Reframe this saying that with such a large sample, not getting to perfect is normal

Margaret Atwood is almost mocking men for there need to be special- and the hero of a situation. This links heavily to Atwoods feminist undertone for her writtings.

The siren is enticing the sailors with words of honey, to trick them to jump off the ship.

I think the two final lines give the ending that the song worked into tricking the person. The poem leads with the idea that this person is special and can hear the song, but in the end gets tricked and meets the fate of many before them.

The song is far reaching and very intoxicating.

An extension of this is Conway's law which may tend to force us to create work which not only reflects us, but which becomes us.

The relationship with tools becomes a two way street between us and the tool as well as the tools which interpose all of us (humanity).

What might this mean for our relationship with Artificial Intelligence? Will our laziness with respect to thinking turn us into non-thinkers who rely on a tool not actually able to think?

(via me at https://old.reddit.com/r/typewriters/comments/1wjidrf/what_words_could_we_use_to_better_describe_the/pamjdv3/)

reply to Kirk Jackson aka u/NashvilleTypewriter at https://old.reddit.com/r/typewriters/comments/1wjidrf/what_words_could_we_use_to_better_describe_the/pal3wez/

This is great. I find it to be the case as well.

Watching other spaces like note taking applications (and methods), stationery, planners, notebooks, pencils, pens, fountain pens, etc. it repeats itself over and over. Some will really love a particular object which seems to work for them while others don't. Fora are filled with people asking, what do you like? how do you use it? when instead they ought to be attempting to figure out what they like and how they would use it.

Some turn their love of a particular thing into a religion (Apple vs. IBM comes to mind) which also tends to make others doubt their own choices and what might work best for them. Instead of experimenting around a bit to see what works for them, they take the supposed "magic" propounded by others and wrongly try to make it work for themselves. This is one of the primary reasons I tell potential new typewriter stewards to visit shops or type-ins so that they can put their hands on a variety of machines to see which "speaks" or works best for them and their needs. There are so many people limping along out there on dreadfully maintained machines because they haven't tried others (or even cleaned, oiled, and well-adjusted ones) and don't know what they're missing.

I was reading/listening to a series by APM recently called Sold a Story about how some academics and scientists created a "new method" for teaching reading and sold it as a products to school systems all over the country. While it managed to work for a terrifically small population, it was broadly wrong for the majority of people. Decades of harm was done to children and the educational space because everyone went with what these experts said (and which may have seemed logical) rather than doing some work, trusting testing and results, or continuing to experiment with a variety of strategies. Similarly, while computers and writing software may work for many, some of the older tried-and-true methods out there using tools like pencils, pens, loose paper, index cards, notebooks, notepads, and typewriters can provide dramatically different affordances in ways of working with them which are actually better for some of the things one might wish to accomplish with them. Some tools have the ability to speak to you in ways that others do not.

(P.S. Thanks for your observation as this may become the kernel of the introduction to the book I'm slowly working on.)

This sentence "learn well and think well" reflects 2 components of education and personal development. Learning well is more then just memorizing facts but encompassing a variety of strategies and approaches that enhance learning. Thinking well shows the ability to analyze information and evaluate your arguments. Focusing on effective learning strategies and promoting critical thinking skill. It's kind of sad to think how critical thinking, creative thinking, problem-solving skills is something has to be encourage by educators in a way so students don't feel or think they can use technology to help them or be given the answer

I like how this quote is saying how learning is supposed to messy and chaotic with encountering difficult concepts but engaging in argument-making is crucial aspects of the learning process. With AI increasing in education, students learning to think for their selves being encourage to ask question, and being able to explain their ideas or viewpoint without any use of AI.

Ausman emphasizing the importance of having experimental learning with students engaging in step by step, hand-on activities, allowing students to gain knowledge and skills and could also enhance their engagement and motivation with their education.

This quote from Tasha Ausman seems pretty accurate with students using AI to help them do the work or just letting it do the work for them because of how most forms give incorrect answers. This highlights the importance of depth of knowledge and expertise in learning environments. It's like student who discover this technology try to look for the easy way out of the work instead of actually doing the learning part.

This sentence by Diana Maliszewski is maybe something that most teachers and educators have thought about and taken into with the increase of AI, especially in classroom learning. To me, it's like teachers are trying to get students to use a critical thinking, use techniques to simulate deep thinking, and to encourage students to ask questions when thinking about the material that's been given to them. Student's should definitely taught how to analyze and critique what they hear but not doubting everything but at least having some focus on the context and the intent behind different types of media. Maliszewski also said, "It's less about the tool, and it's more about the thinking behind using the tool", that seems to emphasizes the importance of critical thinking and mindset in utilizing any technology or resource effectively. Since technology is becoming more efficient in classrooms these days, teaching students techniques for assessing the credibility of online information that includes understanding bias, recognizing misinformation, and verifying facts through reliable sources. I guess a take away from the article could be that if technology and AI going to be contributing more in our lives, especially when it comes to education, to show or teach how to use it responsibly.

Reframe this saying that with such a large sample, not getting to perfect is normal

Read abstract add the end

Lässt sich das z.B. über einen Rückgang der Patentanmeldungen an Hochschulen untermauern? Ein aktueller IW-Kurzbericht (31/2026) konstatiert, dass an "53,5 Prozent aller Kooperationsanmeldungen [..] mindestens ein Unternehmen beteiligt [ist]" https://www.econstor.eu/bitstream/10419/340870/1/1969855312.pdf

Hier ernsthaft die USA als Referentland und den Zeitraum "vor einigen Jahren" zu nehmen, halte ich für sehr problematisch.

Ist dies von Studien in irgendeiner Weise untermauert?

Hier wäre es interessant, wie es denn der Stifterverband mit den Open Science Ansätzen so hält. Offene Daten vermisse ich hier zum Teil.

Wir haben ja z.B. auch die HAWs. Sind diese hier mitgedacht?

Das ist aus der Perspektive der Research Policy Forschung ein ziemlich alter Hut. Etzkowitz und Leydesdorff haben um die Jahrtausendwende ihr Triple-Helix-Modell publiziert: https://doi.org/10.1016/S0048-7333(99)00055-4

Ohne die Forschungsleistung zu schmälern, müsste man doch hier eventuell mal hinterfragen, ob hier die fehlende Transparenz und Nachvollziehbarkeit in privater Hand nicht auch einige Gefahren birgt.

Auch diese sehr verkürzte Darstellung wird der Komplexität der wissenschaftlichen Forschung in keiner Weise gerecht und impliziert eine Zuspitzung, die nicht notwendig ist.

Das erscheint mir eine sehr verkürzte Darstellung der Rollen von Lehrinhalten und Lehrformaten. Zudem ist das wissenschaftliche Subsystem auch ein Teil der Gesellschaft und trägt auch zur Erforschung dieser Bedürfnisse bei.

I can tell that in most of my classes that a lot of what we are learning is just information taken out of the book.

Your peers offer different insights and opinions. Their perspective matters because everyone thinks differently and you may be able to see the same writing thought about in a different way.

This may work for others, but I always find myself losing papers and becoming less organized through the middle of the school year.

going back to our first lesson about min mapping, it helped me understand my ideas a lot better.

Thinking thoroughly about what you research like this can help you better understand your topics and what you are trying to find.

I believe it is important to have credibility for your sources because taking information from an invalid source ruins your credibility as either an author or a speaker. It is important not to spread misinformation especially as an educator in said topics because some people may take it and run with it.

I didn't know Google is algorithm-based, but it'd be safe to assume

I actually just did this regarding a speech I have to give on tuesday.

Google is a good source if you are looking for biased opinions and conjectured "statistics."

How teaching kids to read went so wrong - Sold a Story: Season 1, Episode 1: The Problem<br /> by [[Emily Hanford]] in [[Sold a Story]] from APM Reports

Considering your own opinion before research helps you find a topic that agrees with your stance

Questions should always have multiple answers. Nothing is definite

They are important to me because of the discrimination that may come with them. I think the same issues should be important to other people as well.

definitely issues pertaining to my ethnicity, my gender, and beauty standards.

Visuals help children and even adults understand the material more.

I think this is too much content for a fold. Turn it into a tooltip as well as a link to a different page that has this fleshed out in detail - maybe that page already exists and it's just a matter of linking it

Maybe combine this or align this with the new to this model bit?

This one is short. It could probably just be a tooltip

So many folds at the top. Better than having them unfolded but I feel like there must be a better way of displaying this, perhaps with some horizontal boxes, perhaps some of these should go to external links, etc

I've experienced this firsthand. Sometimes research doesn't mean as much to me unless it pertains to me or a situation ive been in.

Research is more than just finding answers to a question. You can find answers to something that neither you nor anyone else around you has thought about.

The Lempriere document tells the story of hyacinthus and his relationship with apollo and how the hyacinth flower came to be. In that document we see that Apollo uses the hyacinth is used to immortalize hyacinthus due to how much he cared for him. Similarly Elliot says in a later line the hyacinth girl is neither living nor dead just as Hyacinthus was neither living nor dead because of Apollo's caring and loving act.

Lines 30-59 develop two parallel themes: Lance, and the Goblet or Cup as male and female "sex symbols of immemorial antiquity," and Tarot as a system of allotting mortals divine fortunes. Preceding page 8 in Weston's account of semiotics in the Holy Grail legend, descriptions of masculinity and femininity are tied closely to virility and fertility (respectively). Quoted, Weston states that "[as] symbols of immemorial antiquity and world-wide diffusion, the Lance, or Spear, representing the Male, the Cup, or Vase, the Female, reproductive." Weston further states that within the "Grail romances we possess...[is] the fragmentary record of the secret ritual of a Fertility cult."

In these statements Weston justifies analysis by asserting that it is backed by inter-mythological theory; Weston's definitions (of symbol) maintain continuity across various socio-cultural settings, specifically Celtic and Greek. The Cauldron of the Dagda Coire Arsic, known as a "legendary vessel of infinite bound & hospitality in Irish mythology" and the Greek Horn of Plenty.

By calling Madame Sosostris “the wisest woman in Europe” who consults “a wicked pack of cards,” Eliot essentially collapses two contemporary portraits of fortune-telling into one ironized figure. Huxley’s Crome Yellow supplies the name and the basic joke: Mr. Scogan, a man dressed as the elderly “Sesostris, the Sorceress of Ecbatana,” sits in a fairground booth and dispenses darkly comic prophecies for sixpence. Mina Loy’s “At the Door of the House” supplies the atmosphere of commercialised female longing—the card-teller’s rapid catalogue of passion, deception, the Devil, and “intentions little honorable,” ending with women still waiting for “the little love-tale / That never came true.” Eliot fuses both into Sosostris, then places her immediately after the Wagnerian fragments from Tristan und Isolde and the emptied hyacinth-garden vision (itself shadowed by Lemprière’s myth of the beautiful youth killed by the discus and transformed into a flower). The suggestion appears to be that the older, high registers of doomed desire and mythic intensity have been displaced by a modern “wisdom” that is itself a performance: a slightly fraudulent, slightly seedy oracle whose cards still carry traces of older symbols, yet now serve mainly to characterize a culture that can name its own sterility but can no longer believe in genuine revelation.

The paradox reflected by this statement is connected intimately to a large amount of source material. The the human body is rendered as an amorphous shape, almost liquid, able to flow in the manner of water. Antithetically, the nature of the human river is directed against the compulsion of gravity, carrying upwards and cresting the hill rather than falling into compliance and rolling down its slope. In order to effectively attach to the slope, the human body would necessarily be able to establish some form of traction, of adherence that would anchor it and oppose gravitational motion. Bandelaine provides an explanation of this in the form of sap. The sap exists as a "flowing" lively entity that travels the veins of the human body, intermingled anatomically, a stark contrast to the dullness, the predictable patterns of an urban environment. Here, the same is true - the sap takes on a vivid life that resists the predictable motion of the slope, enabling the body to oppose constraints that challenge it. Additionally, Dante makes reference to the wicked souls that fling themselves from the shore, he compares them to autumn leaves falling away during seasonal transition. In this manner, the ejection of the damned into the belly of Hell contrasts vibrantly with common connotations of naturalistic deposition. Shedding of leaves demands lamentation, a mournful tone that encapsulates the loss of a thing that was living, was functional, was fertile. The passage of damned souls resides in opposition, evincing a position of security and justice - sinners are rightfully contained in Hell and represent a damaged vestige of humanity rather than a symbol of fertility. The sap's properties of viscosity and adherence harbor a similar notion. It is a remnant, a liquid that contains the lifeblood of nature, as is reflected by the leaves. However, it is employed in defiance of its customs - to aid the ascendance of humanity against nature's common laws.

In reference to Mina Loy’s “At the Door of the House,” the beginning of “A Game of Chess” presents a woman in a seemingly powerful position. “The Chair she sat in, like a burnished throne” makes Eliot’s woman appear wealthy and queenlike, but the word “like” suggests that this power is only an appearance. Despite possessing everything that should be desirable, she becomes increasingly anxious and insecure. Similarly, Loy’s “thousand women’s eyes” are “riveted to the unrealisable” as they look to a fortune-teller for the love and futures they desire but cannot genuinely attain. In both poems, women appear surrounded by fantasies of fulfillment, yet remain uncertain and dependent upon absent or idealized men. This also parallels Electra waiting at the doorway for Orestes: each woman waits for a male figure at the door (Orestes, the “Man of the Heart,” or, later in Eliot’s poem, Albert) to arrive and change her situation. Therefore, the doorway and the throne both represent forms of suspended power: the women seem important or hopeful, but their lives remain controlled by expectations that may never become real.

Eliot's back-to-back description of the tarot cards makes Madame Sosostris' fortune-telling fast-paced and difficult to follow. Through her abilities, he presents fragments of experience but does not show readers the connecting thread or significance of these cards when read together. His narration draws parallels to Mina Loy's "At the Door of the House," where the narrator takes seemingly unrelated objects such as "wheels," "goblets," and "skeletons," and uses them to make a somewhat coherent prediction about someone's future. In both works, scattered symbols ultimately come together to create a certainty, even if it takes a while to interpret the underlying significance because of the disjointed nature of the predictions. In Crome Yellow Huxley describes this encounter with spiritualism in an almost comic manner. Mr. Scogan imagines absurd "predictions" that he invests his "quiet confidence" in. Ironically, his customers become genuinely frightened by his words because they want to believe that something will happen to them based on the signs they re seeing.

Eliot also adds a nuance to this experience with spirituality with usage of Jessie Weston's discussion of Tarot. Weston connects the origins of Tarot to the Lance (hearts), Lance (diamonds), Sword (spades), and Dish (clubs) tradition. These were originally symbols of human life and fertility. Eliot takes these symbols that were once a part of a meaningful belief system and presents them as mysterious fragments (the same way he does with his biblical references, etc.).

Normally when I hear things about Hysteria I would think about them performing a Lobotomy...Not this.

N'est ce pas un frein pourr retrouver l'information dans le sens oû le titre du document/livre ne reflète pas toujours le contenu exact de l'idée developpée par l'auteur?

La muséologie est-elle donc un mélange à la fois d"un système de connaissances organisées basé à la fois sur les principes de gestion archivistique de part le caractère unique de chaque objet, mais également sur la classification bibliothécaire à cause de la valeur scientifique que représente la documentation de ces mêmes objets?

On peut dire aussi que les documents, en plus de respecter la provenance dans leur classement sont ensuite classés en respectant un ordre de production en fonction de l'évolution de l'activité pour laquelle ils ont été créés.

ICONCLASS serait donc un système hybride d'organisatioon de connaissances?

@All i have another 2 title options: 1. Machine Learning and Time-Series Foundation Models for Day-Ahead Electricity Forecasting in a University Building 2. Beyond Forecast Accuracy: Machine Learning and Time-Series Foundation Models Across University Operating Regimes

Interesting how close in obesity prevalence the ICB with highest and lowest prescribing rate are.

The spread of prescribing rate narrows more than I would have expected. Maybe partly because there are fewer ICB with a large obesity register size?

The use of German is so interesting because the Germans are partly responsible for the destruction of WW1 and they were the enemies of the UK. Their being responsible for the wasteland makes sense in context too. The translation says" To my homeland—my Irish child, where do you dwell?" is interesting as it makes it seem like the Germans are beckoning the Irish to come to their homeland yo die.

Interesting modeling exercise. The attenuated weight loss in T2D is real and clinically important. However, framing the explanation primarily in terms of changes in energy expenditure and the caloric equivalent of urinary glucose excretion keeps the analysis inside the same energy-balance framework whose limitations have been repeatedly noted (e.g., 1,2,3).

Urinary glucose excretion is first and foremost a mass outflow (grams of glucose leaving the system). Treating it principally as an “energy sink” converts a direct mass loss into an indirect energetic term and then uses that term to explain a mass (weight) outcome. A mass-balance perspective would track the grams of glucose excreted alongside other mass flows without the intermediate conversion, thereby avoiding the additional assumptions required by energy-density estimates.

The observation that background SGLT2i therapy further attenuates weight loss is consistent with a larger baseline mass outflow that diminishes as glycemia improves. This can be stated directly in mass units. Whether the same phenomenon is best understood as a change in energy gap or as a change in net mass balance remains an open and consequential question.

References 1. Manninen AH. Mass Balance over Energy Balance: Why Direct Mass Accounting Offers a More Precise and Mechanistically Faithful Framework for Human Body Weight Regulation. Preprints 2026, 2026040690. https://doi.org/10.20944/preprints202604.0690.v4 2. Manninen AH. Implementing the Mass Balance Model (MBM) in Human Nutrition and Obesity Research: Protocols, Analytical Frameworks, and Translational Applications. Preprints 2026, 2026041641. https://doi.org/10.20944/preprints202604.1641.v2 3. Arencibia-Albite F. A numerical and analytical evaluation of the consistency of the energy balance model identifies limitations and systematic relationships between mass intake and body weight dynamics. Front Nutr. 2026 Jul 30;13:1777554. doi: 10.3389/fnut.2026.1777554. PMID: 42597285; PMCID: PMC13468908.

add quotation marks for each phrase to provide consistency within the paragraph

Assessing arguments take time no matter if it’s long or short because there’s lots of steps into determining what your looking for there also the possibility of not finding what your looking for making you dig deeper into the statement so there’s some steps you can follow into finding what it is that your going to need for this

Our mind makes many of our decisions, it proccesses our beliefs, our doubts. We are quick to believe and quick to judge but we need to consider the facts as well sometimes we are wrong and we need to be willing to work with it and grow from it the different experiences but we also have to be open minded to the different possibilities that come with new beliefs .

we tend to believe what we would like to think without any of the facts we like to listen to people of power the ones who influence us to do things without always thinking them through. We see results so we expect those results which is not always the case it might not always work but we want to believe what we see not what happens after .

We all have a right to out opinion and our beliefs we choose to believe what we want wether that’s with or without evidence but that doesn’t mean you should believe everything we are quick to believe everything we see on the internet and while some could be true they are not experts that really know what the product does or what it could do to us it could be a harm without us knowing it and just believing whatever the internet tells us which is why we should always listen to the person who actually knows what the product is not someone who is promoting it

To accept an argument there’s many different factors to consider before believing it as you need a logical reason to believe it and evidence to support it as you go along wether to believe or not believe it

Arguments have lots of different things that make them an argument we have to evaluate all parts to determine many different parts of the “statement” weather it be deductive, in deductive, true or false, reliable or unreliable, weather we should believe it or not we need all these things to be accounted for

meets the governor+ towns people in Eatonville, joe starks becomes the mayor of Eatonville

eLife Assessment

This Review Article provides a comprehensive overview of whole-brain activity changes induced by brain stimulation and effectively summarizes the current state of the field. However, the integrative framework spanning spatial and mechanistic scales, which is presented in the discussion, should be introduced earlier to guide the reader. A more cohesive conceptual framework throughout the manuscript would improve the synthesis of the literature and enhance accessibility.

Reviewer #1 (Public review):

Summary:

This paper is a comprehensive review of perturbation studies, and the state-dependence of the brain's response to perturbation at the circuit, mesoscale, and macroscale level.

Strengths:

The strengths of the paper are the thorough description of many perturbation studies at different levels of organization, and the integration of both experimental and modeling studies. The review clearly communicates the need to consider 1) brain or local-population state, and 2) multiple levels of organization, in order to understand perturbation responses. Another major strength is the ability for the reader to reproduce figures using the EBRAINS platform.

Weaknesses:

The major weakness is that the review does not include a significant integration across scales, and as a result reads like three separate (though comprehensive) reviews. Currently, the only integration across the scales is in a brief conclusion paragraph. I would recommend adding an additional section, in which the overarching picture is discussed. (i.e. a unifying view of state dependence, and what is learned by considering across scales), and more prefacing in the introduction of the overarching message and framework to the review.

Reviewer #2 (Public review):

Summary:

In this review article, the authors discuss the whole brain activity changes induced by brain stimulation. They review the literature on how these activity changes depend on the cognitive state of the brain and divide the results by the scale of the change being induced, from microscale changes across small groups of neurons, up to macroscale changes across the entire brain. Finally, they describe attempts to model these changes using computational models.

Strengths:

The review provides an overview of the results within this sub-field of neuroscience, and the authors are able to discuss a lot of prior results. The framing of the changes in neuronal activity in terms of computational changes is also a helpful approach.

We thank the authors for the updates that they have made in response to our original comments. Their attempts to address many of the comments that we raised have greatly improved the paper. We believe that there are two major points that still require some additional changes:

(1) We raised the concern that the results within each of the three spatial scales did not join together into a cohesive single framework. The authors responded by updating the conclusion section to provide a more conceptual picture linking the different spatial scales. This is much appreciated. However, by placing this framework at the end of the paper, it prevents the reader from using this understanding to building a conceptual model as they progress through the paper. We would ask that the authors intersperse this conceptual picture within the main text, and to then re-emphasize it in the conclusions. This would frame each section in terms of the findings that led directly to it and, therefore, allow the reader to build a conceptual understanding within each section. As one example, we note that the authors have made no changes to the mesoscale processing section. Therefore, when reading that section, it is completely unclear how any of the results seen in the microscale may relate to the changes observed at the mesoscale.

(2) The authors have greatly improved their explanation of the complexity metrics. However, the paper still lacks a conceptual understanding for why "perturbation-based complexity metrics" are a reasonable way to study the state-dependent dynamics? What does studying perturbations provide that studying the spontaneous activity in different states alone, would not provide? Why is this the preferred way to study such dynamical systems? Such a justification would strongly support the analyses reviewed in the paper and would increase the reader's understanding of the methodology.

Author response:

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

eLife Assessment

This Review Article provides a thorough overview of whole-brain activity changes induced by brain stimulation and summarizes the current state of the field. However, it lacks integration across spatial and mechanistic scales, which limits the reader's ability to understand how the different findings relate to one another. In addition, several key concepts are not explained in sufficient depth for non-expert readers. The manuscript would benefit from the development of a cohesive conceptual framework to more clearly synthesize the existing literature.

Thank you for the positive assessment. We fully agree, and as suggested we have added a new conclusion paragraph that outlines a synthesis of the paper and suggests a conceptual framework :

“In this paper, we have reviewed aspects of neuronal responsiveness, from the microscale level of neurons and circuits, the mesoscale level of single brain areas, and the macroscale level of the whole brain. At the microscale, it is apparent that the circuit operating in an asynchronous mode displays the highest responsiveness, as seen in brain slices (D’Andola et al., 2018). The underlying mechanism is that the high levels of synaptic « noise » in asynchronous states set neurons in a high responsive mode, as seen in models of single neurons (Ho & Destexhe, 2000). This higher responsiveness is confirmed at mesoscale, and can be seen for example with Utah-array recordings comparing wake and anesthesia (Dwarakanath et al., 2025). Similarly, propagating waves occur in the asynchronous state in awake monkey (Muller et al., 2014), and sensory inputs evoke more propagating patterns (and higher PCI) in wakefulness with asynchronous states compared to slow-wave states of anesthesia in mice (Montagni et al., 2024). At the whole-brain scale, experiments also find that evoked responses are more complex and propagating compared to slow-wave states (Massimini et al., 2005), a situation which models can reproduce (Goldman et al., 2023; Sacha et al., 2025). Other measures, such as fluidity (Breyton et al., 2024) and reversibility (Camassa et al 2024) also point to the same conclusion. Collectively, these results show that asynchronous and irregular activity states set neurons in a high responsive mode, which in turn impacts network behavior and favors the propagation of activity as mesoscale propagating waves, or macroscale activity patterns that propagate across brain regions. It is therefore not surprising that the best correlate of conscious states is the asynchronous activity (Koch et al., 2016).”

Public Reviews:

Reviewer #1 (Public review):

Summary:

This paper is a comprehensive review of perturbation studies and the state-dependence of the brain's response to perturbation at the circuit, mesoscale, and macroscale levels.

Strengths:

The strengths of the paper are the thorough description of many perturbation studies at different levels of organization, and the integration of both experimental and modeling studies. The review clearly communicates the need to consider (1) brain or local-population state, and (2) multiple levels of organization, in order to understand perturbation responses. Another major strength is the ability for the reader to reproduce figures using the EBRAINS platform.

Weaknesses:

Two major points of improvement should be resolved with the review, in order to make it useful for a broad audience.

The first is that the review does not include a significant integration across scales, and as a result, reads like three separate (though comprehensive) reviews. Currently, the only integration across the scales is in the brief conclusion paragraph. I would recommend adding an additional section, in which the overarching picture is discussed. (i.e. a unifying view of state dependence, and what is learned by considering across scales). This need not be too long, but it should be longer than a single conclusion paragraph.

Thank you for the positive assessment. We fully agree with the excellent suggestion of adding a concluding paragraph where the conceptual framework and overarching picture are presented. Please see the new conclusion paragraph that we added to the paper (copied above in the reply to Editors).

The second major weakness is that there is a lack of clarity on many points throughout, which is needed for the reader to fully understand the results described.

See our answer to the specific comments below for the list of unclear points.

Reviewer #2 (Public review):

Summary:

In this review article, the authors discuss the whole-brain activity changes induced by brain stimulation. They review the literature on how these activity changes depend on the cognitive state of the brain and divide the results by the scale of the change being induced, from microscale changes across small groups of neurons, up to macroscale changes across the entire brain. Finally, they describe attempts to model these changes using computational models.

Strengths:

The review provides an overview of the results within this subfield of neuroscience, and the authors are able to discuss a lot of prior results. The framing of the changes in neuronal activity in terms of computational changes is also a helpful approach.

Weaknesses:

However, the authors are not able to contextualize these results within a single framework, i.e. explaining from first principles how different aspects of stimulus-induced changes interact to generate functional changes in the brain, and how different changes - at distinct spatiotemporal scales - combine to form larger effects. This is a significant weakness in generating a review of the literature, since the authors do not provide a cohesive conceptual framework on which to frame the results. Similarly, the authors do not explain how their different computational models fit together, and how one can get a singular computational understanding of the distinct mechanisms of brain activity changes due to stimulation under different brain states, by combining the results derived from each separate model.

Thank you for the positive assessment. This is an excellent suggestion, actually also requested by Reviewer 1. We have now added a new conclusion paragraph where the conceptual framework is explained (copied above in the reply to Editors).

Major Comments:

(1) The authors have written this review as if it were intended for an audience who is already familiar with the topics. For example, they introduce concepts like complexity, spiral vs planar waves, without much explanation.

Thank you for this helpful comment. We agree that the Introduction should be more accessible to readers who are less familiar with these concepts, and we have therefore revised the text to provide a clearer definition of complexity and a more explicit explanation of propagating wave patterns.

Specifically, we now clarify that complexity can be understood as the richness of the set of accessible states of a system, which in our context can be related to the diversity of slowwave propagation modes and to the high-entropy, desynchronized activity of the awake brain. We also expanded the description of propagating slow waves to distinguish planar from spiral waves and to explain how their relative prevalence changes with anesthesia depth.

In the Introduction we replaced the sentence “New methods … at various scales” with “New methods for characterizing the complexity of network dynamics and their response patterns have emerged, particularly recently (Krohn et al., 2023; Wolf et al., 2018), and are presented here at various scales. Here, complexity is associated with the set of accessible states of a system (Parisi, 2006). In the present context, this notion can be linked to the diversity of slow-wave propagation modes and to the richness (i.e., the entropy) of perturbation-evoked responses in brain activity.”

While in Results (p. 13) the sentence “Spontaneous slow waves … administered (Huang et al., 2010).” has been expanded in “Spontaneous slow waves can also display propagating patterns, as shown in anesthetized mice (Huang et al., 2010; Mohajerani et al., 2010; Pazienti et al., 2022; Stroh et al., 2013). These patterns may take the form of planar waves, which travel across the cortex along a relatively regular front, or spiral waves, which rotate around a central core and therefore produce a more complex spatiotemporal organization. Under relatively deep anesthesia, spiral waves occur more frequently than planar waves, whereas the opposite imbalance is observed as anesthesia is lightened (Huang et al., 2010).”

(2) Regarding complexity, the authors present a quantification termed PCI. However, in the associated box, they state that PCI could be implemented in a number of different ways, using analogous metrics (which are, nonetheless, not identical). Yet the authors simply claim that all these metrics are sufficiently similar to be grouped together as "PCI". The authors do not provide much intuition about this, and they also don't present any other potential quantifications. This makes any interpretation of their results strongly dependent on your understanding of the concept of PCI. It would be helpful to present some other, analogous metric to demonstrate that the results that the authors are focusing on are not somehow tied to the specific computational structure of the PCI metric.

Thank you for pointing out to this inconsistency. We agree that the rationale for focusing on perturbational complexity was not sufficiently introduced in the original version of the manuscript.

Broadly speaking, complexity measures used in consciousness research can be divided into two major classes. The first includes observational measures, which are computed from spontaneous ongoing activity and quantify statistical dependencies within neural time series. The second includes perturbational measures, which quantify the deterministic causal interactions revealed by a controlled perturbation of the system and their spatiotemporal propagation across the network (see Sarasso et al., 2021).

The primary aim of the present Review was to discuss how complexity changes across spatial and temporal scales in response to perturbations. For this reason, we focused on perturbational complexity measures and, in particular, on the Perturbational Complexity Index (PCI), which remains one of the most widely adopted and validated approaches in this category.

As described in Box 2, different implementations of PCI have been proposed. The two most established versions are PCI based on Lempel–Ziv complexity (PCI^LZ) and PCI based on state transitions in principal component space (PCI^ST). Although these implementations differ algorithmically, they were developed to operationalize the same theoretical construct and have been shown to correlate strongly when applied to the same datasets (Comolatti et al., 2019). For this reason, throughout the Review we use the term “PCI” as an umbrella label encompassing these related perturbational complexity measures.

Importantly, all complexity measures discussed in the studies reviewed here belong to this broader class of perturbational approaches. While adaptations of the original algorithms are often required when dealing with different recording modalities and spatial scales, these modifications mainly concern preprocessing and signal representation rather than the underlying theoretical construct being quantified.

To clarify this point, we have revised the Introduction to explicitly motivate our focus on perturbational complexity, to distinguish perturbational from observational complexity measures, and to explain why different PCI implementations can be discussed within a common conceptual framework. We believe that these additions make the rationale of the Review substantially clearer and reduce the impression that the conclusions depend on a specific implementation of PCI.

(3) The authors divide the review into sections organized by the spatial extent of the effects that they are exploring (e.g. from microscale to macroscale). However, they don't bring together these insights into a cohesive structure - for example, by providing potential explanations of the macroscale effects by using the microscale changes.

We agree – and this is now the focus of the newly-added conceptual-framework conclusion paragraph.

(4) The authors completely ignore any aspect of cell-type specificity in their review, despite the known importance of specific cell types at the microcircuit scale. This makes it difficult to map their results onto the true biological system.

We agree that cell-type specificity could be made more explicit. The revised manuscript now clarifies that several models already include cell-type specificity. For example, the AdEx-based models distinguish excitatory regular-spiking or pyramidal populations with adaptation from inhibitory fast-spiking populations without adaptation. This differentiation is not made with other models like leaky or quadratic integrate-and-fire models. At the mesoscale, mean-field approaches can be derived for different structures, such as cortex, thalamus, hippocampus, striatum, or cerebellum, and can incorporate the experimentally experimentally observed firing properties of relevant cell classes.

(5) The authors introduce several different computational models, such as the Hopf model, the AdEx model, and the MPR model. However, they do not provide the reader with a conceptual understanding of the structure of each of these models (except through potentially more complex terminology, e.g. the Hopf model is a "phenomenological StuartLandau nonlinear oscillator"). Additionally, though they present the results of each simulation, they don't provide the reader with intuition about how these models compare against each other, and how best to interpret results derived from each model.

Very good question, and the answer is not easy. If the goal is to capture large-scale phenomena with models as simple as possible, then Stuart-Landau, Hopf, or Jahnsen-Rit may be appropriate. This approach is rather top-down. But if the goal is to assess how microscopic changes (synaptic receptors for example) affect large-scale brain activity, then we need a bottom-up approach, where mean-field models are derived. We can better explain this.

We agree that while the technical definitions of the whole-brain models (Hopf, AdEx, MPR) were provided, a clear conceptual framework comparing their underlying structures, specific trade-offs, and interpretation guidelines was missing. We have substantially revised the "Macroscale" section on Page 22 to provide immediate intuition regarding what each model represents structurally (e.g., macroscopic phenomenology vs. microscopic biological realism). We emphasized the structural assumptions of each of them, as well as the explicit utility in interpreting brain responsiveness. This ensures readers understand exactly why a researcher would choose one model over another depending on the mechanistic question at hand.

(6) In several cases, the authors make statements that they appear to believe to be completely straightforward (and require no justification), but that do not appear so to the reader. For example, they mention: "In wakefulness and REM sleep, ..., the membrane potential is depolarized and close to the spike threshold, which explains why neurons respond more reliably and with less response variability compared with slow-wave sleep". However, this statement is not obvious to the reader and requires explanation (for example, in a system that is close to balance, bringing cells closer to the firing threshold can result in increased response jitter).

We agree that the original statement was an over-simplification of a complex situation. We have revised it to avoid suggesting that depolarization alone monotonically increases reliability. The relevant mechanism is the combination of depolarization, desynchronized high-conductance synaptic input, balanced fluctuations, and reduced tendency to enter long silent Down states. In this regime, weak inputs are more likely to be converted into spikes and propagate through the network. However, too high conductance, excessive noise, can shunt inputs, enhance jitter, or saturate the network. We are now more explanatory.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

As stated in the public review, there is a lack of clarity on many points throughout, which is needed for the reader to fully understand the results described.

Points needing clarification:

(1) sPCI (slice Perturbational complexity index) - is this different from other PCIs in box 2? Regardless, the metric and its interpretation should be briefly explained in the main text.

We use sPCI to refer to the PCI measure adapted for application to cortical brain slices (D’Andola et al., 2017; see Box 2). It relies on the same core algorithm as PCI, namely the Lempel–Ziv complexity of the spatiotemporal pattern of significant responses (Casali et al., 2013) but differs in the preprocessing steps required for slice recordings. We now added this clarification to the text.

(2) Page 9 "by decreasing fast inhibition but also enhancing it" - What does that mean? More info about the model is needed.

Thanks for raising this point, since this sentence was indeed confusing. We have revised it now.

(3) Page 9 "balance between segregation and integration, a crucial ingredient on which sPCI relies" - How is this balance seen in the figure? All I see is sPCI and blockage of GABA.

The comment is correct, and this mention of segregation and integration has now been eliminated.

(4) Figure 3D, Page 11 "two different desynchronized (AI) states in a network of AdEx neurons"- What are the two different states? Why is the response different?

The different AI states correspond to different synaptic strength parameters, we added this precision in the text.

(5) Figure 3B - "Bifurcation diagram showing the different activity regimes displayed by spiking neuron network." Which model? Multiple are cited. This is a general issue throughout where multiple models are mentioned in the text, and it's unclear which is shown in the figure.

We agree with the Reviewer's helpful remark. We have revised the manuscript to explicitly state the types of models depicted in the different panels of Figure 3. Corresponding details have also been incorporated into the relevant text in the Results section (previously pages 10–12)."

A few editorial issues:

(1) The text in many of the figure panels was too small to read. This is a significant issue that must be addressed.

We will fix this at the next round, can you please let us know which figures are not visible?

(2) I recommend reading through for writing flow. E.g. In the first paragraph of the introduction, there are two sentences that start with "importantly, ..." in a row.

Thanks for noting this – this is now fixed.

(3) Figure 1E - How does the color on the left relate to the right? What is the y-axis?

The colour code corresponds to the latency of activation (light blue, 0 ms; red, 300 ms). The Y-axes is the global mean field power (voltage). It has now been included in the figure caption.

(4) Figure 1C - What is the stimulus?

The triangle corresponds to the electrical stimulation of the homotopic area 18 of the contralateral hemisphere. This information is now included in the figure legend.

Definir un poco más extenso (fino)

test

Quitar guiones

Quitar guiones

idem comentário na inicial - figura de enfermeira

em algum momento será falado q é curso 1 de .... ?

essa informação não aparece na descrição inicial do curso

inabilitadas - não aparecem - visualizo até Questão 1 de 5

inabilitada - as 3 abas não são visualizáveis - aparece até caixa Atenção para validação

não aparece por completo: visualízável até Caso 1 - PEP

inabilitado - não aparece o objeto por completo

infográfico acima não visualizável

rever

a imagem mostra enfermeira

I don't like that this doesn't cover the fact that people can use Wikipedia to spread misinformation. Just look at Twitter/X and Reddit, as they are constant hotspots for spreading "Facts" and "Trivia" that aren't even remotely true.

I can't agree with this. I have learned that AI can lie, and as such, it should be a given to take whatever AI says with a grain of salt. Best to stick with academic databases.

For me, the Steely Library database is the ONLY place to start. Academic databases are the quickest and easiest way for students to access information that can always be trusted, especially in this age. I've always used the database for my papers, and I'm not stopping now.

incluir: abordagem, após entre

inabilitado

avaliação de aprendizagem inabilitada - não aparece

inabilitado - não abre

inabilitada - não aparece a sanfona

rever slides da profa com as alterações sugeridas: estes tem erros

Ex: Resolução 195 não trata de prescrição de med. - é a 801/2026

No tratamento candidíase: incluir clotrimazol

A decisão depende do risco de "perder a pessoa" ("excluir") que paciente está exposto

não foi possível abrir as duas rotas acima - inabilitadas não foi possível colar aqui o print da imagem das duas rotas - só dá prá ver parte superior das 2 cabeças, não aprece nem os rostos

These explainers should have backlinks to the section where the thing is actually used.

eLife Assessment

This study presents an important finding regarding the role of oxytocin neurons in thermogenesis and behavioral thermoregulation. The use of numerous converging methods, including behavior, fiber photometry, optogenetics, thermal recordings, metabolic analyses, and more, produces a multi-dimensional dataset delivering findings that provide solid support for the conclusions. The conclusions could be further strengthen by more extensive analyses of behavior and determining whether it is the release of oxytocin (rather than co-release of glutamate) from the PVN that is critical for the transition between behavioral states, nevertheless, the manuscript had many strengths, the findings are novel, and this work opens new doors for understanding the role of the PVT in thermoregulation. This work will be of strong interest to the thermoregulation, social behavior, and oxytocin signaling communities.

Reviewer #1 (Public review):

Summary:

The authors identify and investigate a specific population of PVNOT neurons (oxytocin neurons of the paraventricular hypothalamus) that seem to be involved in both behavioral and autonomic thermoregulation. These cells are activated by social thermoregulatory behaviors, but can influence thermoregulation in both social and social contexts, specifically during transitions and when mice are at low core body temperature (Tb).

Comments on revised version.

The authors have addressed my concerns with clear and reasonable explanations and altered the text accordingly. This has improved the paper, but it still feels in some parts like a patchwork of nice work and discoveries stitched together. Further changes to format, analysis, and some experimental work could hugely improve the manuscript. I see that will surely come from future work, and this is the authors' choice.

Regarding the lack of behavioral analysis, I think it's fair for them to keep it for future studies.

I am happy to see they take and expand the opto inhibition suggestion. Again, that experiment would be nice for this paper, but not crucial.

Regarding discussing Raam et al 2026. It is good that they detail the practical decision of using females. What I meant was that, given that both papers study calcium dynamics around the time when mice engage in social thermoregulatory behaviour, they could have speculated on potential dmPFC-PVN functional connectivity, for example. Or the fact that Raam found that females showed fewer huddling behaviour than males at 5{degree sign}C (however, Vandendoren tested 15{degree sign}C, not 5{degree sign}C). Discussion of these features would be welcome, but maybe all of the current scope.

Overall, this is a very strong paper.

Reviewer #2 (Public review):

This is a very interesting study from Vandendoren and colleagues examining the role of PVN oxytocin neurons during thermoregulatory behaviors, in particular during thermoregulatory huddling. The findings are important and have implications for the thermoregulation field as well as the social/naturalistic behavior field. The findings are compelling and use a combination of state-of-the-art tools (photometry, optogenetics, automated behavior tracking, thermal imaging, and core body temperature measurement), often in combination with each other, to produce a rigorous and high-dimensional dataset.

Comments on revised version.

I appreciate the effort the authors have put into addressing all of my questions, and I have no remaining concerns.

Reviewer #3 (Public review):

Summary:

This study investigates how the activity of hypothalamic paraventricular oxytocin (PVNOT) neurons relates to physiological states in female mice, with a particular focus on behavioral states and thermogenic sympathetic activity. To address this question, the authors combined automated video-based behavioral classification with calcium imaging of PVNOT neuron activity. Sympathetic thermogenesis was inferred from surface temperature changes measured by infrared thermography, and the authors have made their custom analysis scripts available. The authors report that strong, pulsatile activation of PVNOT neurons was "occasionally" observed immediately before transitions from resting to active states. This observation suggests that PVNOT neuronal activity may facilitate the transition from rest to activity. This phenomenon was observed in both pair-housed and individually housed animals. Taken together, these findings raise the possibility that the oxytocinergic system contributes to naturalistic behavior transitions even in the absence of social interactions. However, concerns regarding the selectivity of GCaMP expression in oxytocin-expressing neurons call into question the validity of the recorded PVNOT neuronal activity. The revised manuscript improves the presentation and interpretation of the data. Nevertheless, because the authors have not provided additional experiments or analyses addressing the major methodological concerns, the evidence supporting the central conclusions remains essentially unchanged.

Strengths:

The oxytocinergic neural system is believed to subserve a wide range of physiological functions. Elucidating these roles requires monitoring PVNOT neuronal activity under diverse behavioral contexts, as well as manipulating this activity to establish causal relationships. In this study, the authors present a technically sound experimental framework that integrates behavioral tracking in both individually and group-housed mice with the monitoring and manipulation of PVNOT neuron activity. This setup represents a valuable methodological resource for researchers investigating the physiological functions of oxytocin.

Weaknesses:

(1) Immunohistochemical validation of selective GCaMP expression in oxytocin-expressing neurons showed that only 24-51% of GCaMP-positive neurons expressed oxytocin. As an alternative approach, the authors argue that the similarity between calcium dynamics recorded in virgin and lactating animals supports the identity of the recorded neurons as oxytocin neurons. While this physiological comparison is interesting, it does not constitute direct evidence for cell-type specificity of GCaMP expression. The revised manuscript now acknowledges that in situ hybridization targeting oxytocin mRNA would provide a more reliable validation, but such validation has not been performed. Therefore, uncertainty regarding the identity of the recorded neurons remains, limiting confidence in the interpretation of the calcium imaging data.

(2) Although the authors' interpretation is generally consistent with the data presented, their main conclusions rely heavily on observational findings. Moreover, optogenetic stimulation of PVNOT neurons failed to robustly recapitulate behavioral state transitions (Figs. 6D and S5B). Further interventional experiments remain necessary to rigorously test the authors' interpretation and establish a causal relationship between PVNOT activity and rest-to-active transitions. In particular, loss-of-function approaches targeting the PVNOT system, such as OXTR antagonism, inhibitory optogenetics, or cell-type-specific ablation, remain essential to determine whether perturbation of this system alters behavioral state transitions. Although the authors expanded the Discussion to acknowledge this limitation, the revised manuscript provides no additional experimental evidence addressing it.

Comments on revised version.

I appreciate the authors' efforts to clarify the manuscript and to discuss the limitations more explicitly. Nevertheless, because my major concerns have been addressed primarily through revised interpretation rather than new evidence, my overall assessment of the scientific support for the principal conclusions remains unchanged.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Comments on revised version.

As discussed before, the authors employ a wide range of techniques (FOS IHC, FP for fine scale PVN OXT population dynamics, behavioural analysis, core and surface temperature tracking, physiological recordings to assess AAV specificity, optogenetic activation of PVN OXT neurons, and projection tracing) to address a clear question. The outcomes of these techniques seem to drive the same conclusion that PVN OXT neurons signal transitions from rest to arousal (behavioural and thermogenic) in a state-dependent manner:

- FOS data identifies PVN OXT population activity following behavioural onset

- Ca activity in these cells peaks at behavioural and thermogenic state transitions

- Rump temperature and BAT activity increase at state transition points

- Optogenetic stimulation of these cells recapitulates the thermogenic effects seen during physiological state transitions (in low body temperature animals) with a trending increase in physical activity

Despite the inconclusive IHC results when validating the specificity of their AAV, the virgin female/ lactation experiment is convincing that they are specifically targeting PVN OXT neurons. The rationale for this experiment is clearer in the revised manuscript.

Generally, in terms of the revised manuscript, the authors give strong responses to reviewer comments, either incorporating feedback, or giving clear explanations for the choices they made in the original manuscript. The revised manuscript is clearer about the question the authors aim to address, the reasons for their choice of experiments, and the limitations of the techniques used.

We thank the reviewer for the close attention to the manuscript, the response to reviewers, and the revision, all of which have improved the manuscript.

Criticisms:

I appreciate and agree with the authors' point that this manuscript is more fundamental than simply social basis oxytocin neuron function. This is point is well made by their data, and in the revised text. However, I still believe more behavioural analysis would be welcome to any reader.

They partly justify the lack of behavioural analysis in Figure 6 with the problem of "animal merging" on the SGBS images. However, in Figure 6C, they confirm that, in solo conditions, the SGBS readings are consistent with core body temperature readings. So why not stick to core body temperature, opto stimulate and analyse the social behaviour with DLC (with normal video recordings)?

This is a good suggestion. Because we find that quiescent huddling (paired) bouts were associated with stronger body temperature regulation compared to solo quiescence and other behavioral states, and because PVNOT peak probability and frequency were higher in the paired compared to solo context, these experiments are warranted. We made the following edits to the discussion:

“Future experiments should attempt to disentangle the effects of PVNOT light stimulation on social vs. non-social aspects of these behavioral state transitions; of particular interest would be to examine how light stimulation affects the duration and thermoregulatory control of social huddling.”

The lactation validation still seems out of place in manuscript order. It is a very valuable validation, but it feels more like supplementary data for Figure 1. I feel the authors wanted it as a main figure because of how much work it must have been. In that case, it still makes more sense to include it in Figure 1.

The purpose of the lactation experiment arose from the inadequacy of using histology to test whether AAV-transfected cells were oxytocin-immunoreactive. Because we observed intense oxytocin immunoreactivity in the fibres lining the ventricle, and less reactivity in the cell bodies than what we would have predicted from the Oxytocin-Cre-dependent AAV, we turned to the known physiological relationship between oxytocin-positive neurons and lactation. As such, this study is not associated with Figure 1, which demonstrates our initial, coarse-grained findings relating FOS activity in the PVN and in oxytocin-positive neurons during social thermoregulation.

To your point, it typically does make sense to have the cellular validation “up front” as supporting or background information that enables the downstream experiments. However, what gives this data credibility as a standalone figure is the novel finding that PVNOT neurons display burst-like patterns of activity outside the context of lactation. Previous discussions with experts in the field, along with a review of the literature, unexpectedly led us to the observation that the burst-like patterns we observed during the transition from rest to wake and thermogenesis in virgin females represents a new aspect of oxytocin neuron physiology. Because we wanted to directly compare the new virgin female activity pattern (i.e., Figure 2) with the known lactation activity pattern, we decided it made the most sense to combine the validation aspect with the novel aspect into a standalone figure.

Though their lactation experiment validates that they are targeting PVN OXT neurons, their optogenetic stimulation protocol may not be specifically inducing OXT release from these cells. PVN OXT neurons co-release glutamate but can also release glutamate independently of OXT following lower frequency tonic stimulation. OXT release from PVN neurons requires pulsatile stimulation at a higher frequency (Leithead et al., 2021; Piñol et al., 2014; Lincoln & Wakerley, 1975). In this paper, the authors use a low stimulation frequency (10Hz) and continuous pulse train (20s) to optogenetically manipulate the target PVN population which may bias the cells towards glutamate release over OXT. Therefore, though they find evidence that PVN OXT neurons are involved in driving the transition between states in their other experiments, their optogenetic stimulation may not necessarily involve OXT release/signalling. It may be valuable to separate this out to identify the signalling molecule underlying this behavioural/ thermogenic transition. This could be done by using an opto protocol that recapitulates physiological OXT release.

The authors do however mention that isolating the specific contribution of OXT signalling compared to other co-transmitted molecules was not the aim of this study, so this is not an essential question for this manuscript.

Thank you for this thoughtful point. We agree our optogenetic stimulation experiment should be interpreted as activation of PVNOT neurons rather than as selective evidence for oxytocin release or oxytocin signaling. PVNOT neurons can co-release glutamate (an idea we had also briefly touched upon in the Limitations and caveats section), and the stimulation pattern/frequency may influence the relative engagement of fast glutamatergic transmission versus peptide release. We agree the lactation literature, including Lincoln et al., highlights the importance of high-frequency pulsatile activity for oxytocin release, and that Piñol et al. provide evidence that PVNOT-linked glutamatergic transmission can interact with oxytocin-receptor-dependent modulation of downstream synapses–so thanks for pointing these out.

We made revisions to support our protocol and now acknowledge this important aspect of the neuronal physiology. In Results, we now explain why we selected 10Hz: this frequency was grounded in the study by Fukushima et al. (2022), where 10Hz stimulation of PVNOT terminals in the rMR elicit thermogenic responses and 10Hz stimulation of PVNOT somata produce thermogenesis that’s dependent on oxytocin receptors in rMR.

In the Limitations section, we now cite these three references to include broader context around stimulation frequency and differential release. We emphasize that our optogenetic data demonstrate sufficiency of PVNOT neuron activation, but do not establish whether the downstream thermogenic and behavioral effects are mediated by oxytocin, glutamate, or both. We note that resolving this issue will require future experiments using stimulation-pattern comparisons together with receptor-targeted pharmacology or genetic loss-of-function approaches.

References

Leithead, A. B., Tasker, J. G., & Harony-Nicolas, H. (2021). The interplay between glutamatergic circuits and oxytocin neurons in the hypothalamus and its relevance to neurodevelopmental disorders. Journal of neuroendocrinology, 33(12), e13061. https://doi.org/10.1111/jne.13061

Lincoln, D. W., & Wakerley, J. B. (1975). Factors governing the periodic activation of supraoptic and paraventricular neurosecretory cells during suckling in the rat. The Journal of physiology, 250(2), 443-461. https://doi.org/10.1113/jphysiol.1975.sp011064

Piñol, R. A., Jameson, H., Popratiloff, A., Lee, N. H., & Mendelowitz, D. (2014). Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons. PloS one, 9(11), e112138. https://doi.org/10.1371/journal.pone.0112138

A loss of function experiment to test for sufficiency would be a nice addition to further confirm their claims, but the authors mention that there were technical limitations to their attempts at inhibiting PVN OXT neurons. I appreciate the authors declaring that the DREADDs attempt suffered from unfortunate confounds. But for optogenetic attempts, I don't think they need a closed-loop system to get some useful results. They still can shine the light at "random" moments (that will correspond to random body temperatures) and then separate the data per body temperature.

We thank the reviewer for this constructive suggestion. Such an experiment would strengthen our claims and complement the optogenetic activation (Fig. 6). Reviewer 3 brought up a similar concern.

Building directly on the reviewer’s proposal, we now describe a loss-of-function experiment as an important next step. Optogenetic inhibition of PVNOT neurons can be delivered at pseudo-random times across light and rest phase. Because animals spend extended periods at rest during this phase, a substantial fraction will fall within established rest bouts, which can then be analyzed and stratified by body temperature, as the reviewer notes. The prediction is that silencing PVNOT neurons during rest should prolong the average duration of rest bouts and delay the onset of activity and thermogenesis, relative to matched unstimulated bouts.This provides a direct test of whether PVNOT activity is necessary for the transition from rest to activity. We have revised the Limitations and caveats section to describe this experiment.

“Third, although we show that PVNOT neurons are sufficient to drive thermogenic and behavioral transitions (Fig. 6), we did not perform acute loss-of-function experiments. Such experiments are warranted because decreases in baseline PVNOT calcium activity were associated with transitions toward the onset of quiescence (Fig. 3I-L), suggesting this system may bidirectionally regulate thermo-behavioural state. A tractable next step would be to optogenetically inhibit PVNOT neurons during established rest bouts, delivered at pseudo-random times across the light and rest phase and analyzed post hoc by behavioral state and body temperature; we predict that silencing during rest would prolong the average duration of rest bouts and delay the onset of activity and thermogenesis. Pairing the inhibition with selective oxytocin antagonist (such as L-368,899), would further test whether the thermogenic and autonomic components of these transitions are oxytocin receptor dependent rather than driven by glutamate released by the same neurons.”

Lastly, the mention of Raam et al. 2026 is insufficient. The authors just mention it regarding the potential differences with males, to be explored in future experiments. Even if not using males in the current study doesn't affect the stated conclusions, the fact that they chose females because "their thermo-behavioural states were readily discernible" is a considerable bias. Testing males in this very study might be out of scope, but more discussion is warranted.

We thank the reviewer for this point. We agree that our decision to study females deserves fuller treatment, and we have expanded the Limitations and caveats section accordingly.

We want to be clear about the rationale, because it was methodological rather than an assumption of sex specificity. Our previous study on behavioral thermoregulation in mice (Landen et al., 2024) showed that, during the light/rest phase, females–but not males–display clearly rhythmic episodes of rest and activity that align with transitions between thermoregulatory states, and are therefore well suited to the analyses that form the core of this study. This choice does constrain the generality of our findings to females, but it does not affect the validity of the conclusions we draw, all of which concern PVNOT neurons in females.

At the same time, we agree that whether these mechanisms extend to males is a substantive open question and we now say so explicitly. A direct comparison in males, while beyond the scope of the present study, is an important next step, and the recently defined neural basis of collective thermoregulatory huddling (Raam et al. 2026) offers a useful framework for that work. We have modified the Discussion/Limitations and caveats as follows:

“We focused on females for a practical reason: during the light and rest phase, females show clear, rhythmic bouts of rest and activity, which makes transitions between thermoregulatory states readily discernible and well suited to the analyses around each state transition used here (Landen et al., 2024). This choice constrains the generality of our conclusions, which pertain specifically to females. Because oxytocin signaling can differ between sexes (https://doi.org/10.1016/j.yfrne.2015.04.003), and because the neural control of thermoregulatory behavior may not be identical in males, whether the PVNOT dynamics we describe operate similarly in males remains an open question. Testing males directly was beyond the scope of the present study, but it is an important next step, particularly as the neural basis of collective thermoregulatory huddling has recently begun to be defined (Raam et al. 2026).”

Reviewer #2 (Public review):

Summary:

This is a very interesting study from Vandendoren and colleagues examining the role of PVN oxytocin neurons during thermoregulatory behaviors, in particular during thermoregulatory huddling. The findings are important and have implications for the thermoregulation field as well as the social/naturalistic behavior field. The findings are compelling and use a combination of state-of-the-art tools (photometry, optogenetics, automated behavior tracking, thermal imaging, and core body temperature measurement), often in combination with each other, to produce a rigorous and high-dimensional dataset.

Comments on revised version.

I appreciate the effort the authors have put into addressing all of my questions, and I have no remaining concerns.

Thanks for the comments; they have greatly improved the manuscript.

Reviewer #3 (Public review):

Summary:

This study investigates how the activity of hypothalamic paraventricular oxytocin (PVNOT) neurons relates to physiological states in female mice, with a particular focus on behavioral states and thermogenic sympathetic activity. To address this question, the authors combined automated video-based behavioral classification with calcium imaging of PVNOT neuron activity. Sympathetic thermogenesis was inferred from surface temperature changes measured by infrared thermography, and the authors have made their custom analysis scripts available. The authors report that strong, pulsatile activation of PVNOT neurons was "occasionally" observed immediately before transitions from resting to active states. This observation suggests that PVNOT neuronal activity may facilitate the transition from rest to activity. This phenomenon was observed in both pair-housed and individually housed animals. Taken together, these findings raise the possibility that the oxytocinergic system contributes to naturalistic behavior transitions even in the absence of social interactions. However, concerns regarding the selectivity of GCaMP expression in oxytocin-expressing neurons call into question the validity of the recorded PVNOT neuronal activity.

Strengths:

The oxytocinergic neural system is believed to subserve a wide range of physiological functions. Elucidating these roles requires monitoring PVNOT neuronal activity under diverse behavioral contexts, as well as manipulating this activity to establish causal relationships. In this study, the authors present a technically sound experimental framework that integrates behavioral tracking in both individually and group-housed mice with the monitoring and manipulation of PVNOT neuron activity. This setup represents a valuable methodological resource for researchers investigating the physiological functions of oxytocin.

Thanks for the comments. We are encouraged to hear this framework will open new doors in understanding how the oxytocin system regulates behavior and energy homeostasis.

Weaknesses:

(1) Immunohistochemical validation of selective GCaMP expression in oxytocin-expressing neurons showed that only 24-51% of GCaMP-positive neurons expressed oxytocin. As an alternative approach, the authors demonstrate that GCaMP-expressing PVN neurons in virgin females exhibit calcium peaks during rest-wake transitions with kinetics similar to those observed in PVNOT neurons during early lactation. However, this comparison is based solely on population-level peak profiles and does not provide direct evidence for cell-type specificity of GCaMP expression in oxytocin neurons. This limitation substantially undermines the validity of the optical calcium imaging data. In situ hybridization targeting oxytocin mRNA, rather than immunohistochemistry, may provide a more reliable assessment of expression specificity.

We view our data as showing strong evidence that the recorded neurons include, but may not be limited to, PVNOT neurons for the following two reasons: (1) as the reviewer notes, our longitudinal experiment shows conservation in the physiological and biophysical profile of these neurons in females that went from virgins to parturition and lactation, and (2) as described in Discussion/PVNOT neurons in context of arousal and peptidergic PVN cell-types, non-OT cell-types in the PVN do not show this pulsatile busting profile.

In the “Discussion/Thermal tracking and validation of PVNOT recording specificity” section we had stated “We note that the animals were perfused at ~ZT4–8, before we were aware that somatic OT immunoreactivity in PVN neurons reaches a daily low during the early light phase [56]”. We now add to this the idea, suggested by the reviewer, that “In situ hybridization targeting oxytocin mRNA, rather than immunohistochemistry, may provide a more reliable assessment of expression specificity.”

(2) Although the authors' interpretation is generally consistent with the data presented, their main conclusions rely heavily on observational findings. Moreover, optogenetic stimulation of PVNOT neurons failed to robustly recapitulate behavioral state transitions (Figs. 6D and S5B). Further interventional experiments will be necessary to more rigorously test the authors' interpretation and to establish mechanistic insight into the causal relationship between PVNOT activity and rest-to-active transitions. In particular, loss-of-function approaches targeting the PVNOT system, such as OXTR antagonism, inhibitory DREADDs, or cell-type-specific ablation, will be essential to determine whether perturbation of this system alters behavioral state transitions These points should be addressed in future studies.

Reviewer 1 brought up a similar concern. We have added to the Discussion/Limitations and caveats to address this.

“Third, although we show that PVNOT neurons are sufficient to drive thermogenic and behavioral transitions (Fig. 6), we did not perform acute loss-of-function experiments. Such experiments are warranted because decreases in baseline PVNOT calcium activity were associated with transitions toward the onset of quiescence (Fig. 3I-L), suggesting this system may bidirectionally regulate thermo-behavioural state. A tractable next step would be to optogenetically inhibit PVNOT neurons during established rest bouts, delivered at pseudo-random times across the light and rest phase and analyzed post hoc by behavioral state and body temperature; we predict that silencing during rest would prolong the average duration of rest bouts and delay the onset of activity and thermogenesis. Pairing the inhibition with selective oxytocin antagonist (such as L-368,899), would further test whether the thermogenic and autonomic components of these transitions are oxytocin receptor dependent rather than driven by glutamate released by the same neurons.”

Note: as described in the previous response to reviewers, we have tried inhibitory DREADDs in this system and have concluded that it is of little value because delivering DREADD ligand requires handing the animals for an IP injection—a procedure that disrupts sleep/rest and induces stress hyperthermia.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The authors have answered our criticisms and can proceed as they chose. This is an important paper, and it is the author's choice whether to develop their research here or in a subsequent paper.

Thank you.

Reviewer #2 (Recommendations for the authors):

I thank the authors for citing my pre-print, as suggested by Reviewer 1. The paper has now been published and the authors may like to cite the published version (doi.org/10.1038/s41593-026-02224-0).

Thank you.

Reviewer #3 (Recommendations for the authors):

(1) The authors now interpret their results as indicating that PVNOT activity biases the system toward state transition (from rest to active), rather than acting as a deterministic trigger. This interpretation is reasonable. However, the wording "PVNOT peaks (or neurons) predict transitions to behavioral arousal and thermogenesis" may be misleading. If arousal and thermogenesis occur in more than 80% of cases following PVNOT peaks, then such peaks could reasonably be described as "being predicted". Otherwise, the terminology should be revised for clarity.

We thank the reviewer for raising this question, which touches on a substantive issue in how predictive relationships are characterized. We agree that "predicts" can misleadingly imply a high positive predictive value: i.e., that a large fraction of peaks are followed by transitions.

This is not the claim we intend, nor is it the appropriate statistical criterion. A variable is predictive when it shifts the conditional probability (or, here, the conditional distribution) of the outcome relative to its base rate — the criterion underlying likelihood ratios, relative risk, and signal-detection measures — rather than when it exceeds an absolute occurrence threshold such as 80%. By this standard, a peak can be informative even if transitions do not follow the majority of peaks, provided transitions are substantially more likely (or thermogenically warmer) when a peak precedes them than when one does not.

Our data support precisely this. The logistic regression shows peaks are much more probable immediately before rest offset than at other transitions or at baseline, and our new analysis shows that transitions preceded by peaks carry significantly larger post-offset Tb increases than those without. We are not claiming peaks act as a deterministic trigger, and we agree with the reviewer that they are not present before every transition.

To keep our language aligned with these results, we have revised the wording to avoid "predict" where it could imply high hit-rate determinism, replacing it with comparative phrasing. Accordingly, we have revised the terminology throughout the manuscript: where a claim concerns timing, we now state that peaks “precede” transitions. We have removed “predict”/”predictive” from the section heading, figure legend, introduction and results as follows.

“Then, we discovered that PVNOT calcium dynamics during huddling were associated with increased likelihood of transitions to body warming and arousal.”

“PVNOT neuronal activity precedes transitions towards thermogenesis and behavioral arousal in social and non-social contexts.”

Fig. 3 legend title: “PVNOT peaks are associated with increased likelihood of thermogenic rest-to-active transitions.”

“Thus, PVNOT peaks are at least five-fold more likely to occur near the offset of quiescence/quiescent compared to onset, and signal an increase in physical activity—a correlate of behavioral arousal 53 and a means of increasing metabolic rate and Tb [26]”

“Thus, for nesting and active huddling, PVNOT peaks are two- to three- fold more likely to occur at bout onset than offset.” Dropping flagged word here lol.

“Together these results suggest that elevated PVNOT activity dynamics precede the offset of two rest states (quiescence and quiescent huddling) by approximately 100 seconds, and the onset of two post-quiescence active states (nesting and active huddling) by around 20 seconds, in solo and paired mice respectively.”

“Moreover, PVNOT peaks aligned with the low point of a U-shaped body temperature profile: on average, Tb decreased before, and increased after, the time of the calcium peak in both solo and paired conditions (Fig. 3O,R). Together, these results suggest that PVN^OT peaks occur during a low Tb trough and mark a subsequent rise in Tb.”

(2) Regarding the 400-sec latency of BAT surface temperature increases following optogenetic stimulation, the authors now attribute this delay to slow peptidergic transmission. However, the authors should consider prior findings showing that BAT temperature increased immediately following optogenetic stimulation of PVN→rMR oxytocin neurons in anesthetized rats (Fukushima et al., 2022).

My hunch is that doing this in anesthetized rats gives a stronger signal to noise… not sure if I can back that up though.

At the least we can add a sentence that says “rMR oxytocin neurons immediately increases BAT temperature, while infusion of OXT or NMDA in the rMR results in BAT temperature increases after approximately one minute…” (see Fig. 3,4,5).

We thank the reviewer for redirecting us to Fukushima et al. (2022). We note, however, that in that study the fast-responding variable was BAT sympathetic nerve activity, whereas the BAT temperature itself rose over several minutes following both optogenetic stimulation (their Fig. 4F, quantified at 5 and 10 minutes) and focal rMR infusion of oxytocin or NDMA (their Fig. 5, multiminute traces). This thermal timescale is comparable to the one we observe.

The remaining difference could reflect methodological differences: we stimulated PVNOT somata rather than rMR terminals, measured intrascapular surface rather than BAT temperature directly, and recorded in awake, freely behaving animals (rather than anesthetized animals) in which competing thermoeffector and behavioral processes are active. Consistent with a methodological basis for the delay, focal infusion of oxytocin or NMDA into the rMR in that study increased BAT temperature over roughly a minute (Fukushima et al., 2022). Slow, diffuse peptidergic neuromodulation may further contribute, oxytocin is released from large dense-core vesicles and can act over extended time scales (Ludwig and Leng, 2006; Parmaksiz and Kim, 2025; Qian et al., 2023), although our data cannot isolate this mechanism from the factors above or from fast glutamatergic co-transmission that likely accompanies PVNOT activation (Hrabovszky and Liposits, 2008).

(3) In the previous review, clarification was requested regarding the rationale and histological basis for intravenous FluoroGold injection. While the authors have now added methodological details, they should also incorporate the following explanatory text (previously provided in their rebuttal) into the manuscript for readers unfamiliar with PVN histological analyses:

"Intravenous injection of FluoroGold (FG) was used to histologically differentiate between magnocellular and parvicellular oxytocin neurons in the PVN. Because the posterior pituitary is located outside the blood-brain barrier, i.v. FG is selectively taken up by terminals of magnocellular neurons and retrogradely transported to their cell bodies. This allows us to infer the neuroanatomical identity (magno- vs. parvicellular) of the PVNOT neurons of interest."

We thank the reviewer for this suggestion. We have added the explanatory text to the results subsection, “PVN^OT cellular projections to the rMR”. The text now reads: “rMR cell types in mice, we used FluoroGold (FG to disambiguate magno- vs. parvocellular PVN^OT projections [67] (Fig. S6A-C). Because the posterior pituitary is located outside the blood-brain barrier, intravenous FG is selectively taken up by terminals of magnocellular neurons and retrogradely transported to their cell bodies. This allows us to infer the neuroanatomical identity (magno- vs. parvicellular) of the PVN^OT neurons of interest.”

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Reply to the reviewers

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

In this interesting manuscript the authors present experiments examining the relationship between the obligate intracellular bacterium Chlamydia pneumoniae (Cpn) and the microtubule (MT) cytoskeleton of the host eukaryotic cell using both mammalian cells and yeast as a model. They demonstrate that host microtubule stability contributes to the rate of entry of the bacteria into the cells. Interphase MT architecture is therefore important and correspondingly they show that mitotic cells are less permissive to bacterial entry. Other experiments show that Cpn entry is accompanied by changes to MT stability (measured indirectly via post-translational modifications). Finally they investigate the effects of overexpressing a Cpn virulence factor Cpn0572 in mammalian cells and yeast that they have previously shown to interact with both the actin and MT cytoskeletal networks. Cpn0572 expression induces MT acetylation (stability) which correlates with their previous observations and when expressed ectopically in yeast Cpn0572 suppresses force-dependent MT catastrophe. They propose that chlamydial effectors like Cpn0572 influence MT architecture and stability during Cpn entry, revealing a previously unappreciated role for MT in the bacterial entry process.

• *

Our Response to Summary ____Reviewer #1

We thank the reviewer for the summary of our study. We would like to clarify one point. The mammalian-cell experiments demonstrating that CPn0572 interacts with and alters the MT cytoskeleton, including increased MT acetylation, were reported in our previous work (Höhler et al., 2024; doi: 10.1242/jcs.263450) and are not experiments performed in the present study. In the current manuscript, we use controlled ectopic expression of CPn0572 in S. pombe to analyse its effects on MT dynamics by live-cell imaging and show that CPn0572 reduces catastrophe and depolymerization and suppresses the normal catastrophe response at the cell cortex.

Experimental points

* __Comment 1*__

Fig 1A - to assist interpretation whole cell images similar to those in the supplementary file should be included. It is difficult to relate the images in the small immunofluorescence panels to the phenotypes depicted.* *

Answer to comment 1

We have provided whole cell images of the enlarged images shown in Fig 1A. These are shown in Supp. Fig. S1A. We have added the following statement in the revised results section (line 174): Whole cell images of the zoom images in Fig 1A are shown in Fig S1A.

Comment 2

Cold recovery assay: one wonders what happens to the actin and intermediate filament networks under these conditions and when treated with MT-targetted agents. Some data should be included to rule out that there are additional effects on these systems as off target effects on actin might also influence the data. This is an essential control to support the conclusions drawn.

Answer to comment 2

Cold treatment efficiently perturbs the MT cytoskeleton without causing a comparable gross disruption of the actin cytoskeleton. These images are now included in the revised version of the manuscript (new Supp. Fig. S2). We have added the following sentence to our revised manuscript:(lines 129-130) Cold-treatment did not lead to gross alteration of F-actin organization (Fig. S2A).

Comment 3a

*Is MT repolymerisation synchronous following cold recovery? Its difficult to assess from the included images and sample size. *

Answer to comment 3a

Cold-induced MT depolymerization followed by rewarming results in rapid and highly synchronized MT regrowth in U2OS cells. This has been quantitatively demonstrated by Didier et al. (2008), who showed that MT asters were detectable within 30 s after rewarming and that a centrosome-radiating MT network had reformed in 95% of control U2OS cells within 60 s (Didier et al., 2008; doi: 10.1091/mbc.E06-12-1140). The images shown in our study are representative examples and were not intended to independently quantify the synchrony of MT regrowth.

We have clarified this point in the revised results section as follows: (lines 120–122) “Cold-induced MT depolymerization followed by rewarming results in rapid and highly synchronized re-polymerization of the MT cytoskeleton in U2OS cells [32].”

Comment 3b

Why was this method selected in preference to nocodazole treatment and washout, where synchrony is easier to establish.

Answer to comment 3b

We selected cold-induced MT depolymerization because this approach provides both rapid and synchronized MT regrowth upon rewarming. We do not consider synchrony of MT repolymerization to be inherently easier to establish following nocodazole treatment and washout. In U2OS cells, cold-induced MT depolymerization followed by rewarming is a well-established MT-regrowth assay, and rapid, highly coordinated repolymerization has been demonstrated previously (Didier et al., 2008; doi: 10.1091/mbc.E06-12-1140).

Importantly, the cold-recovery approach is particularly suitable for our experimental question because transfer of the cells from ice to 37°C provides a precisely defined starting point for MT repolymerization and, at the same time, initiates the early infection period. This allows us to analyse C. pneumoniae entry during the first minutes of MT recovery. Nocodazole washout can likewise be used to induce synchronized MT regrowth, but requires drug removal and repeated washing before recovery can be initiated. We therefore chose cold-induced depolymerization as the more appropriate approach for coupling synchronized MT regrowth directly to the very early stages of infection.

Comment 4

Although EB are scored in Fig 1A, they are not shown alongside the microtubules and modified microtubules as described.

Answer to comment 4

We apologize for the misleading representation in Fig 1A. The upper part of the figure is a schematic illustration of the experimental setup, whereas the lower part shows representative microscopic images of the tubulin and acetylated MT phenotype at the indicated time points. The schematic depicts the experimental workflow subsequently used for the infection experiments and was not intended to indicate that EBs were visualized in the images shown in Fig 1A.

We have modified the schematic in the revised manuscript to make this distinction clear and to avoid further confusion.

Comment 5a (please note- we have divided this into several sub comments)

What happens to actin/intermediate filaments following the treatments with Taxol?

Answer to comment 5a

Because actin has a central role in chlamydial entry, we examined whether the Taxol treatment used in our experiments causes a major reorganization of the actin cytoskeleton. U2OS cells treated with DMSO or 10 µM Taxol for 2 h showed prominent F-actin fibres under both conditions, with no obvious gross disruption of the actin cytoskeleton following Taxol treatment (included now as new Fig. S2B). We have added the following sentence to our revised manuscript:(lines 202-204): Under the conditions used, Taxol treated cells showed prominent F-actin fibres with no obvious gross disruption of the actin cytoskeleton (Fig S2B).

We did not analyse intermediate filaments. To our knowledge, there are no data implicating intermediate filaments in chlamydial entry. In C. trachomatis, their reorganization has been described during later inclusion development (Kumar and Valdivia, 2008, doi:10.1016/j.chom.2008.05.018), while in C. pneumoniae-infected cells alterations of vimentin and keratins 8/18 were detected at 48-72 h post-infection (Savijoki et al., 2008, doi:10.1111/j.1574-695X.2008.00488.x).

Comment 5b

Is this specifically targeting the MT under these conditions?

Answer to comment 5b

Taxol directly binds β-tubulin within polymerized MTs and stabilizes the MT lattice (Xiao et al., 2006, doi:10.1073/pnas.0603704103). Nevertheless, because actin and MTs are functionally interconnected, we experimentally assessed the actin cytoskeleton under the exact Taxol conditions used in our infection assay and did not observe a major alteration of F-actin organization.

The purpose of the Taxol/Tubacin comparison was specifically to distinguish MT stabilization from increased tubulin acetylation per se. Taxol stabilizes MTs and consequently increases their acetylation, whereas Tubacin inhibits HDAC6-mediated tubulin deacetylation and increases MT acetylation without stabilizing MTs (Haggarty et al., 2003, doi:10.1073/pnas.0430973100). Under our experimental conditions, both treatments produced a comparable increase in acetylated MTs, whereas only Taxol generated MTs resistant to cold-induced depolymerization. These data are already shown in Fig 2 and Fig S3 and described in the Results (lines 195-220).

Thus, both Taxol and Tubacin increase MT acetylation, but only Taxol stabilizes the MT network and only Taxol increases EB internalization. This is the basis for our conclusion that the long-lived MT state, rather than acetylation alone, is associated with enhanced C. pneumoniae entry.

Comment 5c

How toxic are the treatments and how were they titrated - this does not seem to be included.

Answer to comment 5c

The concentrations and treatment times are already given in both the Fig 2 legend and the Materials and Methods. U2OS cells were treated with 10 µM Taxol or 10 µM Tubacin for 2 h at 37°C before infection (Fig 2, lines 222-232; Materials and Methods, lines 675-686).

These conditions were not established by a de novo dose-response titration in the present study but were selected on the basis of established short-term treatments for manipulating MT stability and acetylation in U2OS cells. Importantly, Jansen et al. used the same conditions - 10 µM Taxol for 2 h and 10 µM Tubacin for 2 h in U2OS cells - to experimentally distinguish stable from acetylated MT populations (Jansen et al., 2023, doi:10.1083/jcb.202106105). Tubacin as an inhibitor of HDAC6-dependent tubulin deacetylation was originally characterized by Haggarty et al. (2003, doi:10.1073/pnas.0430973100).

We additionally verified the intended differential effects of these treatments in our own U2OS cells: both Taxol and Tubacin increased MT acetylation, whereas only Taxol protected MTs against cold-induced depolymerization (Fig 2 and Fig S3; lines 199-209).

We did not perform a separate quantitative cytotoxicity assay. However, treatment was limited to 2 h, we observed no obvious signs of acute cellular deterioration or major changes in cell morphology, and the new F-actin analysis shows no gross disruption of the actin cytoskeleton under the Taxol conditions used.

Comment 6

line 156 - it is unclear what 'microtubule subsets' are referred to here and how the authors arrive at the fact that ~9% of MT are acetylated.

Answer to comment 6

We apologize that the term “microtubule subsets” was not sufficiently defined. By this term, we referred to microtubules distinguished by post-translational modification, in this case acetylated versus non-acetylated MTs. U2OS cells vary considerably in the abundance of acetylated MTs, ranging from cells containing few to cells containing many acetylated MTs.

We have changed the wording in the results section accordingly.

Comment 7

Can the effects of taxol be modulated by changing the bacterial load (MOI)? The dose dependency of taxol is considered but not the reciprocal i.e. whether the effect can be suppressed by increasing the number of bacteria.

Answer to comment 7

We understand the proposed experiment to mean varying the bacterial load (MOI) at a constant taxol concentration to determine whether the taxol-dependent increase in entry becomes less apparent at higher MOIs.

While such an experiment could test how the magnitude of the taxol effect depends on bacterial input, increasing the MOI is not mechanistically reciprocal to the taxol treatment. Taxol alters a host-cell property before addition of C. pneumoniae EBs by stabilizing MTs and increasing the population of long-lived/acetylated MTs. Our experiment therefore addresses whether this pre-existing MT state influences bacterial entry. Increasing the number of bacteria does not reverse or otherwise alter this host-cell state.

Moreover, at high MOIs, a reduced relative difference between control and taxol-treated cells could result simply from saturation of available entry sites or cellular uptake capacity rather than from suppression of the taxol effect. We therefore consider an MOI titration in taxol-treated cells difficult to interpret with respect to the specific question of whether a pre-existing stabilized MT state promotes C. pneumoniae entry.

Comment 8

Figure 1 shows limited co-localisation between EB and MT. Are the authors certain that this is not stochastic? How many EB align with F-actin stress fibres on intermediate filaments under similar conditions. This might be interesting and correct, but controls are lacking to demonstrate specificity, which would make the data more convincing.

Answer to comment 8

We addressed the possibility that the observed EB–MT co-localization reflects stochastic overlap by quantifying the fraction of the cellular area occupied by MTs at the 10-min time point. At this stage of MT recovery, MTs occupied approximately 14% of the cellular area, whereas 35% of internalized EBs co-localized with MTs. Thus, EB–MT association occurred substantially more frequently than expected from MT area coverage alone. Using the MT-covered cellular area as the probability of random overlap, the observed frequency was significantly higher than expected for a random spatial distribution (exact binomial test, p The association was also strongly biased toward a specific MT population. Of the MT-associated EBs, 71% were associated with acetylated MTs, although acetylated MTs represented only approximately 9% of the total MT population under these conditions. This strong enrichment further argues against stochastic overlap.

We deliberately performed this analysis at 10 min after shifting the cells back to 37°C, when MT re-polymerization is still incomplete and individual MT filaments are clearly distinguishable. This minimizes apparent co-localization resulting simply from the dense MT network present in untreated interphase cells.

We do not consider F-actin to provide an equivalent negative control for this question. F-actin remains extensively distributed under these conditions and, importantly, actin is directly involved in chlamydial entry; EB association with actin would therefore be biologically expected rather than a measure of nonspecific cytoskeletal overlap. We did not analyse intermediate filaments. We consider the comparison between the observed EB–MT association and the quantitatively determined probability of random overlap to provide the more direct test of stochastic association.

We have revised the Results section accordingly (lines 154-166): “Next, we analysed the subcellular localization of internalized EBs. At the 10-min time point (Fig. 1A), MT re-polymerization was still incomplete and individual MT filaments were clearly distinguished. Approximately 14% of the cellular area was occupied by MTs, whereas 35% of internalized EBs co-localized with MTs (Fig. 1E, F). Thus, EB association with MTs occurred at a substantially higher frequency than expected from MT area coverage alone. Consistently, comparison with a random spatial distribution using the fraction of MT-covered cellular area as the probability of random EB–MT overlap showed that the observed association was significantly higher than expected by chance (exact binomial test, p __ __

Comment 9

Figure 3. The cell cycle block relies upon RO-3306 which shifts the mitotic cell population from 5% to 36%. Would a thymidine block and release to synchronise the population yield a higher proportion of cells in mitosis? Did the authors consider this approach and exclude it for a defined reason?

Answer to comment 9

Achieving the highest possible proportion of mitotic cells was not the primary requirement for our experiment. Rather, we required a sufficient number of cells entering mitosis within a defined time window after release.

Thymidine arrests cells at the G1/S transition, and cells must subsequently progress through S and G2 before entering mitosis. In contrast, the CDK1 inhibitor RO-3306 arrests cells directly at the G2/M transition and therefore allows rapid and temporally defined entry into mitosis following washout. We therefore considered RO-3306 more suitable for our experimental design. Importantly, our analysis does not rely on the entire synchronized population being mitotic. Following RO-3306 release, mitotic and non-mitotic cells were identified and analysed separately at the single-cell level. Under our conditions, approximately 36% of the population was mitotic, providing sufficient numbers of mitotic cells for quantification of C. pneumoniae infection. Thus, increasing the overall percentage of mitotic cells would not alter the basis of our comparison between mitotic and non-mitotic cells.

Comment 10

Many properties change in mitotic cells in addition to MT architecture. A particular consideration is the profound reorganisation of the actin cytoskeleton and changes in the composition of the plasma membrane, which might also influence the rates of Cpn entry. It is very technically difficult to show that these effects are specifically due to the MT changes and consequently this experiment, while interesting might have many alternative interpretations.

• *

Answer to comment 10

We agree with the reviewer that mitosis involves extensive cellular reorganization in addition to the replacement of the interphase MT network by the mitotic spindle, and that the experiment in Fig 3 cannot by itself attribute the reduced C. pneumoniae entry specifically to changes in MT architecture. Indeed, we already considered this issue in the Results section. We note that endocytosis is generally reduced during early mitosis and is reactivated from anaphase onwards. At the same time, receptor-specific internalization pathways can remain active during mitosis, including EGFR uptake, which is particularly relevant here because EGFR is utilized by C. pneumoniae for host-cell entry. In addition, Fig 3D shows the mitotic reorganization of the actin cytoskeleton by rhodamine-phalloidin staining.

Thus, we agree that changes in actin organization, membrane trafficking and other mitosis-associated cellular properties may contribute to the reduced infection efficiency observed in mitotic cells. Our intention with this experiment was not to establish that the reduction in EB entry is caused exclusively by loss of the interphase MT array. Rather, we asked whether C. pneumoniae entry is altered in a physiological cellular state in which the interphase MT architecture is absent and replaced by the mitotic spindle. We find that mitotic cells remain permissive to EB entry, but infection efficiency is strongly reduced compared with interphase cells.

The MT-specific conclusions of our study are therefore based primarily on the experiments in Figs 1 and 2, in which MT composition and stability are directly analysed or manipulated. The mitotic-cell experiment provides complementary evidence showing that a cellular state lacking the normal interphase MT architecture is associated with strongly reduced EB entry, but we agree that this experiment alone cannot distinguish the contribution of MT reorganization from other mitosis-associated changes.

To make this limitation explicit, we have changed the final sentence of this part of the results section to (lines 276-278):" Thus, EB entry is strongly reduced in mitotic cells, a cellular state characterized by loss of the interphase MT architecture but also by broader changes in cytoskeletal organization and membrane trafficking."__ __

Comment 11a (please note- we have divided this into several sub comments)

While the overexpression experiments in cells and yeast are interesting, these come with caveats about the dose of the effector and the relevance of the system to the pathological process.

Answer to comment 11a

The S. pombe experiments were designed to analyse the effects of CPn0572 on MT dynamics under controlled expression conditions, rather than to reproduce the infection process. CPn0572-mCherry is expressed from a single genome-integrated copy under an inducible TetO promoter, and MT dynamics are analysed after only 1 h of induction. This minimizes dosage heterogeneity and allows early effects of CPn0572 on the MT cytoskeleton to be analysed in living cells. This rationale is already described in the manuscript (lines 384-391).

The manuscript also explicitly acknowledges that TetO-driven expression cannot reproduce the spatially restricted delivery of an effector by the bacterial secretion system; rather, it provides a tractable system in which the consequences of CPn0572 appearance in a eukaryotic cell can be analysed (lines 577-583).

The relevance of the S. pombe system for analysing MT dynamics is addressed in detail in our response to Comment 15. Importantly, CPn0572 has independently been shown to associate with and stabilize MTs in mammalian cells in the infection (Höhler et al., 2024, doi:10.1242/jcs.263450). Thus, the S. pombe experiments are used to resolve how CPn0572 alters MT behaviour, not as a surrogate for C. pneumoniae infection.

Comment 11b

*Could Chlamydia trachomatis TARP that interacts with actin but not MT be used as a control here? *

Answer to comment 11b

We do not consider C. trachomatis TarP an appropriate matched negative control for CPn0572. Although both proteins belong to the TarP family and both modulate actin, their activities toward the actin cytoskeleton are not equivalent. TarP and CPn0572 show distinct patterns of subcellular localization and F-actin association, and CPn0572 additionally binds preassembled F-actin and protects it from cofilin-mediated destabilization (Jewett et al., 2006, doi:10.1073/pnas.0603044103; Jewett et al., 2010, doi:10.1371/journal.ppat.1000997; Zrieq et al., 2017, doi:10.3389/fcimb.2017.00511). CPn0572 additionally associates with MTs (Höhler et al., 2024, doi:10.1242/jcs.263450).

This distinction is important because the actin and MT cytoskeletons are functionally interconnected (Dogterom and Koenderink, 2019, doi:10.1038/s41580-018-0067-1). Consequently, differences in MT behaviour following expression of TarP and CPn0572 could not be attributed specifically to the presence or absence of MT-binding activity. TarP therefore would not constitute a control differing from CPn0572 only in its ability to target MTs.

Comment 11c

While there are interesting effects of CPn0572, which in part relate to the other phenotypes identified in the work, the link between the activities of Cpn0572 overexpression and the infection process are currently weak, beyond the fact that this is one of a number of effectors what have the capability of manipulating the actin and/or MT cytoskeletal networks. The current presentation is therefore speculative.

Answer to comment 11c

We believe that this concern reflects a misunderstanding of how the CPn0572 experiments are positioned within the manuscript. The study addresses three consecutive but distinct questions: (1) whether the pre-existing state of the host MT network influences C. pneumoniae entry; (2) whether C. pneumoniae itself alters the host MT network during early infection; and (3) how a chlamydial protein with MT-modulating activity can alter MT dynamics. CPn0572 is used for the third question as one experimentally tractable example of a chlamydial MT modulator. We do not propose that CPn0572 alone accounts for either the entry phenotype or the infection-induced increase in MT acetylation.

This three-part logic is already stated in the original manuscript. In the Introduction, we first define the permissive host MT state, then describe the infection-induced increase in MT acetylation, and finally introduce controlled expression of CPn0572 to analyse its effect on MT stability (lines 96-104).

The distinction between the first two parts is made particularly explicit in the Discussion: “The preferential infection of cells containing acetylated MTs needs to be distinguished from the increase in MT acetylation observed 1 hr after chlamydial infection.” The manuscript then states that the former represents a host-cell property present before infection, whereas the latter demonstrates that C. pneumoniae remodels the host MT cytoskeleton during infection (lines 531-536).

Likewise, CPn0572 is explicitly introduced as one example with which to investigate how chlamydial proteins might alter MT dynamics. The relevant results sections states that several C. pneumoniae proteins are likely to jointly manipulate the MT cytoskeleton and that CPn0572 was analysed “to start to understand how MTs might be modulated by chlamydial proteins” (lines 374-381). We then show that CPn0572 reduces MT catastrophe and depolymerization and suppresses the normal catastrophe response at the cell cortex, thereby increasing MT persistence (lines 403-410).

The manuscript furthermore explicitly argues against a single-effector model. We state that the infection-induced increase in MT acetylation is likely to result from “multiple EB-associated effectors that together remodel the host MT network” (lines 572-574).

To strengthen this concept experimentally, we have added an experiment in the revised version of the manuscript. We now analysed the combined activity of two chlamydial MT-modulating proteins namely CPn0572 and CPn0443. Thus, if we have two independent MT modulations, we would expect a phenotype intermediate between those produced by either protein alone. CPn0443 was originally identified as a C. pneumoniae protein that strongly alters the interphase MT cytoskeleton by destabilizing it (Wevers et al., 2023, doi:10.3390/ijms24087618) which is opposite to the function of CPn0572.

We therefore asked what happens when these two opposing chlamydial MT modulators are present in the same cell. CPn0572 increases MT occupancy, whereas CPn0443 strongly reduces MT occupancy and longitudinal MT organization. Importantly, simultaneous expression produces an intermediate phenotype: CPn0572 partially counteracts both the CPn0443-induced reduction in MT occupancy and the loss of longitudinal MT organization. These new data (Fig. 7) directly demonstrate that two chlamydial MT modulators can interact at the level of the same cellular MT network. They therefore provide additional experimental support for the concept that host MT remodelling may reflect the combined activities of multiple chlamydial proteins rather than the action of CPn0572 alone.

Thus, the manuscript neither establishes nor claims a one-to-one causal relationship between CPn0572 and the infection-induced MT phenotype. Rather, a pre-existing host MT state affects bacterial entry, C. pneumoniae subsequently remodels the host MT network, and CPn0572 is used as one example to determine how a chlamydial MT-modulating protein can alter MT dynamics.

Comment 12

The Discussion is extensive, and could be reduced to deal with the key findings presented in the work and potentially to address some of the limitations.

__ ____Answer to comment 12__

We have shortened the discussion.

The original discussion already discusses the limitations and boundaries. For example, we state that Taxol-induced MT stabilization may not fully recapitulate the properties of naturally acetylated MTs, that the proposed contribution of MT-dependent membrane trafficking to EB entry remains to be tested, and that the infection-induced MT phenotype is likely to reflect the combined activity of multiple chlamydial proteins rather than a single effector. We also explicitly note that TetO-driven ectopic expression of CPn0572 does not reproduce the spatially restricted delivery of an effector during infection.

In addition, following the reviewer’s specific concern regarding the mitotic-cell experiment, we have clarified this in the revised results section (see comment 10).

Reviewer #1 (Significance (Required)):

* This is an interesting and potentially important study, which will be of interest to researchers studying Cpn, related Chlamydiae and obligate intracellular bacteria, and more generally to those studying the entry of bacterial pathogens into host mammalian cells. Bacterial effectors like Cpn0572 are also of interest to the cell biology community, as studying their activities can reveal novel insights into the regulation and dynamics of the cytoskeleton, relevant to fields including immunology, developmental biology and cancer biology.*

* The manuscript addresses key unresolved questions - for example, it tries to reconcile the potential role for the MT cytoskeleton in bacterial entry, which has been suspected but overtaken by studies of the actin cytoskeleton, where cause and effect and more straightforward. The work investigates role for the posttranslational modification of MT and how this can be reprogrammed by pathogens. Finally, it offers an opportunity to study the interplay between the actin and MT networks and how this might be bridged. This is not well understood in mammalian cells.*

*Notwithstanding the comments above, the individual experiments presented are largely well executed and support the individual conclusions drawn. The weakness of the study is that it is descriptive and correlative. It is an assembly of interesting, but potentially differentially related, experiments examining MT during Cpn infection, essentially in three separate sections i) stability of cellular MT being important for Cpn infection, ii) assessing changes to MT modifications during Cpn infection, iii) the effects of a particular effector amongst many on these processes. While broadly self-supporting in that they all address Cpn and MT, they are presented as cohesive, although the direct relationships between these different topics remains somewhat subjective.

The reviewer actively researches interactions between bacterial pathogens and the host cytoskeleton.*

Our answer to Reviewer 1 (significance)

We appreciate the reviewer’s positive assessment of the interest and potential importance of the study. We would, however, like to clarify both the conceptual connection between the experimental sections and what we consider an important aspect of the novelty of the work.

Bacterial entry into mammalian cells has overwhelmingly been studied as an actin-driven process. Although MTs have been implicated in several bacterial infection cycles, their functions have been studied much less extensively and mainly in post-entry trafficking and later stages of infection. A defined role for different MT states during bacterial entry has remained largely unexplored.

Our central finding is therefore not simply that MTs contribute to C. pneumoniae infection. We show that, within the same mammalian cell population, cells with a particular pre-existing MT state are preferentially infected. Increasing amounts of acetylated/long-lived MTs correlate with increasing entry efficiency, whereas detyrosinated MTs do not, and the Taxol/Tubacin experiments further distinguish MT stability from acetylation itself. To our knowledge, a pre-existing MT state has not previously been identified as a determinant of differential host-cell permissiveness to bacterial entry.

The subsequent experiments build directly on this finding. Having established that a long-lived interphase MT state favors entry, we ask whether C. pneumoniae itself modifies this state and show that early infection increases MT acetylation in a viability-dependent manner. We then use CPn0572 as one mechanistically tractable early effector to ask how a chlamydial MT stabilizer can generate increased MT persistence and show that it suppresses catastrophe and reduces depolymerization.

Thus, while the study does not establish a single linear molecular pathway, the experiments are not an assembly of differentially related observations. Together, they identify a previously unrecognized host-cell MT state that determines permissiveness to bacterial entry, show that Chlamydia subsequently remodels this cytoskeletal system, and provide mechanistic insight into how an early chlamydial effector can generate a persistent MT state.

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

Evidence, reproducibility, and clarity

* Summary

The manuscript by Schenk et al examines how the microtubule state of U2OS cells affects the ability of Chlamydia pneumoniae to enter the host cell. The authors test whether two tubulin post-translational modifications, detyrosination and acetylation, affect bacterium entry and find that cells with higher levels of acetylation display more internalized Chlamydia particles. They also test whether the tubulin state or the post-translational modification is the important factor for Chlamydia entry and find that stabilization of microtubules with taxol treatment is sufficient for increasing the number of internalized particles. They show that entry is higher in interphase cells than mitotic cells. Finally, they show that the Chlamydia protein CPn0572, which was previously shown to alter microtubules in mammalian cells, can alter microtubule dynamics in yeast cells. Overall, this is a straight-forward set of experiments that add information about how the state of microtubules in cells impacts the entry step of Chlamydia infection.*

Response to Summary

We thank the reviewer for this accurate summary of the main findings of our study.

Major comments

Comment 13

In general, the claims and the conclusions are supported by the data. The data in Figures 1–4 address very specific questions and are straightforward. The only issue is that the microtubule immunofluorescence does not look very good. Especially the total tubulin staining. In many cells, it doesn't even look filamentous. Generally, methanol fixation preserves microtubule structures much better than PFA.

__Answer to comment 13 __

We agree that methanol fixation can provide a sharper visualization of filamentous MTs. However, the choice of fixation also depends on the biological question being addressed. Importantly, a study specifically examining fixation effects in Chlamydia trachomatis-infected cells showed that alcohol-based fixation can induce cellular shrinkage and distortion, whereas formaldehyde fixation is used to better preserve overall cellular architecture and the spatial relationships between cellular components (Kokes and Valdivia, 2015; doi: 10.1371/journal.pone.0139153). This consideration was particularly important in our experiments, because our analyses required assessment of the spatial relationship between chlamydial EBs and the host-cell MT cytoskeleton during the early stages of infection.

PFA fixation has also been used in previous studies examining early Chlamydia–host cell interactions, including studies in U2OS cells and during early C. pneumoniae infection (Nans et al., 2014; doi__: _10.1111/cmi.12310_; Mölleken and Hegemann, 2017; doi: 10.1371/journal.ppat.1006556__).

We acknowledge that the total-tubulin staining appears less sharply filamentous in some cells. Nevertheless, MT structures relevant to our analyses are distinguishable under the experimental conditions used, and identical fixation, staining and imaging conditions were applied across the respective experimental groups. Importantly, as also noted by the reviewer, the quantitative data in Figures 1–4 support the conclusions drawn from these experiments.

Comment 14

Its not clear why the inside/outside staining was only used in Fig 2. How do the authors know that the particles in the other figures are inside vs outside the host cell?

__Answer to comment 14 __

Inside/outside staining was used in Fig 2 because these experiments were designed specifically to quantify EB internalization. In contrast, Fig 1 examines changes in MT post-translational modifications, while Fig 3 examines the organization of the MT and actin cytoskeletons. For these experiments, inside/outside staining was technically not feasible because the complete staining combination would require five fluorescence channels, whereas our microscopy setup allows a maximum of four.

We therefore used a spatial approach to assign EB localization in Figs 1 and 3. We initially tested a plasma membrane marker as a means of defining the cell boundary. However, the permeabilization required for subsequent tubulin immunostaining resulted in additional intracellular staining of this marker, preventing an unambiguous identification of the plasma membrane in the final samples. We therefore used the outer boundary of the cytoplasmic α/β-tubulin signal to delineate the cellular area in interphase cells. For the mitotic cells analyzed in Fig 3, the prominent cortical F-actin signal provided a clear definition of the cell boundary.

Each optical section of the complete confocal z-stack was examined individually, and EBs were classified according to their three-dimensional position relative to the delineated cellular area. Thus, whereas Fig 2 uses inside/outside staining to directly distinguish internalized from extracellular EBs, EB localization in Figs 1 and 3 was assigned on the basis of their spatial position within the cellular volume.

Comment 15a (please note- we have divided this into several sub comments)

The use of S. pombe to test the effects of CPn0572-mCherry on microtubule dynamics seems an odd choice. It is not clear whether these findings are relevant to the story since yeast cells are very different from mammalian cells.

Answer to comment 15a

We consider S. pombe a highly appropriate system for analysing the effect of CPn0572 on MT dynamics for four reasons: (1) its simple and exceptionally well-characterized interphase MT cytoskeleton allows changes in MT bundle dynamics to be resolved particularly clearly; (2) fundamental components of the MT system are evolutionarily ancient; (3) yeast-based approaches are established for identifying functions of chlamydial proteins; and (4) Chlamydiae are themselves an ancient lineage of intracellular bacteria, making conserved eukaryotic cellular processes plausible targets for their effectors.

(1) S. pombe interphase cells contain only a small number of well-defined MT bundles whose dynamics and behaviour at the cell cortex can be followed directly and quantitatively in living cells (Drummond and Cross, 2000, doi:10.1016/S0960-9822(00)00570-4; Sawin and Tran, 2006, doi:10.1002/yea.1404). This makes changes in MT bundle dynamics considerably easier to resolve than within the dense MT network of mammalian cells.

(2) Yeast model systems have been exceptionally successful in uncovering fundamental principles of eukaryotic cell biology, as exemplified by Nobel-Prize-for- Medicine winning work on cell-cycle control, vesicle trafficking and autophagy. The tubulin-based MT cytoskeleton is likewise evolutionarily ancient: α-, β- and γ-tubulins and diverse MT motors were already present in the last eukaryotic common ancestor, before diversification of the major eukaryotic lineages (Wickstead and Gull, 2011, doi:10.1083/jcb.201102065). Many years ago, our own work provided a direct example of functional conservation: S. pombe Mal3 belongs to the EB1 family of conserved MT plus-end-tracking proteins that regulate MT dynamics, and human EB1 can substitute for Mal3 in S. pombe (Beinhauer et al., 1997, doi:10.1083/jcb.139.3.717).

__(3) __Several yeast-based approaches successfully investigated chlamydial proteins. A systematic Saccharomyces cerevisiae expression screen identified C. trachomatis proteins that affect yeast cellular functions or target eukaryotic organelles (Sisko et al., 2006, doi:10.1111/j.1365-2958.2006.05074.x), and subsequent yeast-based screening identified chlamydial proteins targeting lipid droplets (Kumar et al., 2006, doi:10.1016/j.cub.2006.06.060). In our own S. pombe screen, 13 of 116 tested C. pneumoniae proteins strongly altered the interphase MT cytoskeleton (Wevers et al., 2023, doi:10.3390/ijms24087618).

(4) Chlamydiae have a long evolutionary history of interaction with eukaryotic cells. The last common ancestor of pathogenic and symbiotic Chlamydiae was already adapted to intracellular survival approximately 700 million years ago and possessed a type III secretion system (Horn et al., 2004, doi:10.1126/science.1096330). It is therefore plausible that chlamydial effectors exploit ancient, conserved features of eukaryotic cell biology, including the MT cytoskeleton.

We therefore do not use S. pombe as a model for mammalian infection itself, but as a tractable system in which effects of CPn0572 on fundamental MT properties can be resolved clearly. Importantly, relevance to mammalian cells is independently supported by our previous demonstration that CPn0572 associates with and stabilizes MTs in mammalian cells (Höhler et al., 2024, doi:10.1242/jcs.263450).

Comment 15b

Furthermore, the Fleig group has already shown that CPn0572 binds to microtubules when ectopically expressed in mammalian cells and causes their stabilization and bundling. It would be useful to see if CPn0572 expression increases acetylation when expressed in mammalian cells

Answer to comment 15b

This experiment has already been performed. Ectopic expression of CPn0572 resulted in an approximately threefold increase in acetylated α-tubulin compared with control cells (Höhler et al., 2024, doi:10.1242/jcs.263450).

Comment 15c

And test whether it directly alters microtubule dynamics using reconstitution assays.

Answer to comment 15c

Reconstitution experiments with purified CPn0572 and tubulin could test whether CPn0572 is sufficient to alter MT dynamics in a minimal in vitro system. However, CPn0572 modulates both the actin and MT cytoskeletons, and such an assay would not establish how its MT effects arise in the cellular context, where additional host components or interactions between the two cytoskeletal systems may contribute. The aim of the present study was to determine whether CPn0572 affects MT organization and dynamics in living cells. Together with our previous demonstration that CPn0572 associates with and stabilizes MTs in mammalian cells (Höhler et al., 2024, doi:10.1242/jcs.263450), the S. pombe experiments establish that CPn0572 alters MT behaviour in a cellular context. Dissecting whether this activity is mediated by a direct interaction with tubulin/MTs or involves additional host factors will require a separate biochemical analysis and is beyond the scope of the present study.

Comment 15d

Furthermore, its role in Chlamydia infection could be tested by deleting the gene from the Chlamydia genome.

Answer to comment 15d

A CPn0572 deletion could address the contribution of this effector to C. pneumoniae infection. However, targeted gene-deletion approaches such as those available for C. trachomatis have not been established for C. pneumoniae (Shima et al., 2018, doi:10.1128/mSphere.00412-18; Wan et al., 2023, doi:10.3389/fimmu.2023.1209879). Thus, deletion of CPn0572 is currently not technically feasible.

Moreover, CPn0572 is a TarP-family effector that modulates both the actin and MT cytoskeletons (Höhler et al., 2024, doi:10.1242/jcs.263450). Consequently, even if a CPn0572 deletion mutant were available, any resulting infection phenotype would reflect the combined loss of its cellular activities and would not by itself establish the specific contribution of its MT-modulating function.

*Minor comments

*

Comment 16

The exact antibodies used for immunofluorescence and western blot should be listed. Some tubulin antibodies are not very good and the reader needs to know that the results are reliable.

__Answer to comment 16 __

We have revised the Materials and Methods section to provide the exact antibodies used for all immunofluorescence and Western blot analyses, including the respective supplier, catalogue number and antibody dilution. In addition, for experiments in which different α-tubulin antibodies were used, we now specify the antibody and its host species for each individual experiment, allowing unambiguous identification of the antibody used.

Comment 17

Fig. 4 – it would be nice to validate the increase in acetylation by immunofluorescence.

Answer to comment 17

We thank the reviewer for this valuable suggestion. We have now independently validated the infection-induced increase in MT acetylation by immunofluorescence microscopy. Representative fluorescence images together with the corresponding quantitative analysis have been added to the revised Fig. 4G, H. Consistent with the Western blot analysis, immunofluorescence showed a significant MOI-dependent increase in acetylated α-tubulin 1 h post infection.

The corresponding text has been added to the revised manuscript (lines 329–332):

“To independently validate the infection-induced increase in MT acetylation observed at 1 hpi, we additionally analysed acetylated MT levels by immunofluorescence microscopy. Consistent with the Western blot results, immunofluorescence analysis showed a significant MOI-dependent increase in MT acetylation (Fig. 4G, H).”

This independent analysis confirms the increase in MT acetylation observed by Western blotting and supports our conclusion that C. pneumoniae infection induces increased MT acetylation.

Comment 18

The term inside-out staining is confusing. I think the authors mean inside/outside staining.

Answer to comment 18

We agree that “inside/outside staining” more accurately describes the staining approach used in our experiments. We have therefore replaced “inside-out staining” with “inside/outside staining” throughout the revised manuscript.

Reviewer #2 (Significance (Required)):

* This study provides new information about how Chlamydia alters the microtubule cytoskeleton to enter mammalian cells. Previous work had shown that Chlamydia utilizes the actin cytoskeleton so this study expands our knowledge of the entry mechanisms. The insights would be more mechanistic if the effects of CPn0572 could be shown in reconstitution assays. The work will be of interest to researchers that study the basic mechanisms of pathogen entry into mammalian cells.*

Our Response to Significance

We thank the reviewer for this assessment. The point concerning a CPn0572 reconstitution assay is addressed in our response to Comment 15c above.

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Referee #2

Evidence, reproducibility and clarity

Summary

The manuscript by Schenk et al examines how the microtubule state of U2OS cells affects the ability of Chlamydia pneumoniae to enter the host cell. The authors test whether two tubulin post-translational modifications, detyrosination and acetylation, affect bacterium entry and find that cells with higher levels of acetylation display more internalized Chlamydia particles. They also test whether the tubulin state or the post-translational modification is the important factor for Chlamydia entry and find that stabilization of microtubules with taxol treatment is sufficient for increasing the number of internalized particles. They show that entry is higher in interphase cells than mitotic cells. Finally, they show that the Chlamydia protein CPn0572, which was previously shown to alter microtubules in mammalian cells, can alter microtubule dynamics in yeast cells. Overall, this is a straight-forward set of experiments that add information about how the state of microtubules in cells impacts the entry step of Chlamydia infection.

Major comments

In general, the claims and the conclusions are supported by the data. The data in Figures 1-4 address very specific questions and are straight-forward. The only issue is that the microtubule immunofluorescence does not look very good. Especially the total tubulin staining. In many cells, it doesn't even look filamentous. Generally, methanol fixation preserves microtubule structures much better than PFA.

Its not clear why the inside/outside staining was only used in Fig 2. How do the authors know that the particles in the other figures are inside vs outside the host cell?

The use of S pombe to test the effects of CPn0572-mCherry on microtubule dynamics seems an odd choice. It is not clear whether these findings are relevant to the story since yeast cells are very different from mammalian cells. Furthermore, the Fleig group has already shown that CPn0572 binds to microtubules when ectopically expressed in mammalian cells and causes their stabilization and bundling. It would be useful to see if CPn0572 expression increases acetylation when expressed in mammalian cells. And test whether it directly alters microtubule dynamics using reconstitution assays. Furthermore, its role in Chlamydia infection could be tested by deleting the gene from the Chlamydia genome.

Minor comments

The exact antibodies used for immunofluorescence and western blot should be listed. Some tubulin antibodies are not very good and the reader needs to know that the results are reliable.

Fig 4 - it would be nice to validate the increase in acetylation by immunofluorescence

The term inside-out staining is confusing. I think the authors mean inside/outside staining

Significance

This study provides new information about how Chlamydia alters the microtubule cytoskeleton to enter mammalian cells. Previous work had shown that Chlamydia utilizes the actin cytoskeleton so this study expands our knowledge of the entry mechanisms. The insights would be more mechanistic if the effects of CPn0572 could be shown in reconstitution assays. The work will be of interest to researchers that study the basic mechanisms of pathogen entry into mammalian cells.

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Referee #1

Evidence, reproducibility and clarity

In this interesting manuscript the authors present experiments examining the relationship between the obligate intracellular bacterium Chlamydia pneumoniae (Cpn) and the microtubule (MT) cytoskeleton of the host eukaryotic cell using both mammalian cells and yeast as a model. They demonstrate that host microtubule stability contributes to the rate of entry of the bacteria into the cells. Interphase MT architecture is therefore important and correspondingly they show that mitotic cells are less permissive to bacterial entry. Other experiments show that Cpn entry is accompanied by changes to MT stability (measured indirectly via post-translational modifications). Finally they investigate the effects of overexpressing a Cpn virulence factor Cpn0572 in mammalian cells and yeast that they have previously shown to interact with both the actin and MT cytoskeletal networks. Cpn0572 expression induces MT acetylation (stability) which correlates with their previous observations and when expressed ectopically in yeast Cpn0572 suppresses force-dependent MT catastrophe.They propose that chlamydial effectors like Cpn0572 influence MT architecture and stability during Cpn entry, revealing a previously unappreciated role for MT in the bacterial entry process.

Experimental points

1. Fig 1A - to assist interpretation whole cell images similar to those in the supplementary file should be included. It is difficult to relate the images in the small immunofluorescence panels to the phenotypes depicted.
2. Cold recovery assay: one wonders what happens to the actin and intermediate filament networks under these conditions and when treated with MT-targetted agents. Some data should be included to rule out that there are additional effects on these systems as off target effects on actin might also influence the data. This is an essential control to support the conclusions drawn.
3. Is MT repolymerisation synchronous following cold recovery? Its difficult to assess from the included images and sample size. Why was this method selected in preference to nocodazole treatment and washout, where synchrony is easier to establish.
4. Although EB are scored in Fig 1A, they are not shown alongside the microtubules and modified microtubules as described.
5. Relating to point 2, what happens to actin/intermediate filaments following the treatments with taxol? Is this specifically targetting the MT under these conditions. How toxic are the treatments and how were they titrated - this does not seem to be included.
6. line 156 - it is unclear what 'microtubule subsets' are referred to here and how the authors arrive at the fact that ~9% of MT are acetylated.
7. Can the effects of taxol be modulated by changing the bacterial load (MOI)? The dose dependency of taxol is considered but not the reciprocal i.e. whether the effect can be suppressed by increasing the number of bacteria.
8. Figure 1 shows limited co-localisation between EB and MT. Are the authors certain that this is not stochastic? How many EB align with F-actin stress fibres on intermediate filaments under similar conditions. This might be interesting and correct, but controls are lacking to demonstrate specificity, which would make the data more convincing.
9. Figure 3. The cell cycle block relies upon RO-3306 which shifts the mitotic cell population from 5% to 36%. Would a thymidine block and release to synchronise the population yield a higher proportion of cells in mitosis? Did the authors consider this approach and exclude it for a defined reason?
10. Many properties change in mitotic cells in addition to MT architecture. A particular consideration is the profound reorganisation of the actin cytoskeleton and changes in the composition of the plasma membrane, which might also influence the rates of Cpn entry. It is very technically difficult to show that these effects are specifically due to the MT changes and consequently this experiment, while interesting might have many alternative interpretations.
11. While the overexpression experiments in cells and yeast are interesting, these come with caveats about the dose of the effector and the relevance of the system to the pathological process. Could Chlamydia trachomatis TARP that interacts with actin but not MT be used as a control here? While there are interesting effects of Cpn0572, which in part relate to the other phenotypes identified in the work, the link between the activities of Cpn0572 overexpression and the infection process are currently weak, beyond the fact that this is one of a number of effectors what have the capability of manipulating the actin and/or MT cytoskeletal networks. The current presentation is therefore speculative.
12. The Discussion is extensive, and could be reduced to deal with the key findings presented in the work and potentially to address some of the limitations.

Significance

This is an interesting and potentially important study, which will be of interest to researchers studying Cpn, related Chlamydiae and obligate intracellular bacteria, and more generally to those studying the entry of bacterial pathogens into host mammalian cells. Bacterial effectors like Cpn0572 are also of interest to the cell biology community, as studying their activities can reveal novel insights into the regulation and dynamics of the cytoskeleton, relevant to fields including immunology, developmental biology and cancer biology.

The manuscript addresses key unresolved questions - for example, it tries to reconcile the potential role for the MT cytoskeleton in bacterial entry, which has been suspected but overtaken by studies of the actin cytoskeleton, where cause and effect and more straightforward. The work investigates role for the posttranslational modification of MT and how this can be reprogrammed by pathogens. Finally, it offers an opportunity to study the interplay between the actin and MT networks and how this might be bridged. This is not well understood in mammalian cells.

Notwithstanding the comments above, the individual experiments presented are largely well executed and support the individual conclusions drawn. The weakness of the study is that it is descriptive and correlative. It is an assembly of interesting, but potentially differentially related, experiments examining MT during Cpn infection, essentially in three separate sections i) stability of cellular MT being important for Cpn infection, ii) assessing changes to MT modifications during Cpn infection, iii) the effects of a particular effector amongst many on these processes. While broadly self-supporting in that they all address Cpn and MT, they are presented as cohesive, although the direct relationships between these different topics remains somewhat subjective.

The reviewer actively researches interactions between bacterial pathogens and the host cytoskeleton.

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Reply to the reviewers

The authors do not wish to provide a response at this time.

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Referee #3

Evidence, reproducibility and clarity

Microtubule dynamics depend on the concentration of soluble αβ-tubulins. When cells detect an increase in soluble αβ-tubulin, they trigger degradation of tubulin mRNAs via a process termed tubulin autoregulation. In this pathway, the ribosome-associated factor TTC5 recognizes nascent amino-terminal autoregulatory MREC and MREI motifs in αβ-tubulins. Upon recognition of the nascent tubulin chain, TTC5 recruits the adaptor protein SCAPER, which in turn engages the CCR4-NOT complex to promote mRNA decay. While this mechanism has been well characterized for α- and β-tubulin transcripts, how cells regulate the abundance of the core microtubule nucleator γ-tubulin remains poorly understood. Here, Assaf et al. show that γ-tubulin-encoding mRNAs are also downregulated through the same tubulin autoregulation pathway (the TTC5-SCAPER-CCR4-NOT axis) in response to elevated soluble αβ-tubulin. They demonstrated that disruption of this pathway, through knockout or mutation of TTC5, SCAPER, or CNOT11, leads to increased γ-tubulin mRNA levels following treatment with the microtubule destabilizer combretastatin A-4 (CA4). Furthermore, mutation of the autoregulatory MPREI motifs in TUBG1 and TUBG2 (TUBGR3H) results in a modest increase in γ-tubulin protein levels. This elevation enhances centrosomal γ-tubulin during mitosis, increases microtubule nucleation capacity (as measured by microtubule regrowth after cold treatment), and ultimately reduces mitotic fidelity.

Major comments:

1. The authors concluded that tubulin autoregulation-associated mitotic defects are largely driven by elevated γ-tubulin protein levels. However, it is somewhat surprising that such a modest increase in γ-tubulin protein level (1.10-, 1.18-, 1.23-fold in TTC5 KO, TUBGR3H, TTC5 KO+TUBGR3H cells, respectively) leads to chromosome alignment and segregation defects. Given that γ-tubulin is a relatively abundant protein, with only a small fraction localized at centrosomes [PMID: 27539480], it remains unclear whether this magnitude of increase is sufficient to account for the observed mitotic defects. To more directly test whether a modest increase in γ-tubulin is sufficient to impair mitotic fidelity, it would be informative to perform live-cell imaging of γ-tubulin-GFP and chromosomes in cells moderately overexpressing wild-type or R3H γ-tubulin (as in Figure 3A), in the presence and absence of siTUBG1. This approach would help determine whether a comparable increase in γ-tubulin levels is sufficient to induce chromosome missegregation. Alternatively, the authors should consider tempering or revising their conclusion.

Minor comments:

1. On page 4, the authors state that measuring TUBG pre-mRNA and mRNA levels allows for distinguishing transcriptional (pre-mRNA) from post-transcriptional (mRNA) regulation, citing reference [33: PMID: 15367667]. While this approach is appropriate, it is unclear why reference [33: PMID: 15367667] is cited here, as it does not appear to directly describe this methodology. Please provide a more relevant reference or clarify the rationale for this citation.
2. For the immunoprecipitation shown in Fig. 3B, an appropriate negative control is needed. For example, a parental cell line lacking γ-tubulin-FLAG expression should be included to assess background binding.
3. Comparing γ-tubulin localization at centrosomes between Fig. 3C, D and Fig. 4B, the difference between parental and mutant cell lines (TTC5 KO, TBUGR3H and TTC5KO + TBUGR3H) appears more pronounced in Fig. 4B. It would be helpful if the authors could quantify centrosomal γ-tubulin localization in Fig. 4B to facilitate a direct comparison. In addition, could the authors comment on whether the cold treatment used in Fig. 4B might influence the soluble αβ-tubulin levels? If so, this could potentially enhance tubulin autoregulation in parental cells, leading to reduced γ-tubulin mRNA levels, while this response would be impaired in the mutant cell lines. Such an effect might contribute to the increased difference in centrosomal γ-tubulin observed under these conditions.

Significance

The manuscript provides significant and new mechanistic insights into microtubule regulation by identifying γ-tubulin as a target of the microtubule autoregulation pathway. It further suggests a new model that cells coordinately adjust both microtubule building blocks and nucleation capacity in response to changes in soluble tubulin pools through a common molecular machinery. The data are clearly presented, the experiments are rigorous, including the well-controlled cell lines, and the manuscript is well written and easy to follow. Based on its quality, novelty, and significance, I strongly support publication.

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Referee #2

Evidence, reproducibility and clarity

In this manuscript, Assaf et al. address an important question by investigating the post-transcriptional-regulation of gtubulin mediated by the tubulin autoregulation mechanism and its functional role in microtubule nucleation and chromosome segregation. Autoregulation of a/b-tubulin has been shown previously, including by the current authors, but whether other tubulin genes are regulated in a similar manner was unknown. Using cell culture models and treatment with microtubule-destabilising/stabilising drugs, they demonstrate that cells regulate gtubulin levels in response to changes in soluble a-b tubulin levels. This regulation depends on the tubulin autoregulation mechanism that the authors previously identified for a/b-tubulin and the authors identify a similar motif in the N-term of gtubulin that is recognized by TTC5. By using a R3H g-tubulin mutant, which specifically disrupts the binding of TTC5, they examine the consequences of deregulated gtubulin biosynthesis. They showed that loss of g-tubulin mRNA regulation moderately increases overall gtubulin levels, which is nevertheless sufficient to enhance microtubule nucleation and induce mitotic defect.

Specific points:

• As pointed out by the authors in the discussion, there needs to be an explanation for how regulating just the mRNA levels of g-tubulin (and not other complex components) can influence the overall protein levels of g-TuRC in order to achieve a functional output for the regulation. The authors provide a nice explanation for when g-tubulin levels drop - this would potentially expose Ubi sites on GCPs that would lead to their ubiquitination and degradation. First, can the authors show that the GCP protein levels are also decreased, like g-tubulin, when they increase the pool of a/b-tubulin dimers? Second, what do the authors think happens when g-tubulin levels are increased? How does this lead to an increase in GCP levels? I don't think this is essential to answer, and certainly not experimentally, but if there is no simple answer then the authors should at least acknowledge this in the discussion.
• Statistics - In several cases (eg. Fig 3D), the authors compare multiple conditions to one control and use Mann-Whitney or t-tests. The need to use one-way ANOVA with correction for multiple comparisons. This is also true when they compare different conditions to each other, while also comparing to controls. The statistical analysis should be done in a single ANOVA analysis, not with multiple different individual tests. E.g. Fig 4C.
• N numbers: In many of the experiments, the authors perform 3-4 biological replicates, plotting the value from each replicate e.g. only 3 or 4 values. In Fig 3D and 4C, however, where they examine g-tubulin levels at centrosomes and microtubule nucleation after cold treatment, they plot the individual centrosome values from each of the replicates. Given that there may be variability between the replicates, and the number of centrosomes are not equal between replicates, it would be better to plot the average value from each replicate, which would better match how data is plotted in other experiments. For example, in Figure 4C, perhaps there is not really a significant difference in microtubule nucleation between TTC5 KO and TTC5 KO + TUBGR3H. This result is a bit odd considering the levels of centrosomal g-tubulin are not different (Fig 3D). The authors try to address this in the discussion (without mentioning the result in the results), but I am not fully convinced by their arguments.
• In the IP shown in Figure 3B, a negative control, such as a construct expressing the FLAG tag alone, is necessary to confirm the specificity of the interactions.
• In Figure 4B, the authors should include an image that is representative of their quantification. Based on the current image, we would conclude that nucleation is reduced in the TTC5 + TubGR3H condition compared to TubGR3H alone, which is not consistent with the quantification shown in Figure 4C.
• In Figure S7A, the authors show that all cell lines show similar timing; however, this is not evident from the examples shown in Fig 5B. This is likely because there is a lot of variation in cell division timing and the authors chose to show example images from cells that happened to have different timings. However, this may appear confusing to readers. I t may be better to include the timing graph in the main figure, along with single images to highlight phenotypes (rather than time series for each condition). The time series images could be moved to supplementary.
• The authors conclude that « partial depletion of g-tubulin restored mitotic fidelity to levels comparable to those of control cells » however in fig 5E, and 5F they only statistically compare the siRNAi control to the siTUBG1 for each genotype (which shows a reduction). But to say that they go back down to control levels, they should also statistically test the difference with the parental siRNAi control.

Minor comments:

• Figure 1C : the line above "SCAPER KO" + should be only on the last 2 columns
• Figure 1E : It would improve clarity if the authors indicated in the figure that the immunoprecipitation was performed using TTC5
• Figure 5E-F : It is not clear from the graph whether the parental cells were also transfected with the siRNA, although this appears to be the case based on the figure legend.
• In the legend of Figure S4E, the cell lines appear to be TTC5 KO and TUBGR3H, rather than mutant TTC5 as indicated in the legend

Significance

The manuscript is well written, the data is well presented and overall the data supports the conclusions being drawn. The results and conclusions are significant and will be of interest to a broad readership.

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 #1

Evidence, reproducibility and clarity

Microtubule nucleation and dynamics are essential for proper microtubule organization and for diverse cellular functions, including cell division. Nucleation is templated by a ring of y-tubulins within the γ-tubulin ring complex (yTuRC) and therefore depends on the cellular availability of γ-tubulin. Nucleation rates are regulated not only by yTuRC activators but also by the pool of soluble αβ-tubulin available for microtubule polymerization. Cellular αβ-tubulin abundance is controlled by a previously identified autoregulatory pathway that fine-tunes αβ-tubulin mRNA stability in response to changes in soluble tubulin levels. Unexpectedly, y-tubulin transcripts were found to be downregulated in parallel with αβ-tubulin under conditions of elevated soluble tubulin, prompting Assaf et al. to investigate whether y-tubulin abundance is regulated by the canonical tubulin autoregulatory mechanism. By combining transcriptomic reanalysis, targeted genetic perturbation, biochemical interaction assays, and functional cell biological approaches, the authors show that γ-tubulin expression is regulated similarly to αβ-tubulin through a post-transcriptional mechanism in response to soluble tubulin levels, identifying the TTC5-SCAPER-CCR4-NOT axis as essential for the decay of γ-tubulin mRNA, as previously shown for αβ-tubulins. The authors also claim tha loss of γ-tubulin mRNA regulation leads to increased γ-tubulin protein levels and enhanced microtubule nucleation, which ultimately affects mitotic fidelity. Interestingly, they show that just subtle changes in γ-tubulin levels are sufficient to compromise mitotic fidelity, suggesting that γ-tubulin-mediated nucleation is a particularly sensitive control point for mitosis.

Major comments:

• Interpretation of TUBG2 transcript data (Fig. 1A, Fig. S1): the authors state that "a similar trend was observed for TUBG2, although the changes in transcript levels were more variable across cell lines." Given that TUBG2 mRNA is expressed at very low levels in non-neuronal cells, conclusions drawn from these datasets are inherently less reliable. This is also reflected by the fact that the authors do not pursue TUBG2 regulation further, in contrast to the detailed analysis of TUBG1. In addition, the trends observed for TUBG2 do not appear as consistent as for TUBG1. I therefore suggest toning down claims regarding TUBG2, or clearly stating that these observations are preliminary. If the authors wish to strengthen this point, experiments in neuronal cells - using microtubule-stabilizing or -destabilizing drugs, or cold-induced changes in soluble αβ-tubulin - would be more appropriate to assess TUBG2 regulation.
• To improve clarity and focus, I suggest reorganizing Figure 1 and Supplementary Figure 1- move TUBG1 transcriptomic data from Fig. S1A to Fig. 1A and TUBG2 data from Fig. 1A to the Supplementary Figures. This would emphasize the main γ-tubulin analyzed throughout the manuscript under conditions of altered soluble αβ-tubulin, while relegating the less robust data to supplementary material. In this context, Fig. 1E could be moved to Fig. S1, while Fig. S1B could be promoted to Fig. 1, as the fold change observed for TUBG1 in rat heart myocardium is relatively large and appears biologically meaningful.
• The final summary of results section 1concludes that "TUBG mRNA is regulated post-transcriptionally in response to changes in soluble αβ-tubulin levels." While the effects of the drugs used are well characterized, microtubule-targeting agents can differ in magnitude and kinetics across cell types. To fully support this statement, it would be important to directly show that soluble αβ-tubulin levels change under the conditions used, for example by biochemical fractionation (polymerized vs soluble tubulin). If this is not feasible, the conclusion should be softened to state that TUBG mRNA responds to microtubule-stabilizing and -destabilizing treatments, rather than inferred changes in soluble αβ-tubulin.
• In Figure 2D, the fold change observed for CNOT11 is less pronounced than for other components of the autoregulation pathway, which is somewhat unexpected given its proposed role as the downstream effector. It would be helpful to clarify whether this regulation is specific to CNOT11, or whether other CCR4-NOT subunits might compensate or contribute. Relatedly, in Fig. S2C, the increase in TUBG1 pre-mRNA levels in CNOT11-KO cells - although not statistically significant - suggests that baseline transcription or mRNA stability may already be altred. This raises some uncertainty regarding the specificity of CNOT11 in regulating γ-tubulin mRNA decay. Additional discussion or clarification would strengthen the interpretation.
• Quantification of centrosomal fluorescence in fgs. 3D, 4C, S5B-C: Centrosomal γ-tubulin and α-tubulin levels are quantified using integrated density measured within a fixed-size circular ROI. Because ROI area is constant, this approach effectively reflects mean fluorescence intensity, rather than total centrosomal content, unless the ROI fully encompasses all centrosomal signal. Given that centrosome size and γ-tubulin spatial distribution may vary between conditions, a brief justification of how the ROI size was chosen, or a control analysis demonstrating robustness to ROI size (beyond fig S6B), would strengthen the conclusions.
• Fig. 3E: the change in γ-tubulin levels is modest; complementary measurements of global γ-tubulin levels would strengthen this conclusion.
• In Fig. 4B, the representative TUBG-R3H cell appears not only to nucleate more microtubules, but also to display faster microtubule polymerization. This is unexpected, as this mutation is proposed to specifically affect γ-tubulin regulation rather than αβ-tubulin availability. In contrast, TTC5-KO cells, where both γ-tubulin and αβ-tubulin regulation may be affected, would more intuitively show such a phenotype. This discrepancy makes the quantification in Fig. 4C difficult to reconcile with the representative images, where microtubule regrowth (based on α-tubulin signal) appears highest in TUBG-R3H cells. Additional clarification or discussion would be helpful.

Minor comments

1. Introduction: while the authors thoroughly describe how cells respond to excess soluble αβ-tubulin through autoregulation, the manuscript does not address how cells initially sense changes in soluble tubulin levels. Even if this mechanism remains unresolved, briefly acknowledging this conceptual gap or discussing current hypotheses in the field would strengthen the Introduction and better frame the study.
2. Figures and legends
   • Fig. 1 and Fig. S1 legends: reference 34 should be reference 36; ***p < 0.001 is mentioned but not shown; Fig. S1B should include a reference along with the GEO accession number.
   • The sentence "In line with previous results for TUBB transcripts (Fig. S1D)" is ambiguous. Please clarify whether this refers to previously published data only, newly generated data, or a combination. Similar clarification may be needed for Fig. S1C and S1E.
   • Fig. 1D: please briefly comment on why microtubule stabilization with PTX leads to a slight but significant decrease in pre-mRNA levels.
   • Replace "decay in tubulin autoregulation" with degradation of TUBA and TUBB mRNA.
   • Text where the call for Fig. 2E appears should read: "TUBG1 and TUBB mRNAs following CA4 treatment in TTC5-KO cells..."
   • Fig. 2F-H: use γ-tubulin and β-tubulin instead of TUBB abd TUBG; the positioning of the γ-tubulin nascent chain within the TTC5 pocket is not clearly illustrated based on author's claim: "the γ-tubulin nascent chain appears to be positioned deeper into the TTC5 pocket (Fig. 2F-G)", and an electrostatic interaction between γ-tubulin R3 and TCC5 D225 should be represented in 2H as in 2G to support the claim that "...the key electrostatic interactions (...) are predicted to be maintained...". Overall, these figures may need adjustement or clarification.
   • Fig. S3B: tubulin should be replaced by α-tubulin.
   • Fig. S3B-C: immunoblots should be accompanied by quantification of the five biological replicates.
   • Fig. S4F-G: these results are compelling and could be moved to the main figure.
   • The metaphase plates shown in Fig. 3C appear relatively homogeneous across conditions, which contrasts with the mitotic defects quantified later (e.g. Fig. 5C). Including representative examples with clearer chromosome alignment or segregation errors, particularly for TTC5-KO cells, would better illustrate the reported phenotypes.
   • In Figs. 3D, 4C, 5B-C, 6C, please specify whether each dot represents a single centrosome or the mean of both centrosomes per cell. Ideally, each dot should correspond to the mean value per cell.
   • Fig. 3E: the change in γ-tubulin levels is modest; complementary measurements of global γ-tubulin levels would strengthen this conclusion.
   • Fig. 4B y-axis should specify α-tubulin fluorescence intensity.
3. Discussion
   • The Discussion would benefit from explicitly acknowledging limitations, such as the relatively - The statement "among γ-TuRC components, only γ-tubulin mRNA is subject to autoregulation" should be rephrased, or additional γ-TuRC subunits should be tested to support this claim.
4. Methods
   • Consider separating imaging procedures from analysis into distinct sections (e.g., Immunofluorescence and Microscopy data analysis).
   • Correct 2.4 µm (not µM).
   • Clarify ROI selection and background subtraction strategy, as discussed above.
5. Optional / stylistic
   • Use consistent placement of "n.s." and asterisks in bar plots.
   • Fig. 2A: if CA4 is included in the schematic, it should be mentioned in the legend. Alternatively, the model could depict a generic increase in soluble αβ-tubulin dimers.
   • Fig. S3A may be unnecessary; instead, consider summarizing homology percentages between αβ-tubulin and γ-tubulin, highlighting higher N-terminal and lower C-terminal conservation to further motivate shared autoregulation.

Significance

This study extends the concept of tubulin autoregulation beyond αβ-tubulin by identifying γ-tubulin as an additional target of the same post-transcriptional regulatory pathway. By doing so, it highlights a coordinated mechanism that links control of microtubule building blocks with regulation of microtubule nucleation capacity, which is central for maintaining proper microtubule organization and mitotic fidelity. The work therefore contributes to a more integrated view of how cells balance microtubule mass, number, and organization during cell division. The tubulin autoregulation pathway involving TTC5, SCAPER, and the CCR4-NOT complex has been well characterized for αβ-tubulin, and previous studies had already established that γ-tubulin levels are tightly controlled, with both overexpression and depletion leading to mitotic defects. While the extension of this regulatory mechanism to γ-tubulin is important, it builds on existing concepts rather than introducing a fundamentally new regulatory pathway. In this sense, the study refines and extends current knowledge by providing mechanistic insight into how γ-tubulin abundance is regulated. The finding that among γ-TuRC components only γ-tubulin mRNA appears to be subject to autoregulation raises interesting questions regarding the specificity and functional consequences of this selective regulation. Although the work does not introduce a completely novel concept, its detailed analysis of γ-tubulin autoregulation and its functional impact on microtubule nucleation and mitotic fidelity will be of interest to the microtubule and cell division research communities.

My expertise lies in microtubule nucleation and minus-end regulation, with a focus on γ-TuRC function and the in vitro reconstitution of its regulation and activity.

é isso mesmo? rever onde consta esse termo nos mod. e as observações deixadas p/ equipe de produção - remover na versão final

na ilustração sobre: HPV persistente: da infecção à lesão precursora Uma sequência causal para leitura clínica; os intervalos variam entre pessoas e não formam um cronômetro rígido. onde consta: e não formam um cronômetro rígido substituir por: a depender da resposta de cada organismo.

na ilustração, onde consta: risco de infertilidade tubária e gravidez ectópica substituir por: risco de infertilidade e gravidez tubária

Após a abordagem sobre os mecanismos de infecção,

onde consta: Comparação concluída Os quatro agentes não seguem a mesma lógica biológica Substituir: lógica biológica por fisiopatologia

os slides estão com erros rever as sugestões de alterações em slides, pela profa: ex: Fig. 2 - substituir lisse por lise gravidez ectópica = gravidez tubárica

na imagem mostra enfermeira

la

rever se é isso mesmo: se de acordo com as alterações nos slides da profa. Janize

reflita sobre

é isso mesmo?

isso vai ser removido?

isso vai ser removido?

nesse momento: indisponível

qual podcast?

nesse momento está indisponível

deixo a obs: nesse momento vídeoaula indisponível

na versão final: remover as descrições/lembretes p/ equipe de produção, inclusive nos slides - revisão em todos os slides, de todos os módulos

Assim ao assistir a vídeoaula, considere que os indicadores epidemiológicos mais recentes do seu território servirão como o fio condutor, na contextualização do conteúdo abordado.

interpretar

needs units

hola

Hola

La quid de la cuestión

eLife Assessment

The manuscript presents a primary important finding, namely that microbial riboflavin-derived MR1 ligands are pharmacological activators of human MAIT cells and that MR1 ligand stimulation enhances MAIT-mediated tumor killing across multiple tumor models. While the evidence presented is generally solid, there are some limitations of the in vitro and in vivo models used, as well as some overstatements about the broader applicability of the research, which should be revised.

Reviewer #1 (Public review):

The manuscript from Zhu et al. identifies microbial riboflavin-derived MR1 ligands as potent pharmacological activators of human MAIT cells and provides evidence that MR1 ligand stimulation can enhance MAIT-mediated tumor killing across multiple solid tumor models. The study is conceptually interesting and supported by a broad combination of human primary samples, tumor cell lines, 3D models, SC transcriptomics, and xenograft experiments. Overall, the data largely support the central conclusion that MR1 ligand stimulation can strongly activate human MAIT cells and enhance anti-tumor cytotoxicity. However, the broader conclusions concerning endogenous MAIT mobilization, tumor specificity, and translational potential are not yet fully supported by the current data and should either be moderated or addressed with additional experiments.

Comments:

(1) The authors use one-way ANOVA throughout the manuscript, but this may not be appropriate for some analyses, particularly when multiple experimental factors are present and their interaction effects need to be considered. For example, Figure 3f appears to involve multiple factors, for which a two-way ANOVA may be more appropriate. Similar issues may apply to other panels.

(2) In Figure 3f, the authors show data from patients #1 and #2 and state that the experiment is representative of three experiments. What does the reported "n=4" represent in this figure?

(3) There appears to be a discrepancy between Figure 3f and Supplementary Figure 3b. The two panels appear to use the same treatment conditions and the same label, and both appear to use patient #1 samples, yet the reported values are different. Please clarify the experimental design and explain the reason for this discrepancy.

In addition, the gating strategy used to define live tumor cells should be clearly described in the figure legend and/or Methods. The authors define "live tumor cells" as MR1/5-OP-RU tetramer-CD45- cells. However, in primary liver tumor samples, the CD45-/tetramer- population may contain other non-hematopoietic cells, such as fibroblasts, and therefore may not exclusively represent tumor cells. The authors should clarify whether additional tumor-specific markers or other criteria were used. The gating strategies for the relevant flow cytometry experiments should be provided in the Supplementary figures.

(4) I have some concerns regarding the claims of "selective activation of anti-tumor inflammatory pathways rather than generalized cytokine release" and "avoiding induction of tumor-supportive mediators." The authors show that MAIT cells stimulated with 5-OP-RU can substantially reduce tumor cell viability. Therefore, the cellular composition of the co-culture is likely to change considerably during the assay, which may affect the absolute levels of cytokines and other soluble mediators detected. For example, reduced tumor cell numbers could lead to lower production of tumor-derived factors such as VEGF, potentially confounding the interpretation that these mediators are not induced by MAIT activation. The authors should consider whether cytokine measurements have been normalized to viable cell numbers or otherwise account for differences in tumor cell abundance.

(5) The in vivo tumor models may show substantial variability between independent experiments. Rather than presenting a single representative experiment, the authors should consider showing pooled data from all independent experiments, with the total number of mice clearly indicated.

(6) Why did the authors use an MR1-overexpressing tumor cell line for the in vivo studies rather than the parental cells with endogenous MR1 expression, together with MR1-KO cells as a negative control? The authors demonstrate that MR1 is detectable across multiple tumor cell lines and that endogenous MR1 expression is sufficient to support MAIT-mediated killing in vitro. Moreover, MR1 overexpression substantially enhances tumor cell susceptibility to MAIT-mediated killing. Therefore, it is unclear whether the strong therapeutic efficacy observed in vivo reflects physiologically relevant MR1 expression or is driven by artificially elevated MR1 expression. An in vivo comparison using parental and MR1-KO tumor cells would substantially strengthen the translational relevance and establish whether the therapeutic effect can be achieved at endogenous levels of MR1.

(7) How is tumor specificity of MAIT achieved ? The authors propose that MAIT-cell activation by MR1 ligands provides an antigen-independent approach for tumor targeting. However, MR1 is broadly expressed and is not tumor specific. While the relative sparing of T and B cells in Figure 7B provides some evidence of cell-type selectivity, this does not establish tumor versus normal tissue specificity. It remains unclear whether activated MAIT cells can discriminate tumor cells from other normal MR1-expressing cells and tissues. This raises an important question regarding the potential systemic toxicity of MAIT cells activated by systemic administration of 5-OP-RU. In particular, could other MR1-expressing cells be targeted when a large number of MAIT cells are simultaneously activated? The authors should consider assessing systemic toxicity in vivo, for example by examining serum ALT/AST levels and tissue pathology, and/or by evaluating the effects of MAIT + 5-OP-RU in tumor-free animals. At least, the potential specificity and safety limitations of systemic MR1 agonism should be discussed.

Reviewer #2 (Public review):

The manuscript by Zhu et al. describes MAIT cell activation by riboflavin metabolites presented by MR1. The authors provide solid evidence for this activation and anti-cancer functional consequence using an array of selected cell lines, primary ex vivo and engineered xenograft models. Broadly, the results are thorough and well controlled, and provide a highly informative insight into the metabolite-MAIT-cancer cell interactions. However, the majority of this work is undertaken using models that preferentially express key targets, and whilst still useful, the (current) broader implications of this research are overstated. Additionally, the suggested MAIT modulation of the tumor microenvironment requires clarification.

Major Comments:

(1) In Figures 2b-d, the authors suggest microbial metabolite stimulation of PBMC cultures increased MAIT cell frequency up to 60%. Whilst their flow data is compelling, the frequency of one population can be influenced by changes in other populations. A form of absolute or relative-to-total count should be used.

(2) The statements regarding cytokine induction in Figure 4e are too strong; many of those inflammatory cytokines are not automatically and consistently tumour-suppressive. The line 299 '...were not induced' may just reflect death of tumor cells. It would be useful to include tumour cell-only controls in Figure 4.

(3) Figure 7 is interesting, but the authors' conclusion that MAIT+5-OP-RU controls the tumor microenvironment is not robustly supported by their evidence.

a) It is not clear how CD14+ cells established a sustained suppressive environment.

b) It is not clear how the peritoneal addition of microbial metabolites 'significantly enhanced MAIT-mediated tumor control'. The authors show that the addition of 5-OP-RU reduced the number of GFP-expressing tumour cells present in peritoneal lavage fluid. There is limited evidence to suggest this occurs through MAIT cells or MR1 in this figure.

c) It is difficult to draw conclusions from peritoneal lavage flow when some experimental groups received cells IP, but then all groups were equally assessed for key populations, and all data are presented as frequencies. The authors should use absolute counts (or similar) to appropriately show changes in cell populations to account for varying total/live/cd45+ cell compartments.

d) It would be necessary at a minimum to include 5-OP-RU-only controls, and ideally include MR1 blocking or the cancer line with MR1 removed. Alongside this, the authors should substantially reduce the strength of their statements on microbial metabolite-MAIT suppression of the tumor microenvironment.

Author response:

Reviewer #1 (Public review):

The manuscript from Zhu et al. identifies microbial riboflavin-derived MR1 ligands as potent pharmacological activators of human MAIT cells and provides evidence that MR1 ligand stimulation can enhance MAIT-mediated tumor killing across multiple solid tumor models. The study is conceptually interesting and supported by a broad combination of human primary samples, tumor cell lines, 3D models, SC transcriptomics, and xenograft experiments. Overall, the data largely support the central conclusion that MR1 ligand stimulation can strongly activate human MAIT cells and enhance anti-tumor cytotoxicity. However, the broader conclusions concerning endogenous MAIT mobilization, tumor specificity, and translational potential are not yet fully supported by the current data and should either be moderated or addressed with additional experiments.

We thank the reviewer for the positive feedback. We will address all comments and suggestions point by point.

Comments:

(1) The authors use one-way ANOVA throughout the manuscript, but this may not be appropriate for some analyses, particularly when multiple experimental factors are present and their interaction effects need to be considered. For example, Figure 3f appears to involve multiple factors, for which a two-way ANOVA may be more appropriate. Similar issues may apply to other panels.

We thank the reviewer for this valuable comment. We will carefully review the statistical analyses and revise the tests as appropriate, including the use of two-way ANOVA where multiple experimental factors are present.

(2) In Figure 3f, the authors show data from patients #1 and #2 and state that the experiment is representative of three experiments. What does the reported "n=4" represent in this figure?

We thank the reviewer for this valuable comment. We will revise the figure legend to clearly define what the reported n = 4 represents.

(3) There appears to be a discrepancy between Figure 3f and Supplementary Figure 3b. The two panels appear to use the same treatment conditions and the same label, and both appear to use patient #1 samples, yet the reported values are different. Please clarify the experimental design and explain the reason for this discrepancy.

In addition, the gating strategy used to define live tumor cells should be clearly described in the figure legend and/or Methods. The authors define "live tumor cells" as MR1/5-OP-RU tetramer-CD45- cells. However, in primary liver tumor samples, the CD45-/tetramer- population may contain other non-hematopoietic cells, such as fibroblasts, and therefore may not exclusively represent tumor cells. The authors should clarify whether additional tumor-specific markers or other criteria were used. The gating strategies for the relevant flow cytometry experiments should be provided in the Supplementary figures.

We thank the reviewer for this valuable comment. Figure 3f (patient #2) and Supplementary Figure 3b (patient #1) were generated using samples from different patients. We will clarify this in the revised manuscript and provide the relevant gating strategies in the Supplementary Information.

(4) I have some concerns regarding the claims of "selective activation of anti-tumor inflammatory pathways rather than generalized cytokine release" and "avoiding induction of tumor-supportive mediators." The authors show that MAIT cells stimulated with 5-OP-RU can substantially reduce tumor cell viability. Therefore, the cellular composition of the co-culture is likely to change considerably during the assay, which may affect the absolute levels of cytokines and other soluble mediators detected. For example, reduced tumor cell numbers could lead to lower production of tumor-derived factors such as VEGF, potentially confounding the interpretation that these mediators are not induced by MAIT activation. The authors should consider whether cytokine measurements have been normalized to viable cell numbers or otherwise account for differences in tumor cell abundance.

We thank the reviewer for this important comment. We agree that differences in tumor cell abundance may affect cytokine measurements. We will moderate our claims accordingly and acknowledge this limitation in the revised manuscript.

(5) The in vivo tumor models may show substantial variability between independent experiments. Rather than presenting a single representative experiment, the authors should consider showing pooled data from all independent experiments, with the total number of mice clearly indicated.

We thank the reviewer for this valuable comment. We will provide pooled data from all independent in vivo experiments and clearly indicate the total number of mice.

(6) Why did the authors use an MR1-overexpressing tumor cell line for the in vivo studies rather than the parental cells with endogenous MR1 expression, together with MR1-KO cells as a negative control? The authors demonstrate that MR1 is detectable across multiple tumor cell lines and that endogenous MR1 expression is sufficient to support MAIT-mediated killing in vitro. Moreover, MR1 overexpression substantially enhances tumor cell susceptibility to MAIT-mediated killing. Therefore, it is unclear whether the strong therapeutic efficacy observed in vivo reflects physiologically relevant MR1 expression or is driven by artificially elevated MR1 expression. An in vivo comparison using parental and MR1-KO tumor cells would substantially strengthen the translational relevance and establish whether the therapeutic effect can be achieved at endogenous levels of MR1.

We thank the reviewer for this important comment. We agree that comparison with endogenous MR1 expression would strengthen the translational relevance of our findings. We will include new in vivo experiment comparing parental tumor cells.

(7) How is tumor specificity of MAIT achieved? The authors propose that MAIT-cell activation by MR1 ligands provides an antigen-independent approach for tumor targeting. However, MR1 is broadly expressed and is not tumor specific. While the relative sparing of T and B cells in Figure 7B provides some evidence of cell-type selectivity, this does not establish tumor versus normal tissue specificity. It remains unclear whether activated MAIT cells can discriminate tumor cells from other normal MR1-expressing cells and tissues. This raises an important question regarding the potential systemic toxicity of MAIT cells activated by systemic administration of 5-OP-RU. In particular, could other MR1-expressing cells be targeted when a large number of MAIT cells are simultaneously activated? The authors should consider assessing systemic toxicity in vivo, for example by examining serum ALT/AST levels and tissue pathology, and/or by evaluating the effects of MAIT + 5-OP-RU in tumor-free animals. At least, the potential specificity and safety limitations of systemic MR1 agonism should be discussed.

We thank the reviewer for this important comment. To further evaluate the potential safety concerns associated with systemic MR1 ligand stimulation, we will include a new experiment assessing the effects of MAIT cells plus 5-OP-RU in tumor-free animals. We will also discuss the potential specificity and safety limitations of systemic MR1 agonism in the revised manuscript.

Reviewer #2 (Public review):

The manuscript by Zhu et al. describes MAIT cell activation by riboflavin metabolites presented by MR1. The authors provide solid evidence for this activation and anti-cancer functional consequence using an array of selected cell lines, primary ex vivo and engineered xenograft models. Broadly, the results are thorough and well controlled, and provide a highly informative insight into the metabolite-MAIT-cancer cell interactions. However, the majority of this work is undertaken using models that preferentially express key targets, and whilst still useful, the (current) broader implications of this research are overstated. Additionally, the suggested MAIT modulation of the tumor microenvironment requires clarification.

We thank the reviewer for the positive feedback. We will address all comments and suggestions point by point.

Major Comments:

(1) In Figures 2b-d, the authors suggest microbial metabolite stimulation of PBMC cultures increased MAIT cell frequency up to 60%. Whilst their flow data is compelling, the frequency of one population can be influenced by changes in other populations. A form of absolute or relative-to-total count should be used.

We thank the reviewer for this valuable comment. We will provide absolute cell counts and/or normalized data to more accurately assess changes in MAIT cell frequency.

(2) The statements regarding cytokine induction in Figure 4e are too strong; many of those inflammatory cytokines are not automatically and consistently tumour-suppressive. The line 299 '...were not induced' may just reflect death of tumor cells. It would be useful to include tumour cell-only controls in Figure 4.

We thank the reviewer for this valuable comment. We agree that the statements regarding cytokine induction should be interpreted more cautiously. We will revise the relevant claims.

(3) Figure 7 is interesting, but the authors' conclusion that MAIT+5-OP-RU controls the tumor microenvironment is not robustly supported by their evidence.

(a) It is not clear how CD14+ cells established a sustained suppressive environment.

We thank the reviewer for this valuable comment. We will include additional experiments to further characterize the contribution of CD14+ cells to the observed suppressive environment.

(b) It is not clear how the peritoneal addition of microbial metabolites 'significantly enhanced MAIT-mediated tumor control'. The authors show that the addition of 5-OP-RU reduced the number of GFP-expressing tumour cells present in peritoneal lavage fluid. There is limited evidence to suggest this occurs through MAIT cells or MR1 in this figure.

We thank the reviewer for this valuable comment. We will include additional T-cell and T-cell + 5-OP-RU control groups to further determine the contribution of MAIT cells to the observed tumor control.

(c) It is difficult to draw conclusions from peritoneal lavage flow when some experimental groups received cells IP, but then all groups were equally assessed for key populations, and all data are presented as frequencies. The authors should use absolute counts (or similar) to appropriately show changes in cell populations to account for varying total/live/cd45+ cell compartments.

We thank the reviewer for this valuable comment. We will provide absolute cell counts, in addition to frequencies, to account for differences in total and viable CD45+ cell numbers.

(d) It would be necessary at a minimum to include 5-OP-RU-only controls, and ideally include MR1 blocking or the cancer line with MR1 removed. Alongside this, the authors should substantially reduce the strength of their statements on microbial metabolite-MAIT suppression of the tumor microenvironment.

We thank the reviewer for this valuable comment. We will include additional T-cell and T-cell + 5-OP-RU control groups and will substantially moderate our statements regarding microbial metabolite-mediated modulation of the tumor microenvironment.

good people aren't good tourists

england + colonization

only knowing colonizer's language to express wrong of colonization

language and colonization -- can only speak back in the criminal's tongue

comparing tourists to animals / irony

rude guests

cont.

why natives can't "get over" colonization

english + colonization + turning everything into england + no where else to go (can't be tourists)

natives + tourists

nature + tourist

tourist tranformation -- pg 14-17

tourist transformation

sun relation w tourist

Document de Synthèse : Projet Stratégique du RNMA (2025-2030)

Résumé Analytique

Le Réseau National des Maisons des Associations (RNMA) engage son nouveau projet stratégique pour la période 2025-2030. Ce plan, fruit d'une coconstruction entre administrateurs, salariés et adhérents, vise à adapter l'action du réseau aux évolutions sociétales tout en réaffirmant ses valeurs fondamentales d'éducation populaire et d'intérêt général.

Les points saillants de cette stratégie incluent :

• La professionnalisation du secteur : Structuration du métier d'Accompagnateur à la Vie Associative (AVA) avec une volonté de reconnaissance officielle (titre RNCP).- L'innovation pédagogique : Déploiement de la plateforme de e-learning EVA et création d'une offre de formation entre pairs.- L'ancrage territorial et scientifique : Renforcement des Observatoires Locaux de la Vie Associative (OLVA) et participation active à des projets de recherche-action sur l'utilité sociale et la coconstruction des politiques publiques.- Un modèle contributif : Une gouvernance horizontale s'appuyant sur des « équipages » (groupes de travail opérationnels) pour favoriser l'implication directe des membres.

I. Fondements et Valeurs du Projet

Le projet stratégique 2025-2030 s'inscrit dans une continuité de valeurs tout en répondant aux défis d'un secteur associatif actuellement « chahuté ».

Valeurs Piliers

• Intérêt général : Chaque décision est motivée par le service aux associations et aux territoires, garantissant une gestion désintéressée et sans profit.- Éducation populaire : Le réseau œuvre pour l'émancipation et l'autonomisation des structures et des individus, en favorisant un accès équitable aux ressources.- Coopération et pouvoir d'agir : L'objectif est de développer l'autonomie des bénévoles et des Maisons des Associations (MDA) pour renforcer la vitalité démocratique.- Développement durable : Intégration explicite des objectifs de développement durable (ODD 17) et des enjeux de transition écologique.

Mission Centrale

La mission du RNMA est de soutenir, accompagner et faire reconnaître ses adhérents comme des espaces ressources indispensables à l'appui et à la mise en synergie des associations locales.

II. Axes Stratégiques Majeurs

Le projet se décline en trois objectifs de transformation, ciblant les citoyens, les associations et les MDA.

Axe 1 : Tisser des liens et créer des synergies

L'enjeu est de passer de l'action individuelle au collectif national pour capitaliser sur les pratiques inspirantes.

• Espaces de rencontre : Organisation de Rencontres Nationales annuelles et de « Blablas MDA » mensuels en visioconférence.- Outils de diffusion : Refonte du centre de ressources numérique, éditorialisation de la newsletter et utilisation accrue de LinkedIn pour faciliter la circulation de l'information.

Axe 2 : Renforcer et promouvoir l’accompagnement associatif

Cet axe vise à professionnaliser les métiers de l'accompagnement pour garantir la pérennité du tissu associatif.

• Reconnaissance du métier AVA (Accompagnateur à la Vie Associative) : Travail sur l'inscription du métier au référentiel de France Travail et création d'un titre professionnel (Bac+3) inscrit au RNCP.- Évaluation de l'utilité sociale : Recherche-action de deux ans visant à doter les MDA d'une méthodologie et d'outils de mesure de leur impact social.- Offre de formation : Lancement prévu en 2026 d'un catalogue de formations hybrides (présentiel/distantiel) axées sur la posture d'accompagnement et la gestion de crise.- Plateforme EVA (E-learning et Vie Associative) : Outil de formation à distance pour les bénévoles et salariés, s'articulant avec les nouveaux dispositifs de certification (Certifaso).

Axe 3 : Valoriser la contribution des associations aux dynamiques territoriales

Il s'agit de démontrer que les associations sont des acteurs majeurs de la cohésion sociale et de l'innovation territoriale.

• Observatoires Locaux de la Vie Associative (OLVA) : Production de données pour mieux comprendre le tissu local et orienter les politiques publiques.- Recherche et Coconstruction : Participation aux projets de recherche « Escape » (sur la coconstruction avec les acteurs publics) et « Aster » (liens entre associations et territoires) pour jeter des ponts entre le monde académique et le terrain.

III. Modèle d'Action et Gouvernance

Le RNMA se définit avant tout comme un réseau vivant où l'expertise est partagée horizontalement.

Structure Organisationnelle

| Instance | Rôle et Fonctionnement | | --- | --- | | Assemblée Générale | Instance souveraine du réseau. | | Conseil d'Administration | Gouvernance horizontale sans bureau, animée par une équipe de membres élus. | | Commissions | Espaces structurels traitant des sujets récurrents (RH, Finances, Plaidoyer, Vie Associative). | | Équipages | Groupes de travail opérationnels et contributifs animés par les salariés sur des thématiques spécifiques (ex: plateforme EVA, OLVA). |

Dynamique Contributive

Le réseau encourage l'implication des permanents des MDA dans les « équipages ». L'objectif est de s'appuyer sur le « pair-à-pair » pour enrichir les outils communs. Le 29 janvier 2025, un séminaire de travail sera dédié à la clarification des rôles et des modalités de participation au sein de ces espaces.

IV. Perspectives et Calendrier Clés

Le déploiement du projet stratégique s'accompagne d'un agenda de mobilisation pour les membres :

• 10-12 décembre 2024 : Rencontres Nationales à Nantes (Thème : les mutualisations).- 16 décembre 2024 : Webinaire sur l'articulation entre la plateforme EVA, le FDVA et Certifaso.- 18 décembre 2024 : Webinaire sur la démarche d'évaluation de l'utilité sociale.- 29 janvier 2025 : Séminaire sur les espaces de coopération et les modalités d'implication dans le réseau.- Février/Mars 2025 : Journée d'échange de pratiques sur les observatoires (OLVA).- 2026 : Lancement du premier catalogue de formation structuré par le réseau.

« Le RNMA est un organisme vivant qui évolue. [...] L'idée est de trouver vraiment les modalités de participation qui soient faisables et réalistes pour tout le monde. » — Synthèse des interventions lors du webinaire stratégique.

eLife Assessment

This study presents a useful finding on using diverse experimental systems to understand how neuromodulatory signals shape glial inflammatory signaling; however, the strength of evidence is inadequate. The astrocyte-enrichment method used may permit contamination by microglia, oligodendrocyte-lineage cells, or neurons, complicating attribution of TNF expression specifically to astrocytes. This concern is compounded by the strong microglial response to Gi manipulation and the lack of quantitative validation of chemogenetic cell-type specificity in vivo. These weaknesses have hindered further evaluation of the claims.

Reviewer #1 (Public review):

In this manuscript, the authors explore whether GPCR signaling in astrocytes affects the production of TNF by astrocytes and, to a lesser extent, microglia. Unfortunately, the method used by the authors to acquire astrocyte-enriched cultures is known to result in meaningful rates of contamination by myeloid cells (microglia and others), oligodendrocyte-lineage cells, and neurons. Alternative methods of generating highly enriched astrocyte cultures, as well as purifying astrocytes with little to no neuronal or myeloid contamination across age and brain regions, have shown no evidence of TNF expression by astrocytes (Zhang et al., J Neurosci, 2014; Zhang et al., Neuron, 2016; Clarke et al., PNAS, 2018). In fact, the paper cited by the authors as demonstrating differences between human and rodent astrocytes found no evidence of TNF expression in immature or mature human astrocytes (Zhang et al., Neuron, 2016). The idea that the majority of the observed TNF transcriptomic signal, at least in culture, comes from myeloid or neuronal contamination also aligns with the authors' observation that myeloid-enriched cultures act identically to astrocyte-enriched cultures.

The authors also use a GFAP virus to drive GPCR signaling in astrocytes and neuronal progenitor cells in their cultures, but, given that these cultures are known to have meaningful contamination by other cell types, such signaling could be due to astrocyte → microglia/neuron signaling or other multicellular pathways that cannot be excluded. Similar concerns mean that we cannot assume the effect of DREADD activation of astrocytes in vivo (Figure 6) reflects a bulk change in TNF expression driven by astrocyte-specific changes rather than by multicellular signaling.

The most compelling evidence for their claim of astrocyte TNF expression comes from the human-induced astrocytes. However, their antibody staining is not sufficient to claim these cells are truly astrocyte-like. Antibody staining is highly prone to non-specificity, as highlighted by the fact that their ALDH1L1 antibody staining appears perfectly nuclear despite ALDH1L1 being a cytoplasmic protein.

To address both the purity concerns of the astrocyte-enriched cultures and the concerns about the astrocyte identity of the induced astrocytes, the authors should perform RNA sequencing. By profiling gene expression in these cultures at the genome-wide level, readers can truly assess the degree of contamination and thus the likelihood of the proposed mechanism (i.e., astrocyte-specific TNF production). Importantly, previous studies have suggested that very little neuronal and myeloid contamination is required to dramatically change cellular responses (Foo et al., Neuron, 2011; Liddelow et al., Nature, 2017).

Reviewer #2 (Public review):

Summary:

Abbasi et al. examine how signaling through the major G-protein pathways (Gs, Gq, and Gi) influences tumor necrosis factor expression in astrocytes and microglia. Using a combination of pharmacological receptor activation, chemogenetic manipulation, primary rodent glial cultures, human induced pluripotent stem cell-derived astrocytes, and an in vivo astrocyte-targeted Gi manipulation, the authors report a broadly consistent pattern in which Gs- and Gq-associated signaling reduces tumor necrosis factor expression, whereas Gi signaling increases it. The study's cross-species and cross-preparation design, spanning astrocytes and microglia as well as in vitro and in vivo systems, provides a potentially valuable framework for understanding how neuromodulatory pathways may regulate glial inflammatory signaling.

Strengths:

A major strength of the study is the breadth of experimental systems used, which includes primary rat glia, human induced pluripotent stem cell-derived astrocytes, and an in vivo manipulation, allowing for comparison across species and levels of biological complexity. The use of chemogenetic receptors in astrocytes provides relatively direct control over Gq and Gi signaling, and these experiments yield consistent effects on both tumor necrosis factor messenger RNA and protein, strengthening the internal validity of the astrocyte findings. The observation that similar directional effects are seen in human-derived astrocytes and in microglial cultures further supports the idea that aspects of this regulatory relationship may be conserved across glial cell types. More broadly, the study addresses an important and timely question about how neuromodulatory signaling pathways interface with glial inflammatory outputs, and it generates a coherent set of observations that could serve as a foundation for more mechanistic work.

Weaknesses:

The central claim that Gs, Gq, and Gi signaling broadly and directly constitute a general regulatory code for tumor necrosis factor expression is more expansive than the current evidence fully supports. In particular, the evidence for Gs-dependent effects is indirect, relying on beta-adrenergic receptor activation and forskolin-mediated adenylyl cyclase stimulation rather than direct manipulation of Gs itself, leaving uncertainty about pathway specificity. More generally, the use of different endogenous receptors to represent each G-protein class in microglia complicates interpretation, since individual receptors may engage additional signaling pathways beyond their canonical G-protein coupling, limiting the extent to which the results can be attributed to G-protein class alone.

The in vivo experiment also does not definitively establish the cellular source of the observed increase in tumor necrosis factor, as measurements are taken from bulk cortical tissue following astrocyte-targeted Gi activation. This leaves open the possibility that the observed changes arise indirectly from other cell types, particularly microglia, which are shown elsewhere in the study to be strongly responsive to Gi-related manipulations. In addition, the specificity of chemogenetic expression in vivo is not quantitatively demonstrated, further limiting cell-type attribution.

There are also important issues related to experimental design and statistical interpretation. Across several experiments, it is unclear whether reported sample sizes reflect independent biological replicates, technical replicates, or imaging fields, which is especially consequential for the human induced pluripotent stem cell-derived astrocyte experiments where donor-level independence is not clearly established. The in vivo design also appears to treat hemispheres as independent observations despite their paired nature, which may inflate statistical independence given the small sample size.

Finally, several conclusions would benefit from more cautious framing. The data support differential regulation of tumor necrosis factor relative to interleukin-1 rather than strict cytokine specificity, and measurements based solely on messenger RNA should not be interpreted as direct evidence of cytokine production. The comparison between glial signaling effects and neuronal excitation or inhibition also juxtaposes fundamentally different biological readouts and should not be interpreted as a direct functional opposition. Overall, while the study provides interesting and potentially important observations, the broader pathway-level and cell-type-specific conclusions are not yet fully established by the current experimental evidence.

Author response:

Reviewer 1 is concerned that our astrocyte enriched cultures have significant contamination of microglia or other myeloid cells, OPCs (and related cells) and neurons. Further, they assert that purified astrocytes do not express TNF.

That astrocytes can’t make TNF directly contradicts our previous paper (Heir, et al., JNeurosci, 2024) showing that the TNF driving homeostatic plasticity is generated by astrocytes. The reviewer seems to want to dispute that paper, which is not really the topic of the current paper (which covers the regulation of TNF production, not whether particular cell types make TNF). The Nedergaard group also saw TNF release from human astrocytes (Wang, et al., 2006). The papers cited by the reviewer (all from the same group) rely on RNAseq data, which has limited depth and cannot distinguish if something is not expressed or simply below threshold. Further, as these datasets were generated from astrocytes isolated from brain (which has normal levels of activity), the astrocytic TNF expression would be very low. Plenty of data supports that astrocytes can express TNF when stimulated (by LPS or other activators), and our previous paper shows that this is also true when neuronal activity is blocked (or absent). But at baseline, astrocyte TNF is quite low and likely undetectable as assayed in those papers.

Here we are using highly purified astrocyte cultures. The reviewer is perhaps unfamiliar with the type of cultures we are using. Given that we use mechanical disruption to remove neurons, followed after 1-2 weeks by shaking to remove microglia, and finally cell passaging, all before experiments, it is surprising that the reviewer thinks there could be neuronal contamination. Neurons cannot survive that procedure, and we do not observe them by morphology or immunostaining, nor see neuronal markers by qPCR.  The microglial contamination is also minimal, as noted in the manuscript, with qPCR for microglia markers is at noise levels (Iba1 Ct value of 34.6), while GFAP shows robust expression (Ct of 16.8; >100,00 fold more than Iba1). But it is possible, if unlikely, that some small number of microglia are making a lot of TNF. However, treating our cultures with the microglia toxin LME (used in Heir, et al., 2024) did not alter our results, further suggesting microglia are not contributing here. Other contaminating cell types (in the OPC lineage, for example) are also possible. However, the majority of cells in our astrocyte-enriched culture are positive for TNF by immunostaining (done while blocking protein export, to prevent any release of TNF). This makes it highly probably that astrocytes are producing TNF (and this production is regulated by g-protein signaling). To verify this, we will show TNF protein in cells co-labeled with astrocyte markers in our upcoming revision of the paper. This will definitively identify astrocytes as producing TNF in these rat cultures. With the human iPSC-derived astrocytes, microglial contamination is not possible (this requires a completely different differentiation protocol). We agree the ALDH1L1 labeling is not as expected, but it is unclear if this is an antibody issue or mis-localized protein. However, the cells also label with S100beta and GFAP, making the astrocyte identity the most likely option by far. We have additional qPCR data showing expression of ALDH1L1 by these cells, in addition to the other astrocyte markers (which will also be added to the revision). The in vivo situation is more complex, and we can’t exclude that astrocyte-DREADD signaling here indirectly alters TNF production in other cells. However, given the direct regulation of astrocyte TNF production in culture, the simplest explanation is that the same is occurring in vivo.

Reviewer 2 was concerned that the Gs data was indirect and the use of pharmacological approaches with microglia. As for Gs signaling, it is a bit unclear what the reviewer is suggesting as an alternative hypothesis. We activate the Gs-coupled beta-adrenergic receptor to reduce TNF levels and get the same effect by activating adenylyl cyclase, the canonical downstream pathway from Gs-coupled receptors. While it is possible that beta-adrenergic receptors could have alternate coupling or that Gs activation acts on additional pathways, it seems odd to argue that Gs would not be working through adenylyl cyclase activation when activating the cyclase yields the same response. Certainly the most parsimonious explanation is that Gs-couple receptors act through adenylyl cyclase to reduce TNF production.

As for the use of pharmacology with microglia, this was the more expedient solution to the difficulty of using AAV virus on microglia. Gathering the necessary Cre and conditional DREADD lines was an impractical solution in terms of time and resources. However, the pharmacology of these receptors is well characterized, as is the g-protein coupling. Given that the results are identical to the results from more specific manipulations in astrocytes, it seems reasonable to conclude that there is a common pattern of GPCR regulation of TNF production. The criticism that non-canonical pathways can be activated by these receptors seems equally true for the DREADDs, as these are just GPCRs with mutated binding sites. If anything, the forskolin experiment is the most specific, yet the reviewer dislikes this approach. The overall consistency of the responses, whether due to DREADD activation, native receptors or direct activation of adenylyl cyclase, is the strongest argument.

This reviewer was also concerned about the limits of in vivo experiments. As noted above, we agree that the in vivo situation is less controlled and indirect effects are possible. However, since the direct action on astrocytes in a defined culture system is identical to what we observe in vivo, the most likely explanation is that the GPCR is having the same effect on TNF production, rather than leading to an unknown secondary signaling which then alters TNF production in microglia (or other cell types).

The remaining concerns about sample size, statistics, etc will be fully addressed in an upcoming revision. All reported n’s are biological replicates. The iPSCs were generated from 3 distinct unrelated individuals.

This caught my attention because it makes me curious because McDonald's is very popular. This also makes me wonder why she ended up deciding to try it.

Évaluation de l’Utilité Sociale des Maisons des Associations (MDA) : Synthèse de la Recherche-Action du RNMA

Résumé Exécutif

Ce document présente les conclusions et la méthodologie issues d'une recherche-action de deux ans (2024-2025) menée par le Réseau National des Maisons des Associations (RNMA).

Soutenu par la Fondation de France et le Fonds Social Européen (FSE+) via l'agence Vise, ce projet visait à définir et mesurer l'utilité sociale des structures d'accompagnement à la vie associative.

Le point central de cette démarche est le concept de renforcement du pouvoir d'agir des associations, décliné à trois niveaux : individuel, collectif et sociétal.

Contrairement aux mesures de performance classiques, cette approche privilégie une analyse qualitative et participative, ancrée dans les valeurs de l'éducation populaire et de l'économie sociale et solidaire (ESS).

Les résultats démontrent que les MDA agissent comme des matrices intermédiaires essentielles, stabilisant le tissu associatif local par un accompagnement de proximité et une posture de "tiers facilitateur".

1. Contexte et Objectifs de la Démarche

1.1 Un projet collectif et ancré

La recherche-action a été initiée par le RNMA en réponse à un appel à projets de la Vise.

Elle a mobilisé six structures d'expérimentation (dont la MDA de Tourcoing et S3A à Hérouville Saint-Clair) et bénéficié de l'appui méthodologique du SIEDEL (Centre international d'études pour le développement local) en tant que "tiers-veilleur".

1.2 Pourquoi évaluer l'utilité sociale ?

Dans un contexte de fragilisation du modèle socio-économique associatif (raréfaction des financements publics, complexification administrative), l'évaluation répond à plusieurs enjeux :

• Politique : Affirmer l'accompagnement comme un acte d'émancipation et non une simple transmission d'outils.

• Légitimité : Rendre visibles les transformations sociales produites par les MDA, au-delà des simples données quantitatives (nombre de rendez-vous ou de formations).

• Pilotage : Offrir un outil de réflexivité pour améliorer les pratiques professionnelles en interne.

2. Le Cadre Conceptuel : Le Pouvoir d'Agir

La recherche a défini l'utilité sociale des MDA à travers leur capacité à générer une "visée transformatrice" sur trois niveaux :

| Niveau de changement | Description | Exemples d'indicateurs | | --- | --- | --- | | Individuel | Développement des capacités des personnes et des structures. | Accès aux ressources, montée en compétences en gestion, autonomie. | | Collectif | Capacité à "faire ensemble" et à s'auto-organiser. | Gouvernance horizontale, coopération inter-associative, mutualisation. | | Sociétal | Place et reconnaissance des associations sur le territoire. | Ancrage territorial, contribution aux dynamiques locales, visibilité. |

3. Méthodologie de l'Évaluation

La démarche proposée aux structures se décline en quatre étapes structurantes :

• Cadrage : Définition du périmètre de l'évaluation, des finalités (plaidoyer ou pilotage) et des ressources disponibles.

• Collecte Interne : Analyse des pratiques par l'équipe et le conseil d'administration. Elle inclut des "récits de pratique" et la cartographie des partenaires et des interactions.

• Collecte Externe :

  • Questionnaires : Envoi à un large public de bénéficiaires pour recueillir des données quantitatives et des effets ressentis.

• Entretiens croisés : Méthode innovante où une MDA interroge les bénéficiaires d'une autre structure (par exemple, Amiens interrogeant les associations de Tourcoing) pour limiter le biais de désirabilité sociale.

• Analyse et Apprentissages : Croisement des données internes et externes pour rédiger un rapport d'utilité sociale et définir un plan d'action.

4. Résultats Transversaux et Enseignements

4.1 Facteurs clés d'utilité sociale

L'analyse des six territoires d'expérimentation révèle des constantes dans l'impact des MDA :

• Rôle structurant : L'accompagnement individuel, basé sur le temps long et la proximité, établit une relation de confiance non normative qui légitime l'expertise de la MDA.

• Catalyseur de collectifs : Les MDA sécurisent les parcours et encouragent l'expérimentation.

Elles transforment les envies individuelles en projets collectifs viables.

• Accessibilité des ressources : La mise à disposition de locaux, de matériel et de conseils en communication est perçue comme un levier fondamental de professionnalisation.

4.2 L'émergence d'une identité commune

Le processus a permis aux professionnels de "mettre des mots sur ce qu'ils font".

Le concept de "visée transformatrice" est désormais central dans le langage des structures participantes, renforçant leur identité liée à l'éducation populaire.

4.3 Points de vigilance

• Hétérogénéité : Les résultats varient selon les configurations partenariales et les moyens humains de chaque MDA.

• Engagement politique : La participation des collectifs aux espaces de décision territoriale reste un domaine où l'effet direct des MDA est plus complexe à mesurer et nécessite un approfondissement.

5. Perspectives et Transfert de la Démarche

5.1 Boîte à outils et ressources

Dès le premier trimestre 2025, le RNMA mettra à disposition une boîte à outils numérique comprenant :

• Le référentiel d'évaluation détaillé et une note méthodologique.

• Des outils ludopédagogiques (jeux de cartes "Action-Vérité", quiz d'auto-positionnement).

• Des modèles types de questionnaires et de grilles d'entretien.- Un guide de bonnes pratiques.

5.2 Accompagnement futur

Le RNMA envisage de proposer des "formations-actions" sur 6 mois pour accompagner de nouveaux collectifs de structures (associatives ou municipales) souhaitant s'approprier cette démarche.

Une articulation est également prévue avec le programme Noura (porté par le Mouvement Associatif, le RNMA et la Fonda) pour changer globalement le regard sur l'évaluation dans le monde associatif, en passant d'une logique de contrôle à une logique d'apprentissage permanent.

Not sure you've made the case for this enough in the intro - I know what you mean, but I think to say this you have to guide the reader through the current problems/fragility with the opioid conversion studies before. I don't think it's unreasonable to have one key recommendation (1)

cite the conversion tables used

should

I think this is really nice, and is your key message? Maybe this could go in the letter to the editor in some form?

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not sure what this is, and doesn't seem immediately obvious from a quick google - bias from a bilinear operation or interpolation? Or is it bilinear basis as in the mathematical form?

recalculation of point estimate and dose range was attempted using a range of conversion values

vary the magnitude of the induced error (??)

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The mathematical...

I think you can make this more direct:

"Studies of perioperative analgesia commonly report differences in opioid requirements between groups using the arthimetic mean of summed TOB. In many cases this is the primary outcome measure used to determine the effectiveness of the intervention."

eLife Assessment

This important study shows that neural responses to visual input during active vision are more closely aligned with self-generated eye movements than with fixation onset. The evidence for neural responses being more aligned to saccade-related events than fixation-related events is convincing, but the evidence for the more novel finding that neural responses are most closely aligned to peak saccade curvature is currently incomplete. This work will be of interest to visual perception and sensory processing researchers.

Reviewer #1 (Public review):

Summary:

This manuscript describes a study examining MEG responses to participants free-viewing natural visual images. The vast majority of our knowledge of visual processing in the brain comes from studies where visual input is presented during fixation and the neural response is measured relative to stimulus onset. Even studies that include eye movements tend to either analyze the data relative to the start of each new fixation, or ignore saccades as noise. The current study simultaneously measures MEG and eye-tracking during active vision, and conducts a variety of analyses testing which of the saccade-related events produce the best alignment to the neural data. Five human participants viewed thousands of complex natural scene images while freely moving their eyes. MEG data were then binned as a function of saccade duration and aligned to different fixation and saccade events. M100 responses were better aligned with the preceding saccade onset than the current fixation onset. An additional analysis showed that when MEG signals were decomposed into independent components, the majority of the components showed more alignment and variance explained from saccade-related events (saccade onset, peak velocity, peak visual motion energy, and peak saccade curvature) compared to fixation-onset-defined events; the strongest performing of these factors was the time of peak saccade curvature. A final analysis compared the similarity of MEG topographies measured from stimulus onset (as would be standard in a static design) to those linked to peak saccade curvature and fixation onset, showing that stimulus onset responses were quite dissimilar to the active vision aligned events.

Strengths:

Overall, I think this is a fundamentally important research question, taking a novel and interesting approach. I very much like the idea behind this study. My enthusiasm is somewhat tempered by the weaknesses described below. However, at the very least I think this study would be valuable as a key launching point for future explorations, and for pushing the field into a much-needed new direction.

Weaknesses:

In its current state, the manuscript seems preliminary/incomplete in terms of both data analysis and engagement with the prior literature.

(1) In terms of the theoretical contribution, there are several potential contributions, some supported more by the data than others, and some more novel than others. In my rough assessment, from most general to most specific:<br /> a. Static vision is not the same as active vision. Supported somewhat by the analyses. Not novel (there are several studies both recent and older making this point, aside from the vaguely referenced sink-source sentence in the discussion), but this is still an understudied/underappreciated area.<br /> b. Neural responses are better aligned to saccade-related events than fixation-related events. Supported pretty compellingly by the analyses, and pretty novel. An important theoretical contribution.<br /> c. Peak saccade curvature is the saccade-related event explaining most variance. An extremely novel finding, but not well supported by the current data. At best, this seems a preliminary, exploratory hint of something to investigate further. It's intriguing but lacking in both empirical support (e.g. is this even consistent across subjects?) and theoretical discussion (what would it mean / what would be the mechanisms of such a link?).

(2) There is a small number of subjects, and for several main analyses, the data are pooled across them. Small N's can be reasonable in cases where there is large data for each subject. But it is standard to show the subjects individually to confirm reliability. Figure 1 does this nicely, but then for the main analyses examining the ICs and variance explained by the different saccade-related events (Figures 2C-F), the data were pooled across subjects. Strong conclusions are being drawn from the pooled data (e.g. highest proportion of explained variance from the peak curvature event), but it's unclear if this is consistent across subjects or potentially dominated by 1 or 2 subjects. Indeed, when the "best" score is presented for each participant (Fig 2E), only 2 of the 5 subjects showed peak saccade curvature as the best. And these results look strikingly different across subjects (P5 doesn't even look anything like an M100 response).

(3) Several parts of the results and methods are hard to follow. I had to read the paper several times to understand it. In many cases, the methods text doesn't even link with the results (e.g. the term "M100" is not anywhere in the methods).

(4) Several parts of the results felt under-explored:<br /> a) The analysis in Figure 1E is very interesting, but it's not reported in enough detail. There are no quantitative results here, just a visual of a distribution and a description of it being broad. I would be particularly interested in seeing the mean alpha reported for the best sensor for each participant (i.e. linking with the rest of that figure).<br /> b) How consistent is the timepoint of peak saccade curvature? It appears to increase with saccade duration, but is it a fixed / consistent percentage of saccade duration? If not, what factors cause it to vary? How similar is this timepoint to the optimal alpha from the analysis in Figure 1E? Would binning the data based on peak saccade curvature instead of saccade duration produce even better alignments for Figure 1D?<br /> c) For the Figure 3 analysis comparing static scene-onset responses to the saccade- and fixation-related responses: I am wondering how much of the difference is actual saccade-related activity vs a true difference in visual processing. It seems the interpretation is that "visual processing", when measured in static contexts, is very different from when measured in active contexts. But what's being compared is not visual processing specifically, but the entire whole-brain MEG response. I think in order to make this conclusion more compelling, there needs to be some way of filtering out these influences. E.g., a study that presents a simulated saccade condition, where a participant keeps their eyes fixated but views snapshots of the visual scene mimicking the exact saccade sequence of another subject.

(5) The discussion felt too thin. See some specific points below. In general, combined with the fact that the results were often hard to follow and sparse, I was left with the impression that this report was being forced into a shorter format than necessary.

(6) How do microsaccades and other types of eye movements fit into this story?

Reviewer #2 (Public review):

Summary:

Although our visual system is continuously analyzing the current visual scene, its processing proceeds in discrete episodes separated by brief eye movements (saccades). It has generally been assumed that the analysis of the next visual snapshot begins in earnest when the eyes land on a new fixated location just after a saccade, but there have been various studies indicating that at least some amount of processing occurs earlier, as the system anticipates the impending eye movement. Here, the authors use magentoencephalography (MEG) measurements to record visually-driven responses and determine at what point exactly the processing of a new visual snapshot begins.

Strengths:

(1) The work is concise and to the point, and the techniques used are a good way to answer the underlying question about visual processing, since they reflect widespread activity in the brain (rather than activity at a particular location or structure).

(2) The use of natural images and extensive data collection from 5 participants is a nice feature of the experimental design which permits characterization of the common effects and of variance across individuals.

(3) The data are analyzed rigorously, but the results are also understood intuitively; for instance, by visual comparison of responses aligned on fixation onset versus saccade onset.

(4) The results provide a clean characterization of when visual analysis begins relative to saccade onset under natural viewing conditions.

Weaknesses:

(1) There were questions about how the scene-onset condition was established, and how data were selected for it.

(2) The significance of the results is slightly overstated; the text would benefit if some of the claims were phrased with a bit more carefully.

(3) In particular, the issue of how motor-related processes (versus stimulus-related content) may determine the processing of the next visual snapshot should be discussed with a bit more nuance.

These are minor weaknesses. Overall, I found the work to be novel and instructive, as it bridges neurophysiological and psychophysical findings in a satisfactory way.

Reviewer #3 (Public review):

Summary:

This manuscript addresses a fundamental question in cognitive neuroscience: which event should serve as the temporal reference for neural processing during natural vision? While fixation onset has traditionally been treated as the analogue of stimulus onset in free-viewing experiments, the authors convincingly demonstrate that this assumption is incomplete.

The study utilizes a remarkable natural-viewing dataset consisting of simultaneous MEG and eye-tracking recordings collected during the exploration of thousands of natural scenes. The authors compare several candidate eye-movement events and evaluate which event best explains the timing of the early M100 response. Across several complementary analyses, saccade-related events consistently outperform fixation onset, with peak saccade curvature emerging as the event that best predicts neural response timing.

Strengths:

A particular strength of the work is that the conclusions do not rely on a single analytical approach. Instead, multiple independent analyses converge on the same interpretation, increasing confidence that the observed timing relationships are robust rather than analysis-specific. The comparison between natural-viewing responses and classical stimulus-onset responses is especially compelling and highlights qualitative differences in their spatiotemporal organization. Of particular conceptual importance, the findings support the broader perspective that perception is intrinsically linked to action and internally generated sensorimotor processes. This aligns well with growing evidence that oculomotor action and active sampling play central roles in perception. The work contributes to an important ongoing shift in how natural vision should be studied experimentally and interpreted theoretically.

Weaknesses:

I identified no major weaknesses in the study. The main limitation is the relatively small number of participants, despite the exceptionally rich dataset. Future work in larger cohorts and across complementary electrophysiological recording modalities will help establish the generalizability of the reported temporal relationships.

Author response:

We would like to thank the editor and reviewers for their constructive and thoughtful feedback. We appreciate the reviewers' assessment that our work addresses a fundamentally important research question through a novel approach. We are also glad the reviewers found our data to be rigorously analysed, and that they valued our focus on the whole cortex rather than localised regions or electrodes. We are encouraged by the overall assessment of our work and welcome the suggestions for improving the manuscript. Below, we summarise how we plan to address the reviewers' comments in our revision:

Analyses

- We will include quantitative results for Fig. 1E that describe the distribution of the optimal alpha values across sensors and participants.

- For Figures 2 and 3 we will include analyses of individual participants in the Appendix.

- We will provide more detailed descriptions on how the timing of peak saccade curvature relates to saccade onset and to the identified optimal alpha value.

Presentation of Methods and Results

- We will phrase our claims and conclusions more carefully and nuanced throughout, ensuring direct coverage by the data and analyses.

- We will revise the currently complex sections of the Methods and Results to improve clarity and readability.

- We will be more explicit about how the data for scene onset were selected.

Revision of the Discussion

- We will extend the Discussion section to address possible mechanisms linking the timing of peak saccade curvature and ERF initiation. We will also provide a more thorough discussion of existing and more recent literature on the topic.

- We will emphasise the main takeaway of the study: the observation that saccade-related processes are more important to the M100 than previously thought, and, reversely, that this component may be less directly related to fixation-locked responses. We will also present our observation of peak saccade curvature as a starting point for future research, as it was not intended as conclusive mechanistic insight into how and why this process relates to early cortical responses.

État des Lieux et Perspectives des Observatoires Locaux de la Vie Associative (OLVA) : Mandat 2026-2027

Résumé Analytique

Ce document de synthèse détaille la démarche des Observatoires Locaux de la Vie Associative (OLVA), pilotée par le Réseau National des Maisons des Associations (RNMA) en partenariat avec l'Institut Français du Monde Associatif (IFMA).

À l'aube de son vingtième anniversaire, la méthode OLVA s'affirme comme un levier stratégique pour pallier l'insuffisance de données locales sur le secteur associatif.

L'enjeu central est de transformer la connaissance en outil d'accompagnement : en objectivant les réalités territoriales, les acteurs publics et associatifs peuvent ajuster les politiques publiques, structurer les projets de mandature et renforcer la coopération territoriale.

Pour la période 2026-2027, la démarche se décline en trois formats modulables (analyse de données existantes, diagnostic simple, ou OLVA complet) et s'enrichit d'une dimension qualitative via le programme de recherche ASTER, visant à saisir les "territoires vécus" au-delà des simples frontières administratives.

1. Missions et Vision Stratégique du RNMA

Le RNMA, tête de réseau regroupant des maisons des associations, des services de collectivités et des structures d'accompagnement, fonde son action sur la conviction que la vitalité démocratique dépend de la solidité du tissu associatif.

Son projet stratégique 2025-2030 s'articule autour de trois axes :

• Animer le réseau : Favoriser le partage de pratiques entre accompagnateurs.

• Reconnaître le métier : Valoriser l'expertise des médiateurs territoriaux et évaluer l'utilité sociale des structures.

• Comprendre la contribution associative : Utiliser la recherche et l'observation pour mesurer l'impact des associations sur les dynamiques territoriales.

2. La Démarche OLVA : Un Outil d'Aide à la Décision

Fondements et Historique

Lancée en 2006, la méthode OLVA adapte les travaux nationaux de Viviane Tchernonog à l'échelle locale.

Elle répond à un besoin critique : si des études nationales existent, les données précises au niveau communal ou intercommunal font souvent défaut.

Objectifs Stratégiques

L'observation n'est pas une fin en soi, mais un processus visant à :

• Objectiver les réalités : Dépasser les intuitions pour s'appuyer sur des chiffres (nombre de bénévoles, budgets, secteurs d'activité).

• Mesurer les évolutions : Comparer les données dans le temps pour évaluer l'efficacité des politiques publiques locales.

• Renforcer le dialogue : Créer une culture commune entre élus, services techniques et dirigeants associatifs.

• Adapter l'offre d'accompagnement : Identifier les besoins précis (formation, locaux, financements) pour coordonner les solutions.

Calendrier et Opportunité Politique

Le début de mandature municipale est identifié comme le moment opportun pour lancer un OLVA.

Cela permet aux élus d'établir un diagnostic de départ, de définir des priorités d'action et de construire une vision partagée pour le reste de leur mandat.

3. Méthodologie et Architecture de l'Observation

La démarche repose sur une approche principalement quantitative, structurée autour d'une enquête par questionnaire de 30 à 40 questions.

Les Quatre Axes de l'Enquête

• Profil des associations : Âge, secteur d'activité, rayonnement géographique.

• Ressources humaines : Nombre de bénévoles, de membres, portrait des dirigeants.

• Moyens de fonctionnement : Emploi (salariés, ETP), ressources financières, locaux.

• Besoins et perspectives : Évolution des effectifs et des financements, attentes vis-à-vis de l'accompagnement.

Les Phases de Mise en Œuvre

Le RNMA propose des "vagues" d'observations synchronisées pour permettre aux territoires de comparer leurs résultats et d'échanger sur leurs pratiques.

| Phase | Durée estimée | Activités Clés | | --- | --- | --- | | Préparation | Septembre - Décembre | Formation, définition des objectifs, mobilisation des partenaires. | | Diffusion | ~ 3 mois | Collecte des réponses (en ligne et/ou papier). | | Analyse | ~ 3 mois | Nettoyage des données, rédaction du rapport, création d'infographies. | | Post-enquête | Continu | Interprétation des chiffres et co-construction d'un plan d'action. |

4. Modalités d'Accompagnement 2026-2027

Pour s'adapter aux ressources humaines et financières variées des territoires, le RNMA propose trois niveaux d'intervention :

• Analyse des données existantes : Travail sur les bases INSEE/Flores.

Limité aux associations employeuses, ce format offre une vision rapide du poids économique sans sollicitation directe des acteurs.

• Diagnostic simple : Questionnaire court et standardisé.

Idéal pour obtenir une photographie globale sans besoin de personnalisation thématique.

• L'OLVA Classique : Questionnaire complet avec possibilité d'ajouter deux thématiques spécifiques (ex: transition écologique, liens avec la municipalité).

Ce format inclut une dynamique inter-acteurs forte et des livrables maquettés.

5. L'Approche Qualitative : Le Programme ASTER

En complément du quantitatif, l'Institut Français du Monde Associatif (IFMA) introduit une dimension qualitative à travers le programme ASTER (Associations, Sociétés et Territoires).

Dépasser les Limites du Chiffre

Si le quantitatif décrit le "quoi", le qualitatif explique le "comment".

L'objectif est de comprendre :

• Les territoires vécus : Comment une association définit son périmètre d'action en fonction des besoins des habitants plutôt que des limites administratives.

• L'ancrage territorial : L'impact réel de la présence physique (locaux) et humaine (bénévoles) sur un quartier.

• Les dynamiques invisibles : Le rôle des collectifs informels ou des petites associations qui agissent "sous les radars" des financements publics mais assurent une intermédiation sociale cruciale.

Méthodes Qualitatives

Le programme ASTER utilise des focus groups, des entretiens et des ateliers participatifs réunissant chercheurs, habitants, élus et agents territoriaux pour analyser les phénomènes de coopération et de compétition au sein de l'écosystème local.

6. Coopération et Soutien Logistique

• Partenariats Institutionnels : Le RNMA collabore avec la DJEPVA et s'inscrit en complémentarité avec des dispositifs comme Guid'Asso pour coordonner l'offre d'accompagnement.

• Ingénierie de Formation : Le RNMA bénéficie de la certification Qualiopi, permettant une prise en charge des coûts de formation par les OPCO pour les structures associatives porteuses d'un observatoire.

• Accompagnement Individualisé : Chaque démarche bénéficie d'un suivi sur mesure (réunions de pilotage, aide à l'interprétation des données) complété par des temps d'échange collectifs entre territoires engagés dans la même vague.

Note : L'interprétation des résultats reste l'étape la plus critique.

Les chiffres ne sont utiles que s'ils sont analysés par les acteurs locaux qui connaissent les spécificités de leur terrain, permettant ainsi de transformer un rapport statistique en une stratégie de développement territorial robuste.

eLife Assessment

This is a valuable manuscript that leverages information already being collected in mosquito surveillance, but that is currently discarded, building on ideas developed during the SARS-CoV-2 pandemic. The work is rigorous, involving ample data from real-world surveillance, independent laboratory testing, and simulation modeling to validate the inference method. The evidence is solid for demonstrating feasibility and biological plausibility.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors were responsive to the previous comments and, where needed, edited the manuscript to improve clarity around assumptions and to highlight specific sensitivity analyses.]

Summary:

This manuscript seeks to make use of information about Ct values from PCR testing of mosquito pools for West Nile virus infection to make inferences about mosquito prevalence and West Nile risk. It does so through analysis of empirical data and simulated data with a realistic agent-based model.

Strengths:

This work is conceptually innovative for mosquito-borne viruses, building on ideas developed primarily during work on SARS-CoV-2. Exploring this topic is worthwhile regardless of the outcome. The use of data, testing in multiple labs, and complementarity of modeling and empirical data analysis are all strengths of the approach.

Weaknesses:

Some of the primary weaknesses include a dependence of the results on relatively narrow model assumptions, and lack of compelling improvement over existing methods. None of these are fatal flaws but are instead modest weaknesses that limit the potential of or excitement about the method.

Reviewer #2 (Public review):

Summary:

The authors extend their previous population-based Ct-value framework for inferring community epidemic trajectories from human infections to vector infections, using mosquitoes as vectors for West Nile virus. They use agent-based modelling to distinguish virus-positive detections arising from non-active infection states from those reflecting active infections, and then apply this framework to mosquito surveillance data from Colorado and Texas.

Overall, this is a well-designed and carefully evaluated study. The manuscript proposes a feasible and potentially valuable framework for vector infection surveillance. The findings are supported by both mechanistic agent-based simulations and applications to real-world mosquito surveillance data, which strengthens the biological plausibility and practical relevance of the proposed approach.

Strengths:

A major strength of the study is its clear methodological extension from human infection surveillance to vector infection surveillance. The agent-based modelling framework provides a useful basis for distinguishing active infections from virus-positive detections that may reflect non-active infection states. The application to surveillance data from two different geographic settings further supports the feasibility of the framework. Overall, the study is carefully designed, and the model schematic and main analyses are generally clear.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript seeks to make use of information about Ct values from PCR testing of mosquito pools for West Nile virus infection to make inferences about mosquito prevalence and West Nile risk. It does so through analysis of empirical data and simulated data with a realistic agent-based model.

Strengths:

This work is conceptually innovative for mosquito-borne viruses, building on ideas developed primarily during work on SARS-CoV-2. Exploring this topic is worthwhile regardless of the outcome. The use of data, testing in multiple labs, and the complementarity of modeling and empirical data analysis are all strengths of the approach.

Weaknesses:

Some of the primary weaknesses include a dependence of the results on relatively narrow model assumptions, and a lack of compelling improvement over existing methods. None of these weaknesses are fatal flaws; they are modest weaknesses that limit the potential of or excitement about the method.

Thank you for the comment

Reviewer #2 (Public review):

Summary:

The authors extend their previous population-based Ct-value framework for inferring community epidemic trajectories from human infections to vector infections, using mosquitoes as vectors for West Nile virus. They use agent-based modelling to distinguish virus-positive detections arising from non-active infection states from those reflecting active infections, and then apply this framework to mosquito surveillance data from Colorado and Texas.

Overall, this is a well-designed and carefully evaluated study. The manuscript proposes a feasible and potentially valuable framework for vector infection surveillance. The findings are supported by both mechanistic agent-based simulations and applications to real-world mosquito surveillance data, which strengthens the biological plausibility and practical relevance of the proposed approach.

Strengths:

A major strength of the study is its clear methodological extension from human infection surveillance to vector infection surveillance. The agent-based modelling framework provides a useful basis for distinguishing active infections from virus-positive detections that may reflect non-active infection states. The application to surveillance data from two different geographic settings further supports the feasibility of the framework. Overall, the study is carefully designed, and the model schematic and main analyses are generally clear.

Weaknesses:

(1) It would be helpful if the authors could provide plots showing variation across locations and over time. This would further support the claim made in the paragraph at lines 101-107.

Thank you for the comment. Our supplementary Material Figures S5 and S6 already included these visualisations. However, we note that these were not referenced in the manuscript. We have now referenced these within the lines:

“First, the variation we observe is consistent across five trapping seasons and two states (Figures S5 and S6).”

(2) Figure 2: The model schematic is clear in terms of workflow, but it would benefit from more information on model parameterization. In particular, it would be helpful to clarify which parameters or migration rates were estimated from the data and which were assumed based on prior literature.

Thank you for the comment. All the parameters are used from the literature and recorded in the Supplementary Material. However, we have now added a note in the caption of Figure 2, referencing the Supplementary Material as below:

“Overall structure of the agent-based model (parameters were derived from the literature; see Supplementary Material S2, S3 and S4)”

(3) Figure 4: I wonder whether the authors examined how changes in the proportion of mosquitoes with static viral-kinetics trajectories would affect the observed bimodal distribution. Relatedly, it would be useful to know whether there is a threshold proportion at which the method becomes less able to distinguish active from static viral-kinetics patterns.

Thank you for the comment. We have conducted this in analysis and have already included the relevant figures in the Supplementary Material, In particular, Figures S14 (in Section S7) and S22. We have referenced Figure S14 where we discuss the proportion of mosquitoes with static viral-kinetics trajectories that would affect the observed bimodal distribution. However, we had not included a reference to Figure S22, where we illustrate the proportions at which the method becomes less able to distinguish active from static viral-kinetics patterns. We have now included this reference in the same line.

“We found that the simulated pooled Ct values aligned well with the observed data when the percentage viral load inherited from birds was 100% and the probability of a productive or non-productive infection in the mosquitoes was 0.5, capturing the bimodal distribution of low Ct values (from productively infected mosquitoes) and high Ct values (from non-productively infected mosquitoes) (Figure 4 (B), (C) & (D); Supplementary Material S7.1, Figure S14 for Ct distributions of viral inheritance probability vs. model change probability and Figure S22 for the accuracy and confidence-interval coverage across different productive infection proportions).”

Conclusion:

Overall, the evidence is reasonably strong for demonstrating the feasibility and biological plausibility of the proposed framework. Some conclusions would be further strengthened by additional sensitivity analyses on key assumptions, especially the proportion of static viral-kinetics trajectories and spatial-temporal heterogeneity across surveillance sites.

Recommendations for the authors:

Reviewer #1 (Recommendations for th e authors):

(1) ll 70-73 - There are a lot of ideas in this sentence. It would be useful to support this with a schematic figure or something like that, which illustrates the conceptual predictions made here. Such a step is necessary given the novelty of what is being explored here.

This portion of the introduction has been simplified to better introduce the core observations that formed our hypothesis:

“The substantial variation in viral quantities observed during cross-sectional entomological surveillance suggests more complex vector/virus interactions, and the precedence set by population SARS-CoV-2 testing in humans suggests that Ct value data for WNV in mosquitoes could inform metrics of disease risk to humans. However, there are substantial differences in the epidemiology and biology of WNV infection in mosquitoes with SARS-CoV-2 in humans.”

We have chosen not to include an additional schematic figure, as the core idea (population viral loads reflect the convolution of infection incidence and within-host viral kinetics) is illustrated in the referenced literature, and the main message of the current manuscript is the explore how this phenomenon is observed in arbovirus vector surveillance.

(2) ll 134-137 - Mosquito species is another factor that could result in wide variation in Ct values due to differences in vector competence and infection kinetics. Given that 2-4 mosquito species are present in these pools with unknown frequencies, this seems like a potentially major source of unexplained variation.

Importantly, Culex pipiens and Culex tarsalis mosquitoes are separated prior to testing for WNV. We have clarified in the legend for Figure 1 that Ct values are presented from pools of either Culex pipiens/restuans/salinarus or Culex tarsalis.

Additionally, we have added the following text to the Materials and Methods section:

“For identification purposes, Cx. Pipiens species mosquitoes are not separated from the Cx. Salinarius or Cx. Restuans, which are nearly identical morphological. However, Cx. Pipiens is far more abundant than either Cx. Salinarius or Cx. Restuans in Nebraska.”

We also show in Figure S22, S23, and S24 that we observe similar variation in Ct values across mosquito species, location, and epi week, and thus we do not think that differences between species play a major impact in our findings.

(3) ll 173-175 - I believe that this is a consequence of the trapping method. Could this please be spelt out a bit more?

Indeed, all of the data in this manuscript were derived from mosquitoes collected in CDC Light Traps that attract host-seeking mosquitoes (i.e mosquitoes looking for a bloodmeal). We are not considering vertical transmission in our model as it has been reported to occur infrequently in laboratory studies. Therefore, WNV-positive mosquitoes collected in CDC Light Traps have been exposed to WNV through a previous blood meal from a bird. We have clarified the text to include this explanation:

“The mosquito pool Ct value data in this study come from specimens collected using CDC Light Traps that are baited with CO2, specifically targeting host-seeking mosquitoes. Vertical transmission is not factored into our model, thus, for a WNV-positive mosquito to be captured in the pool, it must have already obtained one blood meal from an infected bird and be seeking its next blood meal, which introduces a delay between infection and being captured.”

(4) ll 177-178 - Doesn't the temporal trend in Ct values primarily reflect temporal changes in mosquito infection prevalence?

Thank you for the comment. We agree that the temporal changes in mosquito infection prevalence is the main factor influencing the distribution of the Ct values in pools, as the time-since-infection distribution of trapped mosquitoes does not vary sufficiently to lead to trapping mosquitoes at very different points in their viral kinetics trajectory. Our intended point from this sentence was, given the prevalence and pool size, the variation in the viral load of the infected mosquitoes does not vary in time as all infected mosquito are trapped after they have reached a constant high-viral load level. We have now revised this sentence to reflect this.

“As the infected mosquitoes progress from increasing viral load to a high set-point viral load, temporal trends in pooled Ct values primarily reflect time-varying infection prevalence and the number of infected mosquitoes in each pool. The remaining non-temporal variation in pooled Ct values reflect individual-level variation in mosquito set-point viral loads.”

(5) Section 2.2 - It would seem that the assumed viral kinetics in birds would be important to this line of reasoning, given that that determines initial viral load ingested by mosquitoes. I am unclear on what was assumed in the model regarding viral kinetics in birds.

Thank you for the comment. We have discussed the viral kinetics of the birds in detail in Section 5.3 and Supplementary Material S3. However, we agree that we have not explicitly mentioned this in Section 2.2. Therefore, we have added a reference to these sections in the following paragraph:

“The model assumes that the mosquito's initial viral load is proportional to the infector bird's viral load (see Section 5.3 and Supplementary Material S3 for further details on the bird viral kinetics model).”

(6) ll 194-202 - Whilst you have shown that this hypothesis leads to predictions that are consistent with the data, this is a relatively narrow hypothesis, and others are neither discussed nor refuted.

There are two features of the data which we discuss. First, the substantial variation in Ct values across pools. This is described in detail in Section 2.1. The second observation is the bimodal pattern, which L192-202 refers to. While we agree that we have not modelled alternative hypotheses, our point is that the distribution of pooled Ct values is bimodal, and capturing some mosquitoes with very low viral loads is the most plausible explanation for the mode at high Ct values. However, we contend that this is actually a fairly broad hypothesis, as there are many plausible mechanisms generating mosquito infections with low viral loads, which we already discuss (discussion section beginning “This could be explained by a variety of factors…”). No changes have been made to the manuscript.

(7) Section 2.3, first paragraph - The problem with this approach is that these simulations depend on a number of assumptions and parameter settings that are not estimated as part of the model fitting process. Thus, the model is very narrow and contingent on these narrow and not compellingly justified assumptions.

Thank you for the comment. While we agree with the reviewer that this is a potential limitation of our study, we have discussed this in detail in the discussion. As mentioned in the manuscript “the main objective of this study was not to formally fit the multi-scale agent-based model to the data, but rather to understand how individual-level viral kinetics in mosquitoes are reflected in pooled surveillance data, and to demonstrate the use of pooled Ct values in estimating WNV infection prevalence”, we believe the assumptions and model are sufficient to address the research objectives. Furthermore, the fact that simulated Ct value distributions from the ABM can be used directly with the prevalence estimation method to give similar estimates to the existing PooledInfRate package supports the validity of our assumptions, though we agree that this does not necessarily mean all of our assumptions are correct, nor that our model is generalisable to other settings. No changes have been made to the manuscript.

(8) Section 2.3, second paragraph - So the newly proposed method using Ct values does no better than the existing method using binary data?

Thank you for the comment. We agree with the reviewer that our method and the existing PooledInfRate package perform similarly at the estimated prevalence levels of WNV. However, the Ct-based method, as we have discussed and shown, is robust at all prevalence levels where the binary-only method fails, and our method can distinguish the productive and non-productive prevalence.. Thus, while the prevalence estimates are similar under both methods for the current dataset, the novelty lies in the ability to reconstruct prevalence using the data in an entirely different way, and the proof-of-concept for how Ct values may harbour more biological information than treating pools as positive/negative. We believe that these points are sufficiently discussed throughout the manuscript. We have not made any changes to the manuscript.

(9) ll 252-254 - This may only be true because the simulation model and the inference model are identical. If the inference model were misspecified (due, for example, to incorrect assumptions about kinetics, etc), this result would likely weaken.

Thank you for the comment. The difference in robustness between the binary-only method and Ct-based method is not a feature of the method, but rather of how the data is used. At higher prevalence, all pools are likely to have at least one positive mosquito in them, and thus all pools will be positive, removing all information to discriminate between different prevalence levels. In contrast, the Ct-based method is able to still discriminate between prevalence levels even when all of the pools are positive, as there is still information based on whether the positive pools have low or high Ct values. No changes have been made to the manuscript.

(10) ll 272-273 - Again, this is highly dependent on built-in model assumptions.

Thank you for the comment. We agree that the performance of a model can depend on the underlying assumptions and the structure of the model. This is true in general for any model-based inference technique (see White, 1982, for example). Therefore, our simulations, results and interpretations are intended to be evaluated under the model structures and underlying assumptions we have used throughout the manuscript. However, to be explicit, we have now added this line at the end of the paragraph that included the sentence.

“These results are based on the model structure and the underlying assumptions we used and they may be affected by model misspecification, including incorrect assumptions (see White, 1982, for example).”

(11) ll 273-281 - Can this be done with pooled data only, or does it require individual mosquito Ct values? The latter would seem to be less practical to obtain in real-world applications.

Thank you for the comment. As we have cited the related work for SARS-CoV-2, in theory, these methods are applicable when individual Ct values are present. Both pooled data and individual-level data will work, but using pooled data requires the pooling and dilution process to be modelled explicitly. However, as the reviewer mentions, for mosquito surveillance, this is not a practical approach as mosquitoes are always pooled prior to testing to reduce effort and costs. No changes have been made to the manuscript.

Reviewer #2 (Recommendations for the authors):

(1) The supplementary figures do not appear to be ordered according to their first mention in the manuscript, which makes them somewhat harder to follow.

Thank you for the helpful comment. We have now made sufficient changes to the Supplementary Material and updated the references in the manuscript. Where possible, the supplementary figures and sections are now numbered and presented in the order of their first mention in the manuscript.

(2) Lines 71-73: This sentence is somewhat vague, and I was not fully clear on the intended message. The authors may wish to revise it for clarity.

Thank you for the comment. Similar comments have been made by Reviewer #1. We have revised this sentence for clarity.

The this shows that although events from the past cannot be changed the way people understand and interpret them can change over time. New research evidence and perspective can cause a story to reconsider. What happened and why it was important.

because monitoring is costly, so the other shareholders try to make the other shareholders pay for the monitoring.

eLife Assessment

The work presented represents important new evidence linking the cascade of neural processes triggered by memory-based prediction errors. The study uses an impressive collection of approaches and methods to characterize and measure cognitive control, arousal, and memory changes as a function of memory-based violations. The analyses are technically sophisticated and rigorous and, taken together, provide solid evidence that there are multiple processes accompanying prediction errors, and that they differentially relate to successful encoding.

Reviewer #1 (Public review):

This manuscript describes a multi-modal study of associative learning and memory in humans that combines scalp EEG, pupillometry and behavioral analysis to explore the construct of mnemonic prediction errors (MPEs), in terms of their relationship to attention and cognitive control. Across two pooled studies, participants performed associative memory tasks in which they learned the relationship between a cue word (action verb) and subsequent picture (animate or inanimate) with a strong vs. weak (4 or 1 repetitions) encoding manipulation. At test, participants were encouraged to generate a prediction following the cue word to determine whether the subsequently presented picture was a match or mismatch. The timecourse of pupillary responses during match decisions were decomposed using temporal principal components analysis, which identified 6 distinct and overlapping processes. Some of the components (PC3/PC4) exhibited sensitivity to both the strength and mismatch conditions, as well as behavior (both RT and accuracy) and retrieval success on the subsequent trial. Furthermore, relationships were also observed between pupillary responses (specifically for PC4) and both frontal theta and posterior alpha power measures obtained from scalp EEG in Experiment 2, as well as for frontal theta and subsequent learning from mismatch stimuli (assessed using subsequent memory findings from a surprise recognition test). The authors suggest the findings indicate that MPEs elicit changes in attention, arousal and cognitive control which impact subsequent learning.

Strengths:

This manuscript has many strengths, including a clever study design, thoughtful integration of multiple neurocognitive measures, and a set of rigorous and technically sophisticated analyses, which reveal a large set of relationships among the measures and behavior. The findings demonstrating brain/physiology-behavior relationships are particularly important, in that they point to potential functional consequences of MPEs.\

Reviewer #2 (Public review):

Summary:

The authors studied cognitive control and attention in response to mnemonic prediction errors (MPEs): situations in which the external reality violates internal memory-based predictions. The behavioral task first established strong versus weak predictions, and then either confirmed or violated these predictions. The authors examined markers of cognitive control (frontal theta) and attention (posterior alpha suppression, pupil response) while strong and weak predictions were confirmed or violated. They found increased cognitive control (frontal theta) for strong MPEs, which correlated with subsequent memory. Markers of attention (alpha suppression, pupil response) also accompanied strong MPEs but did not correlate with subsequent memory. Pupil response was investigated using an interesting approach that decomposes the response into different components, finding that different components respond earlier or later and show different correlations with MPEs and their strength. The authors also investigated how EEG, reaction time, and pupil responses correlated with one another, providing further insight into the mechanism underlying the response to MPEs. Together, the study points toward multiple control and attention mechanisms involved in MPE response and memory.

Strengths:

The study has a clear behavioral paradigm with multiple measures - behavioral, EEG, and pupillometry that offer an investigation into different aspects of MPE response and memory.

The study is also very comprehensive in looking at multiple phases in processing MPEs: the prediction phase (prior to the violation), the response to MPEs, and subsequent memory of MPEs, all within one study. Specifically, the link between neural mechanisms and subsequent memory is a major advancement, as most prior studies did not include this component. Mechanisms underlying subsequent memory of MPEs are theoretically important, as a primary function of MPEs is to promote learning and memory. As the authors mention, the different neural and pupillary signals are not robustly correlated, suggesting multiple mechanisms underlying MPE detections, which is interesting, offers avenues for future research, and can facilitate a better theory of how MPEs are processed in the brain. Finally, the decomposition of pupil response into different components and their correlation with behavior (RT during match/MPE detection) is interesting.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

This manuscript describes a multi-modal study of associative learning and memory in humans, that combines scalp EEG, pupillometry and behavioral analysis to explore the construct of mnemonic prediction errors (MPEs), in terms of their relationship to attention and cognitive control. Across two pooled studies, participants performed associative memory tasks in which they learned the relationship between a cue word (action verb) and subsequent picture (animate or inanimate) with a strong vs. weak (4 or 1 repetitions) encoding manipulation. At test, participants were encouraged to generate a prediction following the cue word to determine whether the subsequently presented picture was a match or mismatch.

The timecourse of pupillary responses during match decisions were decomposed using temporal principal components analysis, which identified 6 distinct and overlapping processes. Some of the components (PC3/PC4) exhibited sensitivity to both the strength and mismatch conditions, as well as behavior (both RT and accuracy) and retrieval success on the subsequent trial. Furthermore, relationships were also observed between pupillary responses (specifically for PC4) and both frontal theta and posterior alpha power measures obtained from scalp EEG in Experiment 2, as well as for frontal theta and subsequent learning from mismatch stimuli (assessed using subsequent memory findings from a surprise recognition test). The authors suggest the findings indicate that MPEs elicit changes in attention, arousal and cognitive control which impact subsequent learning.

Strengths:

This manuscript has many strengths, including a clever study design, thoughtful integration of multiple neurocognitive measures, and a set of rigorous and technically sophisticated analyses, which reveal a large set of relationships among the measures and behavior. The findings demonstrating brain/physiology-behavior relationships are particularly important, in that they point to potential functional consequences of MPEs.

Weaknesses:

The technical proficiency and complexity of the study and analysis also presents a clear limitation and challenge for interpretation. It is likely that readers, even those that are quite knowledgeable about the methods, constructs, and questions being addressed will often struggle (as this reviewer did) to keep the large set of findings in mind and gain understanding of how they all fit together.

Indeed, it seems like there many threads running together in the paper which make it challenging to find the through-line of the key findings. The authors do address some of the key questions motivating the paper in the Introduction, but the results are somewhat ambiguous with regard to the primary question of the study as to whether the detection of MPEs leads to interaction among cognitive control, attention, and arousal. To their credit, the authors tackle this question through both cross-correlation and formal mediation analyses, and summarize these in diagrammatic figures (Figure 3, Figure 6). Yet it is not resolved whether the results represent a clear answer pointing to independence, or rather a lack of statistical power, or ill-resolved formulation of the mediational relationship. In particular, the cross-correlation suggests that posterior alpha suppression in response to MPEs does precede frontal theta, yet this indirect relationship does not explain the variation in trial-by-trial RTs on mismatches. This suggests a potential model misspecification.

In addition to the primary interaction issue mentioned above (between cognitive control, attention & arousal), the Introduction lays out a number of claims:

(1) That pupil size will be more sensitive to strong than weak MPEs.

(2) That MPE-linked increases in attention (indexed with posterior alpha suppression) and arousal (indexed with pupil size) will be linked to learning.

(3) That MPE learning will vary as a function of prediction strength.

Given the focus on learning, it is somewhat surprising that learning is not included in the mediation models. As the authors indicate in the Discussion, the use of trial-by-trial RT variation to drive the mediation model might be problematic, given that the RTs are sensitive to a range of factors beyond mnemonic prediction strength and also are under competing pressures (longer for mismatches than matches, due to surprise-linked slowing, but also faster following stronger rather than weaker mnemonic predictions). Thus, an alternative possibility might be to use trial-by-trial recognition of mismatches as the outcome variable in mediation models rather than trial-by-trial RT as the independent variable.

A large component of the results (Sections 2 and 3) is devoted to analyses of cue-linked pupil and EEG processes that putatively reflect mnemonic predictions (i.e., occurring before picture probes are presented and match/mismatch detection, i.e., MPEs occur). Yet these Results and the subsequent pupillary PCA components (PC1 and PC5) that are elicited are not well-integrated with the primary themes of the paper or the causal hypotheses. One finding that does seem to figure prominently (in that it is mentioned in Abstract, Introduction & Discussion) relates to the amount of attention allocated to the mnemonic prediction generation. Yet this finding is not well emphasized in the Results themselves. Possibly it refers to the negative relationship between posterior alpha during memory retrieval and the magnitude of pupillary PC3 component, described in Section 3. But it was quite challenging to identify amongst the wealth of results described in this Section as well as the others. More generally, the large amount of findings described across all four lengthy Results sections makes it challenging for readers to discern what are the key ones that the authors would like to highlight.

It is recommended that the authors do another pass through the paper to better highlight the most critical findings that they want to emphasize or which are most interpretable from a mechanistic and causal flow perspective and then de-emphasize or move other findings to the Supplemental Materials. Although the authors are to be commended for such a rigorous and comprehensive set of analyses, there are so many of them and findings, that the key points get buried and the reader needs to struggle potentially unnecessarily to identify the key take-away points.

We thank Reviewer 1 for the helpful feedback on how the manuscript can be further strengthened. We recognize that the rich set of findings can overwhelm the reader, resulting in difficulty discerning the main take aways about the effects of mnemonic prediction errors. We particularly appreciate Reviewer 1’s encouragement to restructure the manuscript so as to focus on the findings reported in Sections 1 and 4 of the original revision; the current revision now focuses on these key observations.

As part of this restructuring, Reviewer 1 also proposed moving the content from Sections 2 and 3 of the original revision to the Supplement. We agree with the Reviewer that the questions addressed in these sections on retrieval-related processes are not the main focus of the paper, but that they are informative in their own right. To avoid their getting lost in the Supplement, we decided that these results would be better served in a separate manuscript and thus we have removed them entirely. 

We acknowledge in the revised manuscript that the mediation and cross-correlation analyses were exploratory and that these specific analyses may not be well powered in the current experiments. With respect to Reviewer 1’s concerns about the specification of the mediation models, we were motivated to test whether MPEs trigger an increase in cognitive control that in turn, triggers an increase in attention and/or arousal (Fig. 4a); as such, we designed the model to assess whether, on strong MPE trials, frontal theta mediates the relationship between prediction strength and attention/arousal. As noted in the manuscript and raised by Reviewer 1, mismatch RT here is an imperfect measure of trial-level prediction strength. Future experiments that selectively elicit strong MPEs and have a more controlled measure of trial-level prediction strength may be better equipped to address these questions about interactions between control, attention, and arousal. We agree with Reviewer 1 that models assessing subsequent memory as an outcome would be desirable. However, given that (a) we did not find strong evidence for interactions at the time of a strong MPE and (b) we only observed a relationship between frontal theta and subsequent memory (but not posterior alpha or pupil), subsequent memory mediation models do not appear to be well justified. Altogether, these findings illuminate open avenues for future research.

Reviewer #2 (Public Review):

Summary:

The authors studied cognitive control and attention in response to mnemonic prediction errors (MPEs): situations in which the external reality violates internal memory-based predictions. The behavioral task first established strong versus weak predictions, and then either confirmed or violated these predictions. The authors examined markers of cognitive control (frontal theta) and attention (posterior alpha suppression, pupil response) while strong and weak predictions were confirmed or violated. They found increased cognitive control (frontal theta) for strong MPEs, which correlated with subsequent memory. Markers of attention (alpha suppression, pupil response) also accompanied strong MPEs but did not correlate with subsequent memory.

Pupil response was investigated using an interesting approach that decomposes the response into different components, finding that different components respond earlier or later and show different correlations with MPEs and their strength. The authors also investigated how EEG, reaction time, and pupil responses correlated with one another, providing further insight into the mechanism underlying the response to MPEs. Together, the study points toward multiple control and attention mechanisms involved in MPE response and memory.

Strengths:

The study has a clear behavioral paradigm with multiple measures — behavioral, EEG, and pupillometry — that offer an investigation into different aspects of MPE response and memory.

The study is also very comprehensive in looking at multiple phases in processing MPEs: the prediction phase (prior to the violation), the response to MPEs, and subsequent memory of MPEs, all within one study. Specifically, the link between neural mechanisms and subsequent memory is a major advancement, as most prior studies did not include this component. Mechanisms underlying subsequent memory of MPEs are theoretically important, as a primary function of MPEs is to promote learning and memory. As the authors mention, the different neural and pupillary signals are not robustly correlated, suggesting multiple mechanisms underlying MPE detections, which is interesting, offers avenues for future research, and can facilitate a better theory of how MPEs are processed in the brain. Finally, the decomposition of pupil response into different components and their correlation with behavior (RT during match/MPE detection) is interesting.

Weaknesses:

The methods are rigorous, and the data support the claims. The weaknesses are minor and are offered here as avenues for future research.

(4) The relationships the authors find between brain measures and pupil components were largely not specific to mismatches/matches. Thus, the specificity of this relationship is untested.

(5) The results with subsequent memory are important and address a major gap in the field that largely did not relate neural effects of MPE to subsequent memory. However, one major limitation of the study is that the authors did not test memory for matches. I understand the logic of avoiding testing matches. Because matches were repeated more times in the study, it’s not a fair comparison and could change participants’ overall criterion for old/new decisions. Future research could address this, e.g., by testing weak matches or potentially using a between-subject design.

We appreciate Reviewer 2’s helpful feedback during the review process and encouraging comments on the strengths of the manuscript. We agree and note in the revision that it would be illuminating for future studies to contrast memory for events that violate and confirm mnemonic predictions.

Comments on revised version

The authors addressed all my concerns. I appreciate the authors’ thoughtful and detailed response.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

It was challenging to read the paper with key findings happening at the time of the MPE presented first, and then to go “backwards in time” to examine process that occurred at preceding time periods (i.e., prior to probe presentation), and then again forward in time to examine learning related processes.

(1) Restructuring the Results

In this regard two distinct recommendations are made:

- Move Sections 2 and 3 to Supplemental Materials, to maintain the focus on the key findings related to detection of MPEs and their effects on subsequent learning.

- An alternative structure would be to present the cue-locked findings first, which relate to mnemonic predictions, then to those that occur when those predictions get violated, and finally to the learning process that occur following MPEs and which can be detected with subsequent recognition tests.

We thank the Reviewer for this encouragement to restructure the manuscript. To address concerns regarding the density of the paper and the cohesiveness of the findings, we removed the content that was in Sections 2 and 3 of the original revision. We will publish those results in a separate paper with additional analyses to more directly address previously raised questions regarding the mechanisms indexed by frontal theta and posterior alpha at the time of retrieval.  

(2) Integration of the summary figures

At the minimum, a recommendation would be to better integrate Figure 6, and maybe various versions of Figure 3, earlier into the text, preferably even in the Introduction, and then repeatedly refer to them throughout the results. For example, in Figure 6, linking the leftmost panel of the figure to Sections 2 and 3 is critical, and the righthand panel to Sections 1 with the rightmost part related to learning explicitly linked to Section 4.

We moved the summary figure up to now be Figure 1; we reference this figure in the Introduction and throughout the manuscript; and we additionally make reference in the figure to the association between frontal theta and PC3 at the time of a strong MPE as well as the cross-correlation outcome. We hope these modifications further aid the reader in identifying the main findings of the manuscript.

(3) Specification of the mediation models

Additionally, for the mediation models it is quite unclear why mismatch RT is treated as the index of MPE magnitude, as this seems to be where the problem may lie in model fitting. Why not think of this as an outcome variable (since would seem to be a causal outcome of the underlying functional processes elicited by mismatch detection)?

We thank the reviewer for these thoughtful comments. Our goal in designing the mediation models in Fig. 4a-c was to test our hypothesis that strong MPEs trigger an increase in cognitive control that, in turn, triggers an increase in attention and/or arousal; thus, attention/arousal should be the outcome and cognitive control should be the mediator. Given that increases in attention and cognitive control were selectively observed for strong and not weak MPEs, the models were restricted to strong MPEs. We therefore needed a trial-level measure of MPE magnitude to determine whether stronger MPEs in the strong condition elicit greater attention/arousal by engaging more cognitive control. As such, we decided to use mismatch RT as a proxy measure of MPE magnitude; we acknowledge in the text that this measure is imperfect. While a model with mismatch RT as the outcome variable is possible, the relative timing of the attention/arousal effects (which largely occur following responses) would render interpretation to be more challenging. Had stronger evidence of indirect effects emerged in Figs. 4b-c, we could have conducted model comparison with RT as an outcome. We acknowledge in the text that these analyses were exploratory and characterization of potential indirect effects will require more data and a more precise measure of MPE magnitude.

Alternatively, examining trial-by-trial recognition memory of mismatches as the relevant outcome variable would also seem to capture the functional process of interest. In this regard, have the authors examined whether trial-by-trial RT on mismatches predicts subsequent recognition of these items? If this direct relationship holds, it could be a target for mediation analyses in itself.

We agree with the reviewer that in theory, a mediation model predicting subsequent memory would be a desirable test of an integrated model of the mechanisms underlying MPE-driven learning. However, the mediation analyses conducted to address the functional relationships at the time of a prediction error (Fig. 4) are not well-powered to begin with; this limitation is raised in the Results and the Discussion. A mediation model predicting subsequent memory would be similarly underpowered; given that there is not strong evidence for indirect effects at the time of a strong MPE, and that only frontal theta – and not posterior alpha nor pupil – predicts subsequent memory, we think that such a mediation model is not well justified to include. Such a model should be more directly tested by well-powered designs in future studies.

(4) Hippocampal theta in the Introduction

The Introduction discusses hippocampal theta as well as frontal theta yet also makes clear that the former is not really well-detected or analyzed using scalp EEG. Consequently, a recommendation would be to remove this paragraph from the Introduction, since it can be misleading and a “red herring” for the reader, and instead only bring up this point in the Discussion section, as a pointer to the need for future research using methods that may be more sensitive to hippocampal interactions with PFC regions.

We appreciate this point and moved discussion of the hippocampus from the Introduction to the Discussion.

eLife Assessment

This manuscript presents an important and timely contribution by incorporating desolvation barriers into coarse-grained models of biomolecular condensates. The findings are convincing, supported by a clear physical model and systematic simulations showing effects on phase behavior, packing, and dynamics. Some clarification and broader context would improve the manuscript, but it provides a foundation that will be of use for developing more realistic coarse-grained interaction schemes.

Reviewer #1 (Public review):

This manuscript is very interesting and timely. By introducing the critical effects of desolvation barriers and solvent (water)-separated minima into the implicit-solvent potentials (of mean force, PMFs) for coarse-grained molecular dynamics simulations of biomolecular liquid-liquid phase separation (LLPS), this work fills a gap that should be apparent to researchers of protein folding in the past couple of decades but has so far escaped deserved attention such that these basic features of aqueous solvation have seldom, though not never, been invoked in recent studies of biomolecular condensates. Although the present paper deals almost exclusively with homopolymers, this work can be a foundation for the future development of a new, more physical coarse-grained interaction schemes for simulating amino acid sequence-dependent effects, which I presume is the authors' ongoing or next endeavor. The results presented in this manuscript are highly valuable.

However, there is room for improvement in the authors' description of (i) the broader impact of effects of desolvation barrier and solvent-separated minimum in the thermodynamics of biomolecular condensates, especially with regard to the ramifications on hydrostatic pressure-dependent effects; (ii) the physical implication of using a 20-parameter hydropathy scale rather than a 210-parameter pairwise amino acid interaction scheme; and (iii) temperature-dependent effects, including the authors' discussion of "enthalpic" and "entropic" contributions. In all these aspects, the authors' discussion should be put in a more comprehensive context of the existing literature. At a few other places, description of the methods and results should be clarified as well.

Comments on revised version

The authors have thoroughly and adequately addressed all my previous concerns and suggestions. The manuscript is now significantly improved in terms of clarity and proper placement in the context of prior works on desolvation effects in protein conformations.

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important and timely question in the molecular simulation of biomolecular condensates. Most residue-level coarse-grained models used for IDP phase separation employ implicit solvent and represent effective interactions through relatively simple pairwise potentials. While these models have been very useful, they usually do not explicitly distinguish direct contacts from solvent-separated interactions, nor do they include an energetic barrier associated with water removal. This manuscript attempts to address that limitation by introducing desolvation-inspired terms into coarse-grained models and examining their consequences for phase behavior, chain conformations, dense-phase packing, and dynamics.

The central idea is physically well motivated. Using a simple homopolymer model, the authors show that increasing the desolvation barrier suppresses phase separation, whereas stabilizing solvent-separated contacts enhances phase separation. They further show that solvent-separated interactions can reduce dense-phase over-compaction, which is a meaningful result given the known challenges in obtaining both accurate single-chain dimensions and realistic dense-phase properties from the same coarse-grained model. The finding that desolvation-like terms can reshape dense-phase packing without simply rescaling the overall interaction strength is interesting and could be useful for future model development. I also found the attempt to connect conformational changes across dilute and dense phases with thermal distance from the critical point to be intriguing. The dynamic analysis, including the FRAP-like simulations and the discussion of kinetic arrest during coarsening, adds another useful dimension to the work.

Overall, I think this is a useful and potentially important contribution.

Comments on revised version.

The authors have addressed my earlier comment regarding conformational changes between the dilute and condensed phases. One small additional suggestion is that they may find two related studies useful in this context: Devarajan et al., Nature Communications (2024), on relationships between dilute-phase conformations and condensate material properties, and Wang et al., Chemical Science (2024), which examines sequence-dependent conformational changes during condensation for both model polyampholyte sequences and naturally occurring IDPs. These studies may provide some complementary context for the discussion. This is simply a literature suggestion and does not affect my overall assessment of the revised manuscript.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

This manuscript is very interesting and timely. By introducing the critical effects of desolvation barriers and solvent (water)-separated minima into the implicit-solvent potentials (of mean force, PMFs) for coarse-grained molecular dynamics simulations of biomolecular liquid-liquid phase separation (LLPS), this work fills a gap that should be apparent to researchers of protein folding in the past couple of decades but has so far escaped deserved attention such that these basic features of aqueous solvation have seldom, though not never, been invoked in recent studies of biomolecular condensates. Although the present paper deals almost exclusively with homopolymers, this work can be a foundation for the future development of a new, more physical coarse-grained interaction scheme for simulating amino acid sequence-dependent effects, which I presume is the authors' ongoing or next endeavor. The results presented in this manuscript are highly valuable.

We thank the reviewer for all these positive comments.

However, there is room for improvement in the authors' description of (i) the broader impact of effects of desolvation barrier and solvent-separated minimum in the thermodynamics of biomolecular condensates, especially with regard to the ramifications on hydrostatic pressure-dependent effects; (ii) the physical implication of using a 20-parameter hydropathy scale rather than a 210-parameter pairwise amino acid interaction scheme; and (iii) temperature-dependent effects, including the authors' discussion of "enthalpic" and "entropic" contributions. In all these aspects, the authors' discussion should be put in a more comprehensive context of the existing literature. At a few other places, the description of the methods and results should be clarified as well. Accordingly, the authors should revise the manuscript to address the following items thoroughly within the revised manuscript (not merely in the response letter) with the additional references mentioned below included in the revised discussion:

(1) In several places, e.g., on line 77 (p.2), the authors appear to suggest that "implicit-solvent representation" is the origin of the deficiency in commonly utilized coarse-grained potentials that this study is aiming to rectify. But desolvation barriers and solvent-separated minima are also features of implicit-solvent representations; they are just features that should be incorporated in more accurate implicit-solvent potentials. This point is stated quite clearly and accurately in the Abstract (p.1) but not consistently in the rest of the text. The authors should check the entire text carefully to ensure that a coherent, accurate perspective is presented.

We thank the reviewer for pointing out this important issue. We agree that implicit-solvent representation itself is not the origin of the deficiency. Our intention is to incorporate desolvation-inspired effective terms within an implicit-solvent coarse-grained framework, because many commonly used implicit-solvent potentials do not directly account for the desolvation features, such as the desolvation barrier and the solvent-separated potential well.

We have revised the Abstract, Introduction, Results, and Discussion to make this distinction consistent throughout the manuscript. The revised text now emphasizes that the model remains an implicit-solvent CG model, but contains additional effective terms inspired by desolvation features observed in all-atom PMFs.

Corresponding changes:

(1) (page 1, lines 17–20) The Abstract identifies the model as an implicit-solvent CG model with added desolvation terms:

"Here, guided by all-atom simulations and experimental measurements, we develop a desolvation-aware implicit-solvent CG model by incorporating residue-level desolvation terms directly into the pairwise energy function and apply it to investigate LLPS of intrinsically disordered proteins."

(2) (page 2, lines 80–81) The Introduction retains the implicit-solvent description of existing residue-level CG models:

"Despite these advances, most residue-level CG models rely on implicit solvent representations, in which individual water molecules are not explicitly represented."

(3) (page 2, lines 87–89) The specific limitation is identified as the absence of a direct account of the multi-step desolvation process:

"More importantly, conventional implicit-solvent CG models used for LLPS usually do not directly account for the multi-step desolvation process that accompanies the transition from a dilute solution to a dense condensate."

(4) (page 4, lines 172–174) The added terms are described as part of a desolvation-inspired effective potential:

"Together, these parameters shape the desolvation-inspired effective potential and modulate the statistical balance between direct-contact and solvent-separated configurations."

(5) (page 15, lines 521–522) The Discussion restates the implicit-solvent model framework:

"To address this challenge, we developed a desolvation-aware implicit-solvent CG framework that incorporates desolvation barrier and solvent-separated terms into the pairwise potential."

(2) In the discussion of the importance of desolvation barriers and solvent-separated minima in the Introduction (pp.1-3), connections should be drawn to recent works that utilize these PMF features to rationalize hydrostatic pressure (P)-modulated effects on biomolecular LLPS, including the P-dependent reentrant phase separation of alpha elastin; see Cinar et al. (2019) Chem Eur J 25:13049 (https://chemistryeurope.onlinelibrary.wiley.com/doi/full/10.1002/chem.201902210) and references therein, especially discussions around Figures 10, 11 & 13 in this reference.

We thank the reviewer for bringing this literature to our attention. We agree that pressure-modulated LLPS provides important context for the physical relevance of desolvation barriers and solvent-separated minima. We have therefore expanded the Introduction and Discussion to connect our model to prior work on hydrostaticpressure-dependent condensate behavior, including pressure-dependent reentrant phase separation of alpha-elastin.

Corresponding changes:

(1) (page 3, lines 93–95) The Introduction connects the PMF features to hydrostaticpressure-dependent LLPS:

"Related studies on hydrostatic pressure effects have further suggested that desolvation barriers and solvent-separated minima can help rationalise pressure-modulated LLPS behaviors, including the pressure-dependent reentrant phase separation of α-elastin Cinar et al. (2019, 2018)."

(2) (page 15, lines 533–537) The Discussion states the pressure-dependent implication conservatively:

"These findings may also provide a useful physical basis for future studies of pressure-dependent condensate behavior, as pressure-induced changes in hydration, solvent-separated states, and desolvation barriers have been proposed to contribute to pressure-modulated and reentrant LLPS Dias and Chan, 2014); Cinar et al. (2019, 2018)."

(3) In the lower panels of Figures 2D, E (p.5), what do the differently colored small circles in the double-minimum free energy profiles represent? Does the color shading have the same meaning as that in the upper panels? If so, what do the positions of the circles on the free energy profile represent? The authors should clarify this.

We thank the reviewer for identifying this ambiguity. The small circles in the lower panels of Figures 2D and 2E are qualitative schematic representations of residue-pair configurations along the effective pair-potential profile. Their blue and green colors distinguish the barrier-variation and solvent-separated-well cases, respectively; they are not a quantitative scale and do not encode temperature or population magnitude. The positions of the circles indicate the direct-contact, barrierregion, or solvent-separated regions, while the density of circles schematically represents the population of configurations.

We have clarified this interpretation in the Figure 2 caption and aligned the Results text with the redistribution among direct-contact, barrier-region, and solvent-separated states.

Corresponding changes:

(1) (page 6, Figure 2D and E lower panels) The schematics distinguish low and high ε_b or ε_ss and use the density and position of the circles to depict populations in the direct-contact, barrier-region, and solvent-separated regions; the blue and green colors distinguish the two parameter families and are not a quantitative scale.

(2) (page 6, Figure 2 caption) The caption defines the population encoding used in the lower panels:

"The lower panels schematically illustrate how changes in ε_b and ε_ss alter the distribution of residue-pair configurations. The small circles indicate schematic populations of residue-pair configurations along the potential profile, with denser circles representing a higher population."

(3) (page 5, lines 210–213) The Results text connects the schematics to redistribution among the three residue-pair states:

"These opposing effects suggest that the desolvation potential regulates macroscopic phase behavior by redistributing residue-pair configurations between direct-contact, barrier-region, and solvent-separated states (lower panels of Figure 2D and E)."

(4) The discussion regarding entropy and enthalpy around Figure 2 is quite confusing as it stands. What do the authors mean exactly by the association of entropy or enthalpy with the desolvation barrier of the solvent-separated minimum? Are they referring to conformational entropy?

We thank the reviewer for pointing out this ambiguity. We agree that our original wording around entropy and enthalpy could be misleading, because it might imply a rigorous thermodynamic decomposition of the PMF. In the revised manuscript, we have therefore clarified that the effect of the desolvation barrier refers to an entropyrelated configurational restriction of residue-pair configurations near the barrier region, rather than the overall conformational entropy of the entire chain. We also replaced the previous "enthalpic stabilization" wording with "effective free-energy stabilization" to avoid implying that the solvent-separated minimum is treated as a purely enthalpic contribution.

Corresponding changes:

(1) (page 5, lines 199–201) The barrier effect is described in terms of the sampled residue-pair population:

"Analysis of residue-residue radial distribution functions showed that higher ε_b suppresses the population of configurations near the barrier region (Figure 2—figure Supplement 1C)."

(2) (page 5, lines 201–202) The entropy-related statement is restricted to configurational sampling near the barrier:

"This reduction in the statistical weight of barrier-region configurations can be interpreted as an entropy-related configurational restriction and thus disfavors phase separation."

(3) (page 5, lines 209–210) The solvent-separated minimum is described as an effective free-energy contribution:

"This solvent-separated minimum provides effective free-energy stabilization for water-mediated configurations and thereby promotes phase separation."

(4) (page 6, Figure 2D and E lower panels) The schematic headings are "Barrier-mediated Restriction" and "Solvent-separated Stabilization", avoiding a strict entropy-enthalpy decomposition.

(5) Do the authors assume that the PMF (effective implicit-solvent potential) is a purely enthalpic term? It appears to be the authors' assumption. If so, the assumption has to be stated clearly in their discussion of "entropy" vs "enthalpy" around Figure 2.

We thank the reviewer for raising this important point. We do not assume that the PMF obtained from all-atom simulations is a purely enthalpic term. The PMF is a free-energy profile that contains enthalpic and entropic contributions. The current manuscript defines the PMF as −k_B T lnP(r), uses the all-atom PMFs to motivate a nonbonded effective coarse-grained potential, and describes the solvent-separated minimum as providing effective free-energy stabilization. We do not perform a rigorous enthalpy-entropy decomposition, and the revised wording avoids implying such a decomposition.

Corresponding changes:

(1) (page 16, lines 594–595) The Methods define the PMF as a free-energy profile obtained from the radial probability density:

"The potential of mean force (PMF) was computed as PMF(r) = −k_BT ln P(r), where P(r) is the radial probability density obtained from the production trajectory."

(2) (page 5, Figure 1 caption) The CG interaction is labeled as an effective potential rather than as an enthalpic PMF decomposition:

"Pairwise effective potential incorporating desolvation-inspired terms. Different curves correspond to different desolvation parameters."

(3) (page 4, lines 172–174) The parameters are described as shaping an effective potential:

"Together, these parameters shape the desolvation-inspired effective potential and modulate the statistical balance between direct-contact and solvent-separated configurations."

(4) (page 5, lines 209–210) The solvent-separated contribution is described using free-energy language:

"This solvent-separated minimum provides effective free-energy stabilization for water-mediated configurations and thereby promotes phase separation."

(6) Closely related to points 3-5 above, it should be stated clearly that the "temperature" used in the authors' simulations does not represent experimental temperature if the authors are using purely enthalpic effective potentials because PMFs are in fact temperature-dependent. This clarification is necessary to avoid misunderstanding. In this regard, it should be noted that temperature-dependent effective interactions have been used for modeling biomolecular condensates in analytical theory (Lin, Song, Forman-Kay & Chan, J Mol Liq 2017, already in the citation list) as well as in coarse-grained molecular dynamics simulations [Dignon et al. (2019) ACS Cent Sci 5:821-830 (https://pubs.acs.org/doi/10.1021/acscentsci.9b00102); Chakravarti & Joseph (2025) Protein Sci 34:e70284 (https://onlinelibrary.wiley.com/doi/10.1002/pro.70284)]. The latter two studies, not cited currently, are particularly relevant and thus should be cited because the authors may wish to incorporate temperature-dependent features in their ongoing or future effort in constructing a more comprehensive coarse-grained interaction scheme for biomolecular LLPS simulation.

We agree with the reviewer that the simulation temperature should be interpreted carefully. In the present simulations, the effective potential is temperature-independent within each chosen parameter set. Therefore, the reduced temperature primarily serves as a model temperature controlling the relative strength of thermal fluctuations, rather than as a direct experimental temperature. We have clarified this point in the revised manuscript and added relevant references on temperature-dependent effective interactions, which represent an important direction for future model development.

Corresponding changes:

(1) (page 4, lines 177–179) The manuscript states that the absolute simulation temperature is not an experimental temperature:

"Because the effective interaction parameters used in the model are temperature-independent, the absolute simulation temperature should not be directly interpreted as an experimental temperature."

(2) (page 4, lines 182–184) The reduced temperature is identified as a model temperature:

"Accordingly, T^* should be interpreted primarily as a model temperature that controls the relative strength of thermal fluctuations, rather than as having a direct quantitative correspondence with experimental temperature."

(3) (page 14, lines 481–485) The FUS LC temperature comparison is framed cautiously:

"It is worth noting that the residue-level coarse-grained models used here employ temperature-independent effective interaction parameters. As a result, the temperature values reported here cannot be interpreted as quantitatively equivalent to experimental temperatures, particularly when they deviate substantially from room-temperature conditions."

(4) (page 15, lines 563–567; continues on page 16, lines 568–569) The Discussion identifies temperature-dependent effective interactions and a corresponding future extension:

"In addition, effective interactions themselves can be temperature-dependent, as demonstrated in analytical theories and coarse-grained simulations of biomolecular condensates Lin et al. (2017); Dignon et al. (2019); Chakravarti and Joseph (2025). Future extensions of the model could therefore incorporate residue-specific and temperature-dependent desolvation parameters derived from bottom-up parameterization or expanded experimental datasets, thereby enhancing predictive accuracy for sequence-dependent LLPS."

(7) In tackling "entropy" vs "enthalpy", it should be noted that the temperature dependence of the effective interactions entails an entropic contribution (which is itself temperature dependent) in addition to conformational entropy. As for the effective potential with desolvation barrier and solvent-separated minimum, it should be noted that the decomposition into entropic and enthalpic contributions at the direct contact, desolvation barrier, and solvent-separated minimum can be dramatically different, see, e.g., MaCallum et al. (2007) PNAS 104:6206-6210 (https://www.pnas.org/doi/full/10.1073/pnas.0605859104) and references therein.

We thank the reviewer for this important clarification. We agree that temperature-dependent effective interactions can contain entropic contributions beyond conformational entropy and that the balance of enthalpic and entropic contributions may differ among the direct-contact minimum, desolvation barrier, and solvent-separated minimum. The present model does not decompose the PMF into temperature-dependent enthalpic and entropic components; accordingly, we have avoided assigning those components to individual PMF features. The Discussion cites explicit-solvent PMF analyses when noting residue-pair and temperature dependence and identifies temperature-dependent desolvation parameters as an important future extension.

Corresponding changes:

(1) (page 15, lines 561–563) The Discussion cites explicit-solvent PMF work when noting residue-pair and temperature dependence:

"Explicit-solvent PMF analyses have shown that desolvation barrier heights and solvent-separated minima can differ substantially among residue pairs and may also exhibit temperature dependence Cinar et al. (2019); Debiec et al. (2014); MacCallum et al. (2007)."

(2) (page 15, lines 563–566) The manuscript states that effective interactions can themselves depend on temperature:

"In addition, effective interactions themselves can be temperature dependent, as demonstrated in analytical theories and coarse-grained simulations of biomolecular condensates Lin et al. (2017); Dignon et al. (2019); Chakravarti and Joseph (2025)."

(3) (page 15, lines 566–567; continues on page 16, lines 568–569) Temperature-dependent desolvation parameters are identified as a future model extension:

"Future extensions of the model could therefore incorporate residue-specific and temperature-dependent desolvation parameters derived from bottom-up parameterization or expanded experimental datasets, thereby enhancing predictive accuracy for sequence-dependent LLPS."

(8) P.7, line 340: The proportionality relation follows directly from the standard FloryHuggins result T_c = T chi(T)/chi_c, thus the proportionality constant is exactly 1/chi_c. Is this the standard relation that the authors are invoking here? The authors should clarify this.

We thank the reviewer for pointing out the missing intermediate steps. Yes, the relation we invoked is based on the standard Flory-Huggins critical condition. In the revised manuscript, we have expanded the derivation to explicitly show how the critical condition chi(T_c) = chi_c leads to the relation between chi(T_sim) - chi_c and the normalized thermal distance (T_c - T_sim)/T_sim.

We also revised the wording to avoid presenting this as a universal law. The relation is now presented as a simulation-supported trend within the present model, rationalized by a simplified linear-response assumption between Delta R_g and the excess interaction strength.

Corresponding changes:

(1) (page 7, lines 263–264) The critical-condition substitution is now shown explicitly:

"At the critical point, χ(T_c) = χ_c, which gives ε_eff = k_BT_cχ_c. Substituting this relation into the expression for χ(T_sim) yields χ(T_sim) = χ_cT_c/T_sim."

(2) (page 7, line 265) The resulting relation is written as Equation (2):

"χ(T_sim) − χ_c = χ_c (T_c − T_sim)/T_sim."

(3) (page 7, lines 266–267) The fixed-chain-length assumption is stated explicitly:

"For systems with the same chain length, χ_c is a fixed constant. Thus, the deviation from the critical interaction parameter is directly related to the rescaled thermal distance (T_c − T_sim)/T_sim."

(9) The study on dynamic consequences on pp.8-11 is interesting, but clarifications are necessary:

(i) The vertical schematic in Figure 4A should be explained in detail in its entirety. As it stands, no explanation is provided either in the figure caption or in the text. In particular, what does "elasticity driven" refer to?

(ii) The top snapshot in Figure 4A is labeled t_sim = 0 ns. Does it mean that the snapshot shown is the only chain configuration that the authors used to start the simulation, and that the snapshot does NOT represent the result of any time evolution, no matter how short the duration is? However, if that is the case, why is this snapshot identified with spinodal decomposition if it is not the product of a time evolution from a more homogeneous configuration?

(iii) Related to (ii) - do the rectangular boxes shown represent the entire simulation box or just part of the box containing the polymer chains? One would imagine that if the top snapshot represents spinodal decomposition, the simulation would have been started at a more uniform distribution a short time prior? Why is this not the case?

(iv) What precisely do the small yellow beads and black-colored springs in the zoomin image of Figure 4E represent?

We thank the reviewer for all these inspiring comments and questions. We agree that the original Figure 4 schematic did not sufficiently explain the sequence of dynamical events and the meaning of several graphical elements. We have therefore revised both the Figure 4 caption and the Results text to make the schematic self-contained and to clarify how it relates to the quantitative analyses in Figure 4F and G.

First, we replaced the phrase "elasticity driven" with a more precise description of "viscoelastic resistance". In the revised text, interfacial tension is described as favoring domain fusion thermodynamically, whereas transient inter-chain network connectivity within dense domains generates viscoelastic resistance to the deformation required for coalescence. This revision avoids implying that elasticity is the driving force and instead identifies it as a resistance that delays domain fusion kinetically during the plateau regime.

Second, we clarified the meaning of t_sim = 0 ns and its relation to spinodal decomposition. The system was equilibrated at a supercritical temperature to obtain a homogeneous one-phase state and was then instantaneously quenched to the target temperature. The label t_sim = 0 ns denotes the first snapshot immediately after the quench. It is not the only initial configuration used in all simulations; the reported kinetic metrics were averaged over six independent slab simulation replicas.

The t_sim = 0 ns snapshot is therefore described as a homogeneous but thermodynamically unstable post-quench state. Spinodal decomposition refers to the subsequent amplification of the initial density fluctuations after the quench, including the development of interconnected density fluctuations within 1-2 ns, rather than to a preceding evolution represented by the t_sim = 0 ns snapshot.

Third, we clarified that the rectangular snapshots in Figure 4A show the entire simulation box viewed along the z-axis. The subsequent snapshots show how post-quench density fluctuations grow and reorganize into dense domains during spinodal decomposition.

Finally, we clarified the symbols in the zoom-in schematic of Figure 4E. Yellow beads now denote residues involved in transient inter-chain contacts, and black springs denote schematic network connections formed by these contacts. These elements are meant to illustrate transient network connectivity and are not additional simulated particles or force-field terms.

Corresponding changes:

(1) (page 10, Figure 4A) The vertical schematic now labels the progression as "Thermodynamic instability", "Kinetic arrest (viscoelastic resistance)", "Domain coarsening (interfacial-tension dominated)", and "Dynamic equilibrium (chain self-diffusion)".

(2) (page 10, Figure 4E) The plateau schematic labels the competing effects as "Interfacial Tension" and "Transient network resistance".

(3) (page 10, Figure 4 caption) The caption defines the snapshots and the vertical schematic:

"Upper snapshots show the simulation box along the z-axis at t_sim = 0, 10, and 500 ns. The vertical schematic summarizes the dynamical progression described in the main text, from the post-quench spinodal instability to kinetic arrest, domain coarsening, and dynamic equilibrium."

(4) (page 11, lines 368–370) The first recorded time point after the quench is defined explicitly:

"Here, t_sim = 0 ns denotes the first snapshot immediately after the temperature quench, corresponding to a homogeneous but thermodynamically unstable nonequilibrium state."

(5) (page 10, Figure 4 caption) The yellow beads and black springs are defined:

"In the zoom-in view, yellow beads denote residues involved in transient inter-chain contacts, and black springs denote schematic network connections formed by these contacts."

(6) (page 12, lines 401–404) The Results explain the physical meaning of the transient network:

"In the zoom-in schematic in Figure 4E, this transient network is represented by connections between residues involved in inter-chain contacts, illustrating how multivalent interactions can resist domain deformation during the plateau regime."

(10) In discussing dynamic effects, it is useful to draw connections to related works on the effect of chain flexibility on "aging" of condensate [Biswas & Potoyan (2024) PRX 45:9222-9245 (https://journals.aps.org/prxlife/abstract/10.1103/PRXLife.2.023011)] and characterization of viscoelasticity in simulations of biomolecular condensates [Tejedor et al. (2023) J Phys Chem B 127:4441-4459 (https://pubs.acs.org/doi/10.1021/acs.jpcb.3c01292)], as the effects of desolvation can be explored further based on these prior works.

We thank the reviewer for these important references. We have added connections to simulation studies of condensate viscoelasticity and aging. The revised manuscript now places our dynamic results in the context of transient network connectivity, chain flexibility, sticker lifetime, desolvation-associated rigidification, and viscoelastic or aging-like material behavior.

We present these connections conservatively as relevant context and as future directions for extending the current model, rather than claiming a new universal dynamic mechanism.

Corresponding changes:

(1) (page 12, lines 397–399) The dynamics section cites simulation-based rheological analyses of condensate viscoelasticity:

"Similar viscoelastic effects have recently been quantified in molecular simulations of biomolecular condensates using rheological analyses of time-dependent material properties Tejedor et al. (2023)."

(2) (page 12, lines 399–401) The manuscript connects condensate aging to chain flexibility, sticker lifetime, and desolvation-associated rigidification:

"Molecular simulations of condensate aging have further highlighted the roles of chain flexibility, sticker lifetime, and desolvation-associated rigidification in promoting more solid-like states Biswas and Potoyan (2024)."

(3) (page 12, lines 423–427) The kinetic interpretation is connected to viscoelastic andaging-like behavior:

"The sensitivity of kinetic arrest and coarsening dynamics to desolvation parameters underscores the importance of incorporating desolvation features into coarse-grained potentials for more physically plausible molecular simulations of LLPS, especially when connecting microscopic interaction lifetimes to emergent viscoelastic or ageing-like material behavior."

(4) (page 16, lines 569–572) The Discussion identifies simulation-based rheological analysis as a future direction:

"An additional direction would be to combine these potentials with simulation-based rheological analyses to quantify how desolvation reshapes condensate viscoelasticity, aging-like maturation, and long-time material relaxation Tejedor et al. (2023); Biswas and Potoyan (2024)."

(11) Much of the present study is based on the original HPS formulation of Dignon et al. (2018). In this regard and also in anticipation of future development of improved interaction schemes, several issues should be stated and discussed, even if briefly:

(i) The original HPS model has a basic shortcoming in accounting for the relative interaction strengths of, among others, arginine vs lysine residues [Das et al. (2020) PNAS 117:28795-28805 (https://www.pnas.org/doi/10.1073/pnas.2008122117)].

(ii) Compared to 210-parameter pairwise interaction schemes, such as KH in Dignon et al. (2018) and Joseph et al. (2021), the 20-parameter interaction scheme is likely too restrictive to account for pairwise amino acid residue interactions [Wessén et al. (2022) J Phys Chem B 45:9222-9245 (https://pubs.acs.org/doi/10.1021/acs.jpcb.2c06181)].

(iii) The height of the desolvation barrier may vary significantly for different amino acid residue pairs, see, e.g., Figure 11 of Cinar et al. (2019) mentioned above (and references therein). The authors should discuss these nuances in the revised version. They may also wish to take them into consideration in future investigations.

We thank the reviewer for the suggestion to clarify these limitations. We have revised the Discussion to acknowledge explicitly the limitations of the 20-parameter hydropathy-scale representation relative to more flexible 210-parameter pairwise interaction schemes for describing amino-acid-pair interactions. We have also added discussion emphasizing that future desolvation-aware models should incorporate residue-pair-specific parameters for the desolvation barrier and solvent-separated potential well.

Corresponding changes:

(1) (page 13, lines 445–446) The scope of the averaged baseline parameterization is stated explicitly:

"This uniform parameterization captures the generic desolvation features of the PMFs but does not resolve residue-pair-specific variations in desolvation energetics."

(2) (page 15, lines 551–554) The Discussion identifies the limitation of the 20-parameter HPS representation, including Arg/Lys interactions:

"In particular, HPS-type models use a 20-parameter hydropathy-scale representation, which is useful for capturing generic IDP phase behavior but is not flexible enough to resolve residue-pair-specific chemical effects, such as the distinct interaction patterns of arginine and lysine residues Das et al. (2020)."

(3) (page 15, lines 554–557) The greater flexibility of 210-parameter pairwise schemes is described:

"More general 210-parameter pairwise interaction schemes, such as KH-type and related residue-pair-specific models, provide greater flexibility for encoding amino acid-pair preferences and capturing sequence-specific interaction heterogeneity Dignon et al. (2018b); Joseph et al. (2021); Wessén et al. (2022)."

(4) (page 15, lines 557–561) The limitation of using one averaged desolvation parameter set is stated:

"Second, the present desolvation model employs a single set of averaged parameters (α_b, α_ss) for all residue pairs. While this simplification is effective for isolating the generic physical consequences of desolvation, it has limitations in describing the pair-specific variations in the desolvation barrier and the solvent-separated minimum."

(5) (page 15, lines 566–567; continues on page 16, lines 568–569) Residue-specific and temperature-dependent parameters are identified as a future extension:

"Future extensions of the model could therefore incorporate residue-specific and temperature-dependent desolvation parameters derived from bottom-up parameterization or expanded experimental datasets, thereby enhancing predictive accuracy for sequence-dependent LLPS."