cryo-EM. With that said, while the nominal resolution is 4.8 Å, I think that with the streakiness the EM structure looks to have worse resolution than that. -We agree that the streakiness of the map is substantial. This is simply a result of the severe anisotropy of the map, which means that the resolution is probably worse than 4.8 Å in the "bad directions" of the map. The supplementary material (S9 Fig) clearly shows the preferred orientations leading to this problem. In the course of this study, we tried several methods to lessen the preferred orientation problem such as using graphene oxide-coated grids and collecting tilted data. However, when we got the crystal structure we saw no point in continuing these efforts. To address the comment of the reviewer, we extended the description of the EM map in the main text to say:

"Due to strong preferred orientations, it was not possible to get an isotropic, high-resolution 3D structure of dAK using cryo-EM. The resulting 3D map had a nominal resolution of 4.8 Å, but a clearly anisotropic appearance probably reflecting lower resolution in the poorly resolved direction (S9 Fig)."

**Referees cross-commenting**

Overall, I agree with Reviewer #1's evaluation, and don't have any further suggestions or thoughts at this time.

Reviewer #2 (Significance (Required)):

Medical relevance: G. intestinalis is a parasite that causes 190 million cases of giardiasis per year. While treatable, there is evidence that giardia are developing a resistance to the main treatment at the moment, metronidazole. Thus, the authors provide a compelling case for the medical relevance of their investigation of Gi dNK for further pharmaceutical development. They provide further evidence for this by showing that several deoxyadenosine analogs bind the dNK and inhibit giardia growth. This work represents a very useful first step into a potential avenue for medical development. It's important to note that clinical studies are not within the purview of this research. However, in the discussion, the authors provide several comments on the promise of this avenue for future research.

Conceptual, technical, and mechanistic relevance: Through biochemical and structural study, the authors provide a compelling framework to understand an enzyme that is very important to the unique lifestyle of giardia parasites. From an evolutionary standpoint, the authors provide insight into how giardia can survive even without major components of de novo DNA synthesis. The authors principally use well-established tools and techniques of the enzymology field. but do so to characterize a unique and previously uncharacterized enzyme system. This enzyme proves to be notable not just for its medical significance, but because it is unique among its family (non-TK1-like deoxynucleotide kinases) in its strong affinity for substrate and tetrameric quaternary structure. One relatively novel technique used in the study is mass photometry, which is a relatively new and exciting way to characterize native proteins at very low concentrations. Using this technique helps the authors overcome a common criticism of structural studies in which the high concentrations or crowding conditions of techniques like crystallography and cryo-EM may be inducing non-physiological oligomers.

In summary, this work represents a meaningful addition to the protein structure-function literature. While it will principally be of interest to basic/fundamental researchers who study the mechanistic detail of protein function and evolution, it also provides a foundation for future translational work and antiparasitic drug design.

Reviewer's background: I received my PhD in chemistry studying the structure and function of another enzyme key to DNA metabolism (except in giardia), ribonucleotide reductase. My background is in structural biology and biochemistry. I do not have sufficient expertise to comment on studies performed on G. intestinalis growth and susceptibility to deoxyadenosine analogs.

• *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary:

Ranjbarian et al. investigated a non-TK1-Like deoxyribonucleoside kinase (dNK) found in the protozoan parasite Giardia intestinalis. They used enzyme kinetic assays on heterologously ex-pressed Gi dNK in E. coli to determine which deoxyribonucleotides were most likely physiological substrates for the enzyme. Their characterization revealed that this Gi dNK has a strong affinity to deoxyadenosine. They further investigated the affinity and activity of the dNK on deoxyadenosine analogues, some of which have known pharmaceutical utility. Finally, using a combination of crys-tallography, cryo-EM, chromatography, and mass photometry, they reveal that unlike other dNKs, Gi dNK forms a tetramer. They characterize important regions required for tetramerization and pos-tulate that this tetramerization evolved to provide Gi dNK with a heightened affinity for deoxy-adenosine.

Major comments and questions:

• The claims in this manuscript are well-supported, and I found no major issues with experi-mental methods.
• The authors provide a structure of tetrameric dNK and suggest that this tetramer leads to the increased affinity to substrate compared to non-giardia dNKs. They also show through mu-tations that removing the novel dimerization regions decreases substrate affinity by 100-fold. However, I was left unclear about why the tetramer would lead to such high affinity for substrate compared to two dimers. This is especially notable, since the authors state that there are no signs of cooperativity, which is a common way that oligomerization may lead to heightened affinity. If the authors have no current evidence explaining this, they can con-sider adding a short amount of discussion speculating on the mechanism and future direc-tions of study.

Minor comments and questions:

• The authors state that dATP acts as a mixed inhibitor and not a simple competitive inhibitor, and that previous studies have shown that this is because the dNTP competes in two loca-tions (line 163). Is it also possible that competitive inhibition + allosteric regulation could be causing this behavior instead?
• In the introduction (line 93), non-TK1-like dNKs are described as "not structurally related to TK1-like". This left me unclear, are they still interrelated among themselves?
• I was left confused by lines 106-116 in the introduction, where the specificities of dNKs in giardia are discussed. This is touched upon again in the discussion, but it was not clear here that there are several deoxyribonucleotides unaccounted for.
• When describing enzyme assays (Line 145), the authors say there is no salt dependence, but there looks to be MgCl2 always included in the assays (presumably for the ATP).
• I was confused by the y-axis of Fig 2. How is enzyme activity lower when dAdo is added? I think I read "enzyme activity" as total substrate depleted, when it is actually referring ex-clusively to the given non-dAdo substrate in each column.
• Lines 239 - 255 and Figure 3 were a little unclear to me. Specifically, I was having trouble following in the text which dimer is in the ASU, which is symmetry related, and matching those terms with which are canonical and non-canonical.
• The authors suggest that in the experiment shown in Figure S9 (Line 285), low activity may be caused by minor impurities. I'm not sure why impurities would lower activity sig-nificantly. Could there be other differences in experimental conditions that are at play in-stead?
• (Optional) From looking at the crystallography stats, I think the authors can potentially push the resolution more. At higher resolutions, Rmerge may become high, but depending on the data collection strategy, Eiger detectors can lead to high Rmerge just out of sheer data redundancy. Cc 1/2 can be a more useful metric in these contexts.
• For Figure S8, the Polder map feature in Phenix are another option for showing ligand oc-cupancy in an unbiased way. Did the authors try this?
• It's disappointing that the tetramers show so much preferred orientation in the cryo-EM. With that said, while the nominal resolution is 4.8 Å, I think that with the streakiness the EM structure looks worse resolution than that.

Referees cross-commenting

Overall, I agree with Reviewer #1's evaluation, and don't have any further suggestions or thoughts at this time.

Significance

Medical relevance: G. intestinalis is a parasite that causes 190 million cases of giardiasis per year. While treatable, there is evidence that giardia are developing a resistance to the main treatment at the moment, metronidazole. Thus, the authors provide a compelling case for the medical relevance of their investigation of Gi dNK for further pharmaceutical development. They provide further evi-dence for this by showing that several deoxyadenosine analogs bind the dNK and inhibit giardia growth. This work represents a very useful first step into a potential avenue for medical develop-ment. It's important to note that clinical studies are not within the purview of this research. Howev-er, in the discussion, the authors provide several comments on the promise of this avenue for future research.

Conceptual, technical, and mechanistic relevance: Through biochemical and structural study, the authors provide a compelling framework to understand an enzyme that is very important to the unique lifestyle of giardia parasites. From an evolutionary standpoint, the authors provide insight into how giardia can survive even without major components of de novo DNA synthesis.

The authors principally use well-established tools and techniques of the enzymology field. but do so to characterize a unique and previously uncharacterized enzyme system. This enzyme proves to be notable not just for its medical significance, but because it is unique among its family (non-TK1-like deoxynucleotide kinases) in its strong affinity for substrate and tetrameric quater-nary structure. One relatively novel technique used in the study is mass photometry, which is a relatively new and exciting way to characterize native proteins at very low concentrations. Using this technique helps the authors overcome a common criticism of structural studies in which the high concentrations or crowding conditions of techniques like crystallography and cryo-EM may be inducing non-physiological oligomers.

In summary, this work represents a meaningful addition to the protein structure-function literature. While it will principally be of interest to basic/fundamental researchers who study the mechanistic detail of protein function and evolution, it also provides a foundation for future transla-tional work and antiparasitic drug design.

Reviewer's background: I received my PhD in chemistry studying the structure and function of another enzyme key to DNA metabolism (except in giardia), ribonucleotide reductase. My back-ground is in structural biology and biochemistry. I do not have sufficient expertise to comment on studies performed on G. intestinalis growth and susceptibility to deoxyadenosine analogs.

Author Response

eLife assessment

This important study provides a new, apparently high-performance algorithm for B cell clonal family inference. The new algorithm is highly innovative and based on a rigorous probabilistic analysis of the relevant biological processes and their imprint on the resulting sequences, however, the strength of evidence regarding the algorithm's performance is incomplete, due to (1) a lack of clarity regarding how different data sets were used for different steps during algorithm development and validation, resulting in concerns of circularity, (2) a lack of detail regarding the settings for competitor programs during benchmarking, and (3) method development, data simulation for method validation, and empirical analyses all based on the B cell repertoire of a single subject. With clarity around these issues and application to a more diverse set of real samples, this paper could be fundamental to immunologists and important to any researcher or clinician utilizing B cell receptor repertoires in their field (e.g., cancer immunology).

We apologize for the long delay in implementing the suggested changes. Some of the co-authors had some personal issues that made it hard to efficiently work on the revision.

We have addressed all the essential points below, as well as all the detailed comments of each reviewer in the following pages.

Due to the journal’s guidelines we have to upload an “all black” version of the manuscript as the main version. We have uploaded a revised manuscript with the changes marked in red as a “Related Manuscript file”, which appears at the very end of the Merged Manuscript File, after all the Figures, and at the end of the list of files on the webpage. We apologize for this inconvenience.

In addition, we have added an extension of HILARy to deal with paired-chain repertoires, and have benchmarked the new method on a recently published synthetic dataset. This new analysis is now presented in new Fig. 5.

Reviewer #1 (Public Review):

Identifying individual BCR/Ab chain sequences that are members of the same clone is a longstanding problem in the analysis of BCR/Ab repertoire sequencing data. The authors propose a new method designed to be scalable for application to huge repertoire data sets without sacrificing accuracy. Their approach utilizes Hamming Distance between CDR3 sequences followed by clustering for a fast, high-precision approach to classifying pairs of sequences as related or not, and then refines the classification using mutation information from germline-encoded regions. They compare their method with other state-of-the-art methods using synthetic data.

The authors address an important problem in an interesting, innovative, and rigorous way, using probabilistic representations of CDR3 differences, frequencies of shared and not-shared mutations, and the relationships between the two under hypotheses of related pairs and unrelated pairs, and from these develop an approach for determining thresholds for classification and lineage assignment. Benchmarking shows that the proposed method, the complete method including both steps, outperforms other methods.

Strengths of the method include its theoretical underpinnings which are consistent with an immunologist's intuition about how related and unrelated sequences would compare with each other in terms of the metrics to use and how those metrics are related to each other.

I have two high-level concerns:

(1) It isn't clear how the real and synthetic data are being used to estimate parameters for the classifier and evaluate the classifier to avoid circularity. It seems like the approach is used to assign lineages in the data from [1], and then properties of this set of lineages are used to estimate parameters that are then used to refine the approach and generate synthetic data that is used to evaluate the approach. This may not be a problem with the approach but rather with its presentation, but it isn't entirely clear what data is being used and where for what purpose. An understanding of this is necessary in order to truly evaluate the method and results.

The reviewer is correct in their understanding of the pipeline. It should be stressed that the lineages used to guide the generation of the synthetic data was done on VJl classes for which the clustering was easy and reliable, and should therefore be largely model independent.

We have added an explanation in the main text of why the re-use of real data lineages inferred by HILARy doesn’t bias the procedure, since it’s done on a subset of lineages within VJl classes that are easy to infer (section “Test on synthetic dataset”).

(2) Regarding the data used for benchmarking - given the intertwined fashion by which the classification approach and synthetic data generation approach appear to have been developed, it is not surprising that the proposed approach outperforms the other methods when evaluated on the synthetic data presented here. It would be better to include in the benchmark the data used by the other methods to benchmark themselves or also generate synthetic data using their data generation procedures.

We agree with the reviewer that a test of the method on an independent synthetic dataset is important for its applicability and to compare to other methods.

We have added a new synthetic dataset from the group that designed the partis method to our benchmark. Our method still performs competitively, on par with partis—which was developed and tested on that dataset—and better than other methods. The results are presented in revised Fig. 4 (panels E-G), and Figure 4–figure supplement 1 as a function of the mutation rate.

In addition, we have used that dataset to benchmark a new version of HILARy that also uses the light chain. We present the results in new Figures 5 and Figure 4–figure supplement 1.

An improved method for BCR/Ab sequence lineage assignment would be a methodologic advancement that would enable more rigorous analyses of BCR/Ab repertoires across many fields, including infectious disease, cancer, autoimmune disease, etc., and in turn, enable advancement in our understanding of humoral immune responses. The methods would have utility to a broad community of researchers.

Reviewer #2 (Public Review):

This manuscript describes a new algorithm for clonal family inference based on V and J gene identity, sequence divergence in the CDR3 region, and shared mutations outside the CDR3. Specifically, the algorithm starts by grouping sequences that have the same V and J genes and the same CDR3 length. It then performs single-linkage clustering on these groups based on CDR3 Hamming distance, then further refines these groups based on shared mutations.

Although there are a number of algorithms that use a similar overall strategy, a couple of aspects make this work unique. First, a persistent challenge for algorithms such as this one is how to set a cutoff for single-linkage clustering: if it is too low, then one separates clusters that should be together, and if too high one joins together clusters that should be separate. Here the authors leverage a rich collection of probabilistic tools to make an optimal choice. Specifically, they model the probability distributions of within- and between-cluster CDR3 Hamming distances, with parameters depending on CDR3 length and the "prevalence" of clonal sequence pairs (i.e. family size distribution). This allows the algorithm to make optimal choices for separating clusters, given the particular chosen distance metric, and assuming the sample in question has been accurately modeled. Second, the algorithm uses a highly efficient means of doing single-linkage clustering on nucleotide sequences.

This leads to a fast and highly performant algorithm on data meant to replicate the original sample used in algorithm design. The ideas are new and beautifully developed. The application to real data is interesting, especially the point about dN/dS.

However, the paper leaves open the question of how this inference algorithm works on samples other than the one used for simulation and as a template for validation. If I understand the simulation procedure correctly - that one takes a collection of inferred trees from the real data, then re-draws the root sequence and the identity of the mutations on the branches - then the simulated data should be very close to the data used to develop the methods in the paper. This consideration seems especially important given that key methods in this paper use mutation counts and overall mutation counts are preserved.

Repertoires come in all shapes and sizes: infants to adults, healthy to cancerous, and naive to memory to plasma-cell-just-after-vaccination. If this is being proposed as a general-purpose clonal inference algorithm rather than one just for this sample, then a more diverse set of validations are needed.

We agree that testing the method on a differently generated dataset is a useful check. We should point out, however, that our synthetic dataset is not as biased as it may seem. In particular, it is based on trees from VJl classes that we predicted are very easy to cluster, which means that they are truly faithful to the data, and not dependent on the particular algorithm used to infer them. The big advantage over this synthetic dataset over others is that it recapitulates the power law statistics of clone size distribution, as well as the diversity of mutation rates. To us, it still represents a more useful benchmark than synthetic datasets generated by population genetics models, which miss most of this very broad variability.

However, to check how the method generalizes to other datasets, we repeated our validation procedure on the dataset used to evaluate Partis in Ralph et al 2022. The new results are discussed in the main text and in new panels of Fig. 4 in the same form as the previous comparisons. We also added a comparison of performance as a function of mutation rate in the new Figure 4–figure supplement 1.

It is unclear how to run the code. The software repo has a nice readme explaining the file layout, dependencies, and input file format, but the repo seems to be lacking an inference.ipynb mentioned there which runs an analysis. Perhaps this is a typo and refers to inference.py, which in addition to the documented cdr3 clustering, seems to have functions to run both clustering methods. However, it does not seem to have any documentation or help messages about how to run these functions.

We have completely overhauled the github to provide a detailed step by step explanation of how to run the code. The code is now easily installable using pip.

The results are not currently reproducible, because the simulated data is not available. The data availability statement says that no data have been generated for this manuscript, however simulated data has been generated, and that is a key aspect of the analysis in the paper.

We have uploaded the simulated data to zenodo, as well as provided scripts in the github to run the benchmarks.

More detail is needed to understand the timing comparisons. The new software is clearly written to use many threads. Were the other software packages run using multiple threads? What type of machine was used for the benchmarks?

All timing comparisons were made based on a single VJl class on a 14 double-threaded CPU computer. HILARy uses all 28 threads, and other methods were run with default settings, with multi-threading allowed.

We have clarified the specifications of the computer.

Reviewer #3 (Public Review):

B cell receptors are produced through a combination of random V(D)J recombination and somatic hypermutation. Identifying clonal lineages - cells that descend from a common V(D)J rearrangement - is an important part of B cell repertoire analysis. Here, the authors developed a new method to identify clonal lineages from BCR data. This method builds off of prior advances in the field and uses both an adaptive clonal distance threshold and shared somatic hypermutation information to group B cells into clonal lineages.

The major strength of this paper is its thorough quantitative treatment of the subject and integration of multiple improvements into the clonal clustering process. By their simulation results, the method is both highly efficient and accurate.

The only notable weakness we identified is that much of the impact of the method will depend on its superiority to existing approaches, and this is not convincingly demonstrated by Fig. 4. In particular, little detail is given on how the other clonal clustering programs were run, and this can significantly impact their performance. More specifically:

We have added a new benchmark to address these concerns, presented in Fig. 4 and in new figure 4 – figure supplement 1 as a function of a controllable mutation rate.

(1) Scoper supports multiple methods for clonal clustering, including both adaptive CDR3 distance thresholds (Nouri and Kleinstein, 2018) and shared V-gene mutations (Nouri and Kleinstein, 2020). It is not clear which method was used for benchmarking. The specific functions and settings used should have been detailed and justified. Spectral clustering with shared V gene mutations would be the most comparable to the authors' method. Similar detail is needed for partis.

In the updated version I use the 2020 version. The 2018 is very similar to simple single linkage so will be removed from the benchmark.

(2) It is not clear how the adaptive thresholds and shared mutation analysis in the authors' method differ from prior approaches such as scoper and partis.

We have changed the paragraph in the discussion section about the benchmark to highlight the innovative aspects and differences with previous approaches.

(3) The scripts for performing benchmarking analyses, as well as the version numbers of programs tested, are not available.

We have added to the github all the scripts used for benchmarking. We have added details about the version numbers in the data and code availability section of the methods.

(4) Similar to above, P. 10 describes single linkage hierarchical clustering with a fixed threshold as a "crude method" that "suffers from inaccuracy as it loses precision in the case of highlymutated sequences and junctions of short length." As far as we could tell, this statement is not backed up by either citations or analyses in the paper. It should not be difficult for the authors to test this though using their simulations, as this method is also implemented in scoper.

We have added this method to our benchmark to support that point. The results are presented in Figure 4 – figure supplement 2.

References

Nouri N, Kleinstein SH. 2020. Somatic hypermutation analysis for improved identification of B cell clonal families from next-generation sequencing data. PLOS Comput Biol 16:e1007977. doi:10.1371/journal.pcbi.1007977

Nouri N, Kleinstein SH. 2018. A spectral clustering-based method for identifying clones from high- throughput B cell repertoire sequencing data. Bioinformatics 34:i341-i349. doi:10.1093/bioinformatics/bty235

We have changed citation [22] to refer to the 2018 paper. The 2020 paper is citation [18].

I think currently most people are spending too much time on social media and paying too much attentions to the photo they are posting. I agree that people tends to posting their good news and photos and I believe it can lead to a skewed perception of reality, where it seems like everyone else is always having a great time, looking perfect, and living their best lives. But it's important for us to remember that the post is just a small piece of their lives and there's more behind the scene.

Nowadays, people from various backgrounds use social media for different reasons. Some may share content to document their lives, while others may post to attract attention and satisfy themselves. In this seamless integration of real life and the internet, I believe it's important to maintain our own healthy mindset and standards. This phrase may also implies that when you encounter internet content that triggers negative emotions, just leave it alone.

Taoism in the ethics world is pretty cool, especially when it comes to social media and coding. It's all about going with the flow and not forcing things to happen, which is honestly quite refreshing. Unlike Confucianism, which emphasizes rituals and ceremonies, Taoism says, "Let's just see what happens," and I like that. It's like being told to take it easy and not push too hard because everything will work out in its own time. And the idea of being like water—soft and flexible but powerful enough to wear down rock—really resonates. It's a good reminder that being adaptable and not overly rigid can help you get further, especially in the tech world where everything changes at the speed of light. Thinking about coding or designing social media content with this mindset may result in more natural, user-friendly outcomes. Instead of simply doing things the same way they've always been done, the goal is to find a more organic, less forced way to make cool things happen.

I think the Taoist view is more in line with my inner thoughts. "Trying to force something to happen can be counterproductive" and I'm sure it's not just me, a lot of people feel that way. The more you try to do something right, the more likely you tend to make mistakes. I often do tell myself to focus and be calm, but the more I think about it the harder it is to get into the zone.

Author Response

The following is the authors’ response to the original reviews.

This study reports important evidence that infants' internal factors guide children's attention and that caregivers respond to infants' attentional shifts during caregiver-infant interactions. The authors analyzed EEG data and multiple types of behaviors using solid methodologies that can guide future studies of neural responses during social interaction in infants. However, the analysis is incomplete, as several methodological choices need more adequate justification.

Reviewer #1

Public Review:

The authors bring together multiple study methods (brain recordings with EEG and behavioral coding of infant and caregiver looking, and caregiver vocal changes) to understand social processes involved in infant attention. They test different hypotheses on whether caregivers scaffold attention by structuring a child's behavior, versus whether the child's attention is guided by internal factors and caregivers then respond to infants' attentional shifts. They conclude that internal processes (as measured by brain activation preceding looking) control infants' attention, and that caregivers rapidly modify their behaviors in response to changes in infant attention.

The study is meticulously documented, with cutting-edge analytic approaches to testing alternative models; this type of work provides a careful and well-documented guide for how to conduct studies and process and analyze data for researchers in the relatively new area of neural response in infants in social contexts.

We are very pleased that R1 considers our work an important contribution to this developing field, and we hope that we have now addressed their concerns below.

Some concerns arise around the use of terms (for example, an infant may "look" at an object, but that does not mean the infant is actually "attending); collapsing of different types of looks (to people and objects), and the averaging of data across infants that may mask some of the individual patterns.

We thank the reviewer for this feedback and their related comments below, and we feel that our manuscript is much stronger as a result of the changes we have made. Please see blow for a detailed description of our rationale for defining and analysing the attention data, as well as the textual changes made in response to the author’s comments.

Recommendations For The Authors

This paper is rigorous in method, theoretically grounded, and makes an important contribution to understanding processes of infant attention, brain activity, and the reciprocal temporal features of caregiver-infant interactions. The alternative hypothesis approach sets up the questions well (although authors should temper any wording that suggests attention processes are one or the other. That is, certain bouts of infant attention can be guided by exogenous factors such as social input, and others be endogenous; so averaging across all bouts can actually mask the variation in these patterns). I appreciated the focus on multiple types of behavior (e.g., gaze, vocal fluctuations in maternal speech); the emphasis on contingent responding; and the very clear summaries of takeaways after each section. Furthermore, methods and analyses are well described, details on data processing and so on are very thorough, and visualizations aptly facilitate data interpretation. However, I am not an expert on infant neural responses in EEG and assume that a reviewer with such expertise will weigh in on the treatment and quality of the data; therefore, my comments should be interpreted in light of this lack of knowledge.

We thank R1 for these very positive and insightful comments on our analyses which are the result of a number of years of methodological and technical developmental work.

We do agree with R1 that we should more carefully word parts of our argument in the Introduction to make clear the fact that shifts in infant attention could be driven by a combination of interactive and endogenous influences. As a result of this comment, we have made direct changes to parts of the Introduction; removing any wording that suggests that these processes are ‘alternative’ or ‘separate’, and our overall aim states: ‘Here, recording EEG from infants during naturalistic interactions with their caregiver, we examined the (inter)-dependent influences of infants’ endogenous oscillatory neural activity, and inter-dyadic behavioural contingencies in organising infant attention’.

Examining variability between infant attention episodes in the factors that influence the length and timing of the attention episode is an important area for future investigation. We now include a discussion on this on page 38 of the Discussion section, with suggestions for how this could be examined. Investigating different subtypes of infant attention is methodologically challenging, given the number of infant behaviours that would need to inform such an analysis- all of which are time consuming to code. Developing automated methods for performing these kinds of analyses is an important avenue for future work.

Here, I review various issues that require revision or elaboration based on my reading of what I consider to otherwise be a solid and important research paper.

Problem in the use of the term attention scaffolding. Although there may be literature precedent in the use of this term, it is problematic to narrowly define scaffolding as mother-initiated guidance of attention. A mother who responds to infant behaviors, but expands on the topic or supports continued attention, and so on, is scaffolding learning to a higher level. I would think about a different term because it currently implies a caregiver as either scaffolding OR responding contingently. It is not an either-or situation in conceptual meaning. In fact, research on social contingency (or contingent responsiveness), often views the follow-in responding as a way to scaffold learning in an infant.

Yes, we agree with R1 that the term ‘attention scaffolding’ could be confusing given the use of this term in previous work conducted with children and their caregivers in problem-solving tasks, that emphasise modulations in caregiver behaviour as a function of infant behaviour. As a result of this suggestion, we have made direct edits to the text throughout, replacing the term attentional scaffold with terms such as ‘organise’ and ‘structure’ in relation to the caregiver-leading or ‘didactic’ perspective, and terms such as ‘contingent responding’ and ‘dynamic modulation’ in relation to the caregiver-following perspective. We feel that this has much improved the clarity of the argument in the Introduction and Discussion sections.

Do individual data support the group average trends? My concern with unobservable (by definition) is that EEG data averages may mask what's going on in individual brain response. Effects appear to be small as well, which occurs in such conditions of averaging across perhaps very variable response patterns. In the interest of full transparency and open science, how many infants show the type of pattern revealed by the average graph (e.g., do neural markers of infant engagement forward predict attention for all babies? Majority?). Non-parametric tests on how many babies show a claimed pattern would offer the litmus test of significance on whether the phenomenon is robust across infants or pulled by a few infants with certain patterns of data. Ditto for all data. This would bolster my confidence in the summaries of what is going on in the infant brain. (The same applies as I suggest to attention bouts. To what extent does the forward-predict or backward-predict pattern work for all bouts, only some bouts, etc.?). I recognize that to obtain power, summaries are needed across infants and bouts, but I want to know if what's being observed is systematic.

We thank R1 for this comment and understand their concern that the overall pattern of findings reported in relation to the infants’ EEG data might obscure inter-individual variability in the associations between attention and theta power. Averaging across individual participant EEG responses is, however, the gold standard way to perform both event-locked (Jones et al., 2020) and continuous methods (Attaheri et al., 2020) of EEG analysis that are reported in the current manuscript. EEG data, and, in particular, naturalistic EEG data is inherently noisy, and averaging across participants increases the signal to noise ratio (i.e. inconsistent, and, therefore, non-task-related activity is averaged out of the response (Cohen, 2014; Noreika et al., 2020)). Examining individual EEG responses is unlikely to tell us anything meaningful, given that, if a response is not found for a particular participant, then it could be that the response is not present for that participant, or that it is present, but the EEG recording for that participant is too noisy to show the effect. Computing group-level effects, as is most common in all neuroimaging analyses, is, therefore, most optimal to examining our main research questions.

The findings reported in this analysis also replicate previous work conducted by our lab which showed that infant attention to objects significantly forward-predicted increases in infant theta activity during joint table-top play with their caregiver, involving one toy object (compared to our paradigm which involved 3;Wass et al., 2018). More recent work conducted by our lab has also shown continuous and time-locked associations between infant look durations and infant theta activity when infants play with objects on their own (Perapoch Amadó et al., 2023). To reassure readers of the replicability of the current findings, we now reference the Wass et al. (2018) study at the beginning of the Discussion section.

Could activity artifacts lead to certain reported trends? Babies typically look at an object before they touch or manipulate the object, and so longer bouts of attention likely involve a look and then a touch for lengthier time frames. If active involvement with an object (touching for example) amplifies theta activity, that may explain why attention duration forward predicts theta power. That is, baby looks, then touches, then theta activates, and coding would show visual gaze preceding the theta activation. Careful alignment of infants' touches and other such behaviors with the theta peak might help address this question, again to lend confidence to the robustness of the interpretation.

Yes, again this is a very important point, and the removal of movement-related artifact is something we have given careful attention to in the analysis of our naturalistic EEG data (Georgieva et al., 2020; Marriott Haresign et al., 2021). As a result of this comment we have made direct changes to the Results section on page 18 to more clearly signal the reader to our EEG pre-processing section before presenting the results of the cross-correlation analyses.

As we describe in the Methods section of the main text, movement-related artifacts are removed from the data with ICA decomposition, utilising an automatic-rejection algorithm, specially designed for work with our naturalistic EEG data (Marriott Haresign et al., 2021). Given that ICA rejection does not remove all artifact introduced to the EEG signal, additional analysis steps were taken to reduce the possibility that movement artifacts influenced the results of the reported analyses. As explained in the Methods section, rather than absolute theta power, relative theta was used in all EEG analyses, computed by dividing the power at each theta frequency by the summed power across all frequencies. Eye and head movement-related artifacts most often associate with broadband increases in power in the EEG signal (Cohen, 2014): computing relative theta activity therefore further reduces the potential influence of artifact on the EEG signal.

It is also important to highlight that previous work examining movement artifacts in controlled paradigms with infants has shown that limb movements actually associate with a decrease in power at theta frequencies, compared to rest (Georgieva et al., 2020). It is therefore unlikely that limb movement artifacts explain the pattern of association observed between theta power and infant attention in the current study.

That said, examining the association between body movements and fluctuations in EEG activity during naturalistic interactions is an important next step, and something our lab is currently working on. Given that touching an object is most often the end-state of a larger body movement, aligning the EEG signal to the onset of infant touch is not all that informative to understanding how body movements associate with increases and decreases in power in the EEG signal. Our lab is currently working on developing new methods using motion tracking software and arousal composites to understand how data-derived behavioural sub-types associate with differential patterns of EEG activity.

The term attention may be misleading. The behavior being examined is infant gaze or looks, with the assumption that gaze is a marker of "attention". The authors are aware that gaze can be a blank stare that doesn't reflect underlying true "attention". I recommend substitution of a conservative, more precise term that captures the variable being measured (gaze); it would then be fine to state that in their interpretation, gaze taken as a marker for attention or something like that. At minimum, using term "visual attention" can be a solution if authors do not want to use the precise term gaze. As an example, the sentence "An attention episode was defined as a discrete period of attention towards one of the play objects on the table, or to the partner" should be modified to defined as looking at a play object or partner.

We thank the reviewer for this comment, and we understand their concern with the use of the term ‘attention’ where we are referring to shifts in infant eye gaze. However, the use of this term to describe patterns of infant gaze, irrespective of whether they are ‘actually attending’ or not is used widely in the literature, in both interactive (e.g. Yu et al., 2021) and screen-based experiments examining infant attention (Richards, 2010). We therefore feel that its use in our current manuscript is acceptable and consistent with the reporting of similar interaction findings. On page 39 of the Discussion we now also include a discussion on how future research might further investigate differential subtypes of infant looks to distinguish between moments where infants are attending vs. just looking.

Why collapse across gaze to object vs. other? Conceptually, it's unclear why the same hypotheses and research questions on neural-attention (i.e., gaze in actuality) links would apply to looks to a mom's face or to an object. Some rationale would be useful to the reader as to why these two distinct behaviors are taken as following the same principles in ordering of brain and behavior. Perhaps I missed something, however, because later in the Discussion the authors state that "fluctuations in neural markers of infants' engagement or interest forward-predict their attentiveness towards objects", which suggests there was an object-focused variable only? Please clarify. (Again, sorry if I missed something).

This is a really important point, and we agree with R1 that it could have been more clearly expressed in our original submission – for which, we apologise. In the cross-correlation analyses conducted in parts 2 and 3 which examines forwards-predictive associations between infant attention durations and infant endogenous oscillatory activity (part two), and caregiver behaviour (part three), as R1 describes, we include all infant looks towards objects and their partner. Including all infant look types is necessary to produce a continuous variable to cross-correlate with the other continuous variables (e.g. theta activity, caregiver vocal behaviours), and, therefore, does not concentrate only on infant attention episodes towards objects.

We take the reviewers’ point that different attention and neural mechanisms may be associated with looks towards objects vs. the partner, which we now acknowledge directly on page 10 of the Introduction. However, our focus here is on the endogenous and interactive mechanisms that drive fluctuations in infant engagement with the ongoing, free-flowing interaction. Indeed, previous work has shown increases in theta activity during sustained episodes of infant attention to a range of different stimuli, including cartoon videos (Xie et al., 2018), real-life screen-based interactions (Jones et al., 2020), as well as objects (Begus et al., 2016). In the second half of part 2, we go on to address the endogenous processes that support infant attention episodes specifically towards objects.

As a result of this comment, we have made direct changes to the Introduction on page 10 to more clearly explain the looking behaviours included in the cross-correlation analysis, and the rationale behind the analysis being conducted in this way – which is different to the reactive analyses conducted in the second half of parts one and three, which examines infant object looks only. Direct edits to the text have also been made throughout the Results and Methods sections as a result of this comment, to more clearly specify the types of looks included in each analysis. Now, where we discuss the cross-correlation analyses we refer only to infant ‘attention durations’ or infant ‘attention’, whilst ‘object-directed attention’ and ‘looks towards objects’ is clearly specified in sections discussing the reactive analyses conducted in parts 2 and 3. We have also amended the Discussion on page 31so that the cross-correlation analyses is interpreted relative to infant overall attention, rather than their attention towards objects only.

Why are mothers' gazes shorter than infants' gazes? This was the flip of what I'd expect, so some interpretation would be useful to understanding the data.

This is a really interesting observation. Our findings of the looking behaviour of caregivers and infants in our joint play interactions actually correspond to much previous micro-dynamic analysis of caregiver and infant looking behaviour during early table-top interactions (Abney et al., 2017; Perapoch Amadó et al., 2023; Yu & Smith, 2013, 2016). The reason for the shorter look durations in the adult is due to the fact that the caregivers alternate their gaze between their infant and the objects (i.e. they spend a lot of the interaction time monitoring their infants’ behaviours). This can be seen in Figure 2 (see main text) which shows that caregiver looks are divided between looks to their infants and looks towards objects. In comparison, infants spend most of their time focussing on objects (see Figure 2, main text), with relatively infrequent looks to their caregiver. As a result, infant looks are, overall, longer in comparison to their caregivers’.

Minor points

Use the term association or relation (relationships is for interpersonal relationships, not in statistics).

This has now been amended throughout.

I'm unsure I'd call the interactions "naturalistic" when they occur at a table, with select toys, EEG caps on partners, and so on. The term seems more appropriate for studies with fewer constraints that occur (for example) in a home environment, etc.

We understand R1s concern with our use of the term ‘naturalistic’ to refer to the joint play interactions that we analyse in the current study. However, we feel the term is appropriate, given that the interactions are unstructured: the only instruction given to caregivers at the beginning of the interaction is to play with their infants in the way that they might do at home. The interactions, therefore, measure free-flowing caregiver and infant behaviours, where modulations in each individual’s behaviour are the result of the intra- and inter-individual dynamics of the social exchange. This is in comparison to previous work on early infant attention development which has used more structured designs, and modulations in infant behaviour occur as a result of the parameters of the experimental task.

Reviewer #2

Public Review

Summary:

This paper acknowledges that most development occurs in social contexts, with other social partners. The authors put forth two main frameworks of how development occurs within a social interaction with a caregiver. The first is that although social interaction with mature partners is somewhat bi-directional, mature social partners exogenously influence infant behaviors and attention through "attentional scaffolding", and that in this case infant attention is reactive to caregiver behavior. The second framework posits that caregivers support and guide infant attention by contingently responding to reorientations in infant behavior, thus caregiver behaviors are reactive to infant behavior. The aim of this paper is to use moment-to-moment analysis techniques to understand the directionality of dyadic interaction. It is difficult to determine whether the authors prove their point as the results are not clearly explained as is the motivation for the chosen methods.

Strengths

The question driving this study is interesting and a genuine gap in the literature. Almost all development occurs in the presence of a mature social partner. While it is known that these interactions are critical for development, the directionality of how these interactions unfold in real-time is less known.

The analyses largely seem to be appropriate for the question at hand, capturing small moment-to-moment dynamics in both infant and child behavior, and their relationships with themselves and each other. Autocorrelations and cross-correlations are powerful tools that can uncover small but meaningful patterns in data that may not be uncovered with other more discretized analyses (i.e. regression).