might touch on the diagrammatic? that psych paper on human perception/recognition.

With modern improvements in AI and other text based programs. It will presumably be increasingly difficult to distinguish between what content is created authentically by a human and what is artificial. With increase usage of AI, the desire for authentic creations full by humans might increase.

A parasocial relationship while undesirable in most cases might in fact be the end goal of some figures like politicians or cult leaders. This dynamic can be manipulated for personal gain by the leader, in order to fulfill their own desires. This can vary from influencing or straight up telling their followers what to do.

I love how this part explains a real-world belief through myth. It connects the natural (wind) and the spiritual (wandering souls), showing how stories gave people a way to understand death and the unseen world. It also keeps the memory of Coyote’s act alive in everyday experience.

This quote shattered my previous assumption that schools are neutral spaces for learning. Before reading this, I thought inequality in education mostly came from outside factors, but the idea that schools actively structure it was a revelation.The teachers explained that tracking helps meet students’ needs, but it actually traps poor students in lower-level courses, limiting their future options.

The emphasis on "the only means" here struck me. It implies how high the stakes are for U.S. public schools.The system’s design, which relies on local property taxes for funding, means schools in poor areas can never truly equalize opportunities, leaving those without generational wealth stuck in a cycle.

ah okay so this guy has an actual reason to state for what happened with the Great Tradition

As an international student who researched U.S. schools extensively before arriving, I experienced this deeply local system firsthand. I noticed huge differences in high school curricula across states: some required four years of math, while others only required three; some emphasized STEM, while others focused on the humanities. Even within the same state, school funding varied drastically, districts with higher property taxes had better facilities and more teachers. Back home, we have a national curriculum that ensures consistency in what students learn, so this local control was confusing at first. I can see its benefits: a rural district might focus on agricultural education to meet local needs, for example. But the downside is clear, students in poor districts don’t get the same opportunities as those in wealthy ones, which undermines the American Dream’s promise of equal starting lines.

Learning about these persistent gaps was a wake-up call for me, as I’d previously heard mostly positive stories about U.S. education reform.This makes me think that progress in education isn’t just about passing policies. It’s about making sure those policies reach the most vulnerable groups.

In my country, the government allocates school resources more centrally to reduce such gaps, so seeing this paradox in action makes me realize how deeply rooted it is in the U.S. system.

Many public schools require community service hours for graduation, which is designed to foster civic responsibility. However, I’ve heard from local friends that this goal is unevenly achieved. Schools in wealthy suburbs can organize high-quality volunteer programs, while schools in poor areas often only offer basic service options, due to limited resources. This means the starting line of the American Dream isn’t the same for all students, even though public schools are supposed to level it.

As an international student, the definition of the American Dream here resonates with what I’ve heard before, but it also makes me reflect on the tension between individual pursuit and collective well-being. In my home country, success is often intertwined with family and community contributions, so the idea that "each person is responsible for their own dreams" feels both empowering and isolating.

RIGHT cause he didnt explain exactly how the shift in education was contributing other than vaguely describing it as fracturing from The Great Tradition. he does so much leaning on this nostalgic idea without fleshing out the differences. i dont recall any actual examples or anything he shared about this

The 1920 poem "Mr. Apollinax" centers itself on a group of scholars and students attending a dinner party. The character of Mr. Apollinax himself is heavily inspired by Bertrand Russell, a mentor and friend to Eliot, who was also a famous logician, though his concepts at times were difficult for even well-versed philosophers and scholars to comprehend. In the poem, the descriptions of Mr. Apollinax and his behavior at the dinner party obtains direct parallels to "Death by Water" and the motif of the ocean itself.

1. "His laughter was submarine and profound / Like the old man of the sea’s" (lines 8-9). In these lines, there are two clear connections to water or the ocean: the description of Mr. Apollinax's laugh as being "submarine", and the equating of such laugh to "the old man of the sea's". Beyond his supreme intellect, Mr. Apollinax is portrayed as a man deriving a great amount of pleasure and fulfillment from the world, as conveyed by his "submarine and profound laugh", indicating that unlike the hollow and despairing voices of TWL, Mr. Apollinax acquires a humor rooted in a genuine "inner richness" and vitality.
2. "Where worried bodies of drowned men drift down in the green silence, / Dropping from fingers of surf" (lines 11-12). These lines stood out to me as referencing the general human condition of the time: people are swept away by the chaotic tide of worldly forces (industrialization, war, pursuit of goods or wealth) that have risen in the rapidly changing times of the early 20th century. The world has become so complicated that it has become nearly impossible for the regular individual to properly navigate it, leading them to "drown". This metaphorical drowning could indicate several things: on a broader scale, the fall of humanity into sin and wrongdoing; on a similar vein, humanity becoming separate from the ideals of religion or spirituality; or the succumbing of mankind to the pursuit of worldly things such as wealth, fame, or material goods. In my mind, a good argument could be made for each of these possibilities.
3. "I looked for the head of Mr. Apollinax rolling under a chair / Or grinning over a screen / With seaweed in its hair" (lines 13-15). Now, this is an interesting detail. It is another example of decapitation (the first major instance being the headless corpse of a sex worker in Le Fleurs du Mal). The head, which literally represents reason and intellect, being separated from the body suggests a collapse of rational order and a breakdown of the mind itself. In the context of TWL, such a disjunction reflects the intellect’s estrangement from emotional or spiritual grounding. The description of the head “grinning over a screen” adds an absurd and grotesque dimension to the scene, transforming what might otherwise be horrific into a moment of unsettling fascination. Ultimately, the tension between humor and horror illuminates a key theme that runs through TWL: intellect, when isolated from the fuller spectrum of human experience (emotion, spiritual faith, and vitality) risks devolving into alienation or madness.

Eliot's choice to mention the Thrush instantly stood out to me because the Hummingbird, which is the other bird he mentioned, has held significant meaning throughout The Waste Land. Similar to the Hummingbird which represents Philomela's lost but prophetic voice, the Thrush is introduced in Chapman's handbook as "This Thrush comes to us in the spring, when the woods are still bare, and lingers in the autumn until they are again leafless." This directly mimics how Eliot has discussed rebirth and spring's involvement with the cycle of death. The Thrush's voice is described as "not remarkable for variety or volume, but in purity and sweetness of tone and exquisite modulation they are unequaled." This description of pureness makes it so the Thrush has a prophetic voice, nothing material, just pure. Especially as Eliot uses "Drip drop drip drop drop drop drop." The "Drip" refers to the Water that has been sought after throughout this segment of the poem, and the purity and saving quality of the bird's song. The thrush's song being described as "While traveling, the Hermit Thrush is not in full voice, and he who would know its song must follow it to the mossy forests, which are its summer home" connects to the journey in the poem. The travelers must follow something deeper to find real renewal, just like you have to follow the thrush to its true home to hear its full song. In Keats's Thrush poem, the Thrush's voice is again described as warm and naturally freeing from knowledge. "O thou, whose only book has been the light / Of supreme darkness which thou feddest on / Night after night when Phoebus was away" describes someone who has learned from darkness itself. The thrush says "O fret not after knowledge. I have none, / And yet my song comes native with the warmth." This natural wisdom without overthinking is what the wasteland needs. The thrush offers instinctive hope, not thought out hope. It just sings, and in singing, it brings the sound of water, the promise of renewal, without needing to understand or explain it.

This is not our first encounter with “Death by Water.” “The Burial of the Dead” begins with “April is the cruelest month, breeding / Lilacs out of the dead land.” Spring’s rain breeds life out of decayed crops, but also out of the struggles of winter and war. Both in the poem and in the greater scope of culture, water is seen as necessary for spiritual renewal and cleansing, physical sustenance, and the regrowth of nature. The Tempest is mentioned throughout the poem and even its title reveals Eliot’s narrative journey. A tempest is a violent storm or an intense turmoil, its root “tempus” meaning time or season. The idea of a tempest itself is a violent and unforgiving turbulence which eventually ends in peace, but not without ravaging disaster. In a tempest, the water known for renewal, rebirth, and the essence of life is a force of violence. In the play, The Tempest, Ariel consoles another character about the believed loss of his father to drowning, saying, “Full fathom five thy father lies;/Of his bones are coral made;/Those are pearls that were his eyes:/Nothing of him that doth fade/But doth suffer a sea-change/Into something rich and strange.” By emphasizing that the father will remain he has just changed to become one with the sea, Shakespeare frames death by water as a spiritual shift instead of an end. Then, back to Eliot, Madame Sosostris twists this line from The Tempest when she reads the card titled “the drowned Phoenician Sailor,” says “Fear death by water,” and reminds the narrator of the line “Those are pearls that were his eyes.” Here, she does not see death as a spiritual transformation, but a loss of humanity which should be feared, emphasizing the pearl eyes as a sign that the sailor’s soul has been lost. Madame Sosotris, as sourced from Huxley, lives under several disguises, though, as a man pretending to be a woman and a poser pretending to be a prophet. Thus, Eliot frames Madame Sosostris as a false representation of the cycle of life, so that he can correct her skewed perception which is widely held by society. Here, death returns to the title but it has changed from “Burial of the Dead” to "Death by Water.” In the first section of the poem, “the dead” were given their own identity, but by this section it has become “death,” a word less connected to the people and more to their state. In the Corinthians, we see the more traditional image of water as spirituality. However, all the other referenced sources show water as death, less as a continuation of the natural cycle and more as a violent and inevitable force. <br /> In these sources, there is a recurring theme of ships being struck head on right before reaching their destination. In The Life and Death of Jason, the characters are spared and turn back, but they do not reach their destination. For Ulysses, he survives his journey and returns home safely to his family, after losing his shipmates to the sea and other challenges. In Dante, right as the characters can see land ahead, a “whirlwind struck the ship head on” and “the sea closed over us.” Eliot’s shift from water as a symbol of rebirth/life to a symbol of death is a continuation of the off-beat nature of the poem, and the awareness versus the denial of one’s fate.

In the "Death by Water" section, the power comes from what it turned out not to be as much as what it became. The ten lines were carved out of a much longer, more narrative draft about a grim fishing voyage. By cutting all the backstory and noise, Eliot and Pound basically transformed a tedious story into a haunting epitaph, almost as if they realized the idea of death was more powerful than the messy process of dying. What's really interesting is how this condensed version seems to be in conversation with other, grander stories of death at sea. There's Dante's Ulysses, who's damned for his ambitious, endless quest for knowledge. And then there's the myth of Hylas, who's almost magically absorbed into the water by lovesick nymphs. But Phlebas isn't a hero or a lover; he's a Phoenician merchant. His world was "profit and loss." His death isn't a tragic punishment or a mythical transformation. it just happens. The water here isn't really saving or damning him; it's just this neutral, indifferent force that processes his body, erasing his identity. The ending suddenly shifts and speaks directly to "you who turn the wheel." It feels like a warning, but I'm not entirely sure what it's warning against. Maybe it's that in our own focus on modern, practical concerns, like turning the wheel of our own lives, looking for the next opportunity, we're all just as vulnerable to being completely undone as Phlebas was. It's not about a grand failure, but a quiet, universal one. The poem seems to be suggesting that this kind of anonymous, materialistic death is the modern condition, and we're all sort of drifting toward it.

kinda interesting that he'd say this. to me this sounds like a flaw. does he approve of this or disapprove? it sounds like he must find it bad but he thinks the tradition is great

immediate thought: okay, what do the moral values look like in this Great Tradition?

even with strong literacy education in elementary schools, in higher education, students struggle with literacy because of the lack of background knowledge on topics.

This is interesting because it shows how Rome stayed strong through challenges.

This quote shows how Rome stayed strong through challenges. Even after being attacked they kept getting more powerful overtime.

It is interesting how even in a big fight between good and evil hero’s can have hidden purpose.

it’s interesting how the Ramayana started as an old story passed down by word of mouth it shows how powerful storytelling was even before people began writing things down.

It’s interesting that the Greek city-states tried to use Philip’s death to gain freedom, but Alexander quickly crushed them, especially by destroying Thebes it really shows how strong and determined he was even at a young age.

This really challenges the traditional psychoanalytic view I’ve always heard about that everything traces back to childhood trauma. Ellis’s perspective feels way more empowering. If your beliefs are the problem, not your past, then change feels more possible. You're not stuck with your history you can retrain your thinking.

Underneath this section, can you add a section about the process? Step 1: request your invite Step 2: Attend a pre-event interview to help me curate the perfect group experience (and make sure you're ready for this experience!) Step 3: 2 Days of action taking Step 4: Attend your Feedback Call to celebrate your wins and cement your aha-moments

Strictly Capped at 14 Attendees Max.

I just found this important enough to highlight

helpful checklist for final draft revisions

when doing research its important to cite your sources so you're not claiming to have come up with concepts yourself.

Research allows you to not only deepen your understanding of a chosen topic but helps you connect to the material through interesting facts.

this shows that ghost guns is actually a huge issue in the city

this notes that there is a significant direct connecttion to the rise of shoots during the pandemic

this shows that there will be some type of significant change to the world

when slavery was about race

stemmin from anti black racism ofc-Haitians and Cubans

This line shows that the gods control human fate.Odysseus's journey home depends on their will - without it, no one can move or even be seen . It highlights the theme of divine power over mortal lives in The Odyssey.

Eurylochos's escape shows the danger of temptation and the importance of self-control by not following. This line highlighted his awareness and circe's cunning.

Greek culture valued Xenia (hospitality); however, instead of welcoming odysseus, the king plotted something cruel, marking a turning point in Odysseus's journey.

a king of Ithaca and Greek leader in the Trojan war who after the war wanders 10 years before reaching home.

Okay, so the "smart" virtue being about people skills and not actual intelligence kind of blew my mind. I've definitely been on teams before where the smartest person in the group was actually the worst team member because they had no idea how to read social situations. When we did that conflict exercise, or well when my team. I'm realizing I probably need to work on this more myself because I tend to just say what I think without always considering how it's landing with everyone else. Does anyone else struggle with knowing when to speak up versus when to just let something go?

This whole "hungry" thing hits different after what we've been doing in class. Lencioni's point that hungry people don't need to be pushed to do more work - they just naturally look for ways to help - that's exactly what made our team project actually work. We have several vocal team members that are willing to passionate explain what we should change or what should be included, and honestly that energy is contagious. It makes the rest of us want to step up too. But I've also seen the other side of this where someone is so hungry that they steamroll everyone else's ideas. The reading doesn't really address that much. I guess the difference is whether your hunger is about making the team better or just about proving yourself?

The way Lencioni breaks down humility here is kind of different from what I expected. I always thought being humble just meant not bragging, but he's talking about something deeper - like actually putting the team first even when you could take credit. This reminds me of when our group was working on the Recipe Lookup app and we had that whole debate about how our backend/database should be. I was worried about having to implement our own database from scratch but I was a stronger supporter of setting up our own database to have total control over what our database does. However, the team was able to find an API that will give us exactly what we need for the application, without all the hassle. What I'm still trying to figure out though is how you balance humility with actually contributing your ideas. Like, if you're too humble, doesn't that mean you might hold back good suggestions?

I absolutely agreed! Quantitative results tell part of the story, and we would need to hear students' voices directly through a qualitative research paradigm.

This aligns with what I have seen in open pedagogy, that is, giving students real agency over their work seems to transform motivation. I wonder if there is a debilitating aspect to choice as well. In other words, does "too much" choice impact learning by overwhelming students? In Csikszentmihalyi's Flow Theory, we learn that there must be a balance of "Autonomy and Structure" in order for students to be in a state of flow, where they are engaged. I wonder how the researchers balanced the students' autonomy vs. structure in both research conditions, and if that played any role in the way the felt about their respective conditions.

how did fear and violence evolve? hunter-gatherer groups 30-40 ppl, nomadic for the group to survive, every person is critically important mutual regard and caring the in- group. survival depends upon the group, not on own where does belonging come from? There the person who distributes the meat is not the hunter, but the person who gave the arrow to the hunter. so the hunter isn't necessarily the one in full control PALEOLITHIC not neolithic. human mind starts changing as you go from nomadic small tribes to secure agricultural civilization HERES THE DEAL: hatred and fear of outgroup is not genetic. so thats pretty good for the potential of us being able to overcome this? Peter is saying mcdermott and hatemi might be wrong with this one

"Trade finance", "corporate payday loans"

The stark inequality is not confined to cognitive skills but is deeply rooted in behavioral and mental health dimensions. It is unjust to attribute low-income students' “low classroom engagement” and “antisocial behavior” solely to individual or family factors. These traits are more akin to survival strategies and psychological responses under the pressure of poverty. Isn't this moral blackmail?

This data has somewhat overturned my understanding. I agree that it powerfully demonstrates that among the factors influencing academic achievement, class disparities may be more significant and fundamental than racial disparities, and the issue of educational inequality must first be examined from the perspective of economic structure.

Here, the abstract concept of “inequality” is immediately concretized into two starkly divergent life trajectories, making me keenly aware that the educational divide extends far beyond mere test scores—it directly connects to the fundamental difference between mere survival and a life worth living.

The comment of legos really helps me imagine the offset views. I vividly remember seeing these views in my most recent lego build! In the example below, is it common practice to cut in center lines?

PMID: 40612488

Gene: MECP2

HGNC: 6990

Case: 13.9-year-old female patient

DiseaseAssertion: Rett, (Z-RTT/PSV: Zappella Rett syndrome/preserved speech variant)

FamilyInfo: Tics were also present in the mother and grandmother, de novo MECP2 variant was detected in the proband, a maternally inherited VoUS class 3 in GABBR1 (p. F692S), was identified in the proband, and segregated in the mother and grandmother

ParentalTest: FullPhase

CasePresentingHPOs: HP:0012760, HP:0000817, HP:5200030, HP:0012171, HP:0100035, HP:0100034, HP:0000739, HP:0100716, HP:0030051, HP:0002136, HP:0001276, HP:0002342, HP:0007010, HP:0000717

CaseHPOFreeRext: the case exhibited social withdrawal, difficulties in relationships, limited verbal communication, midline pinching and hand clapping, skin picking, progressive hypertonia with rigidity of the limbs, impaired her fine motor abilities.

CaseNOTHPOs: NR

CaseNOTHPOFreeText: NR

CasePreviousTesting: Whole Genome Array-CGH yielded normal results

PreviouslyPublished: Not previously published

GenotypingMethod: Exome Genome

Variant: NP_004983.1: c.397C>T; p.Arg133Cys

HGVS: NM_001110792.2:c.433C>T

ClinVarID: 11809

CAID: CA211250

gnomAD: NR

MultipleGeneVariants:

DiseaseEntity: Rett

AlleleOrigin: Germline

Variant: NM_001470.4:c.2075T>C

Mutation: Missense

Zygosity: Heterozygous

CAID: CA3689865

gnomAD: 0.000024 (GnomAD v4.1.0)

PreviouslyPublished: No

Learning does not have to be intentional, intrinsic learning is the best way to learn in my opinion. Like if you are living somewhere and you pick up phrases and terms, you are intrinsically learning

I love this lesson, we don't stop learning, even when we don't try, I can't tell you how many of my daily actions are muscle memory, and I do them off instinct, because I have gone through the same thing every day, so I naturally learn what I will do and need

This is a great example of the other factors we've been talking about in class, tone, body language, etc tells us things that some people don't verbally. We can still see he is mad even though he is communicating he is fine

This makes a lot of sense, there is a famous saying by bob ross, a great painter, and that he say's art is talent it is practice and the same theory applies here

eLife Assessment

This important and creative study finds that the uplift of the Qinghai-Tibet Plateau - via its resultant monsoon system rather than solely its high elevation - has shifted avian migratory directions from a latitudinal to a longitudinal orientation. The authors have expanded and clarified their lines of evidence (including an enlarged tracking set and explicit caveats on species-level eBird inference), such that the central claims are now solid. The conclusions - that monsoon dynamics, rather than elevation per se, are most consistent with observed longitudinal reorientation - illustrates how large, community-sourced and climate-model datasets can inform continent-scale shifts in migratory behavior over time that complement traditional approaches.

Joint Public Review:

The study assesses how the rise of the Qinghai-Tibet Plateau affected patterns of bird migration between their breeding and wintering sites.

This is an interesting topic and a novel theme. The visualisations and presentation are to a very high standard. The Introduction is very well-written and introduces the main concepts well, with a clear logical structure and good use of the literature. The Methods are detailed and well-described, and written in such a fashion that they are transparent and repeatable.

Editorial note: These latest revisions are minor in the sense that they expand on the dataset but do not change the primary results.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

The authors have done a good job of responding to the reviewer's comments, and the paper is now much improved.

Again, we thank the reviewer for positive comments during review.

Reviewer #2 (Public review):

I would like to thank the authors for the revision and the input they invested in this study.

We are grateful for your thoughtful feedback and enthusiasms, which helps us improve our manuscript. 

With the revised text of the study, my earlier criticism holds, and your arguments about the counterfactual approach are irrelevant to that. The recent rise of the counterfactual approach might likely mirror the fact that there are too many scientists behind their computers, and few go into the field to collect in situ data. Studies like the one presented here are a good intellectual exercise but the real impact is questionable. 

We understand your concern about the relevance of the counterfactual approach used in our study. Our intent in using a counterfactual scenario (reconstructing migration patterns assuming pre-uplift conditions on the QTP) was to isolate the potential influence of the plateau’s geological history on current migration routes. Similar approach was widely used to estimate how biogeographic barriers facilitated the divergent vertebrate communities across the world  (e.g., Williams et al. 2024). We agree that such an approach must be used carefully. In the revision, we have explicitly clarified why this counterfactual comparison is useful – namely it provides a theoretical baseline to test how much the QTP’s uplift (and the associated monsoon system) might have redirected migration paths (Gilbert and Lambert 2010, Sanmartín 2012, Bull et al. 2021). We acknowledge that the counterfactual results are theoretical and have explicitly emphasised the assumptions involved (i.e., species–environment relationships hold between pre- and post- lift environments) in the main text (Lines 91- 98). Nonetheless, we defend the approach as a valuable study design: it helps generate testable hypotheses about migration (for instance, that the plateau’s monsoon-driven climate, rather than just its elevation, introduces an east–west shift en route). 

References:

Bull, J. W., N. Strange, R. J. Smith, and A. Gordon. 2021. Reconciling multiple counterfactuals when evaluating biodiversity conservation impact in social-ecological systems. Conservation Biology 35:510-521.

Gilbert, D., and D. Lambert. 2010. Counterfactual geographies: worlds that might have been. Journal of Historical Geography 36:245-252.

Sanmartín, I. 2012. Historical Biogeography: Evolution in Time and Space. Evolution: Education and Outreach 5:555-568.

Williams, P. J., E. F. Zipkin, and J. F. Brodie. 2024. Deep biogeographic barriers explain divergent global vertebrate communities. Nature Communications 15:2457.

All your main conclusions are inferred from published studies on 7! bird species. In addition, spatial sampling in those seven species was not ideal in relation to your target questions. Thus, no matter how fancy your findings look, the basic fact remains that your input data were for 7 bird species only! Your conclusion, “our study provides a novel understanding of how QTP shapes migration patterns of birds” is simply overstretching.

We appreciate the reviewer’s comment here. We would like to clarify that our conclusions regarding longitudinal shifts in migratory distributions are based on distribution models derived from eBird data of 50 species, not merely on migration tracks from seven species. These species-level spatiotemporal models allow us to infer large-scale biogeographic patterns across the Qinghai-Tibet Plateau (QTP).

The original seven tracking species were used specifically for analysing the relationship between migration directions (azimuths) and environmental variables, offering independent support for the patterns revealed in the eBird-based distribution models. Recognising the reviewer’s concern on sample size and coverage, we have now expanded this part by incorporating migration tracks from 12 additional species, derived through georeferenced digitisation of published migratory maps. Importantly, this expansion did not change our conclusions, i.e., the monsoons instead of the high elevations act as a prominent role in shaping the current migration direction of birds in the QTP. While the overall conclusion remains unchanged, the expanded dataset led to slight changes in difference between spring and autumn migration. We have updated the Figure 2 and the corresponding results and conclusions throughout the manuscript. We have also clarified in the Discussion that regions of the QTP with relatively less data might lead to underestimation of some migration routes to make sure readers are aware of these data limitations (Lines 211-218).

The way you respond to my criticism on L 81-93 is something different than what you admit in the rebuttal letter. The text of the ms is silent about the drawbacks and instead highlights your perspective. I understand you; you are trying to sell the story in a nice wrapper. In the rebuttal you state: “we assume species' responses to environments are conservative and their evolution should not discount our findings.” But I do not see that clearly stated in the main text.

Thanks, as suggested we have clearly stated the assumptions of niche conservatism in the Introduction (Lines 91-98).

In your rebuttal, you respond to my criticism of "No matter how good the data eBird provides is, you do not know population-specific connections between wintering and breeding sites" when you responded: ... "we can track the movement of species every week, and capture the breeding and wintering areas for specific populations" I am having a feeling that you either play with words with me or do not understand that from eBird data nobody will be ever able to estimate population-specific teleconnections between breeding and wintering areas. It is simply impossible as you do not track individuals. eBird gives you a global picture per species but not for particular populations. You cannot resolve this critical drawback of your study. 

We agree that inferring population-specific migratory connections (teleconnections) from eBird data is challenging and inherently limited. eBird provides occurrence records for species, but it generally cannot distinguish which breeding population an individual bird came from or exactly where it goes for winter. Our objective is not to determine one-to-one migratory links between specific populations, but to identify general broad-scale directional shifts when birds cross the QTP during their migration. We regret any confusion caused by our earlier wording. To make this clearer, we have now emphasised that our interests focus on the migratory direction and their environmental correlates, rather than population assignments. We have also rephrased the relevant text to explicitly clarify that our study operates at the species level and at large spatial scales (Lines 253–257). We exemplify how distribution of eBird observations and GPS tracking data of four species can be different from each other whilst showing similar migration patterns (Figure S10). We have also explicitly stated in the Discussion that confirming population connectivity would require targeted tracking or genetic studies, and that our eBird-based analysis could only suggest plausible routes and region-to-region linkages (Lines 200-202).

I am sorry that you invested so much energy into this study, but I see it as a very limited contribution to understanding the role of a major barrier in shaping migration.

We thank the reviewer’s honest assessment and understand the concern regarding the scope of our contribution. Our intention was not to provide an exhaustive account of all aspects of the QTP as a migratory barrier, but to address a specific and underexplored question: how the uplift of the plateau and the resulting monsoon system may have influenced the orientation of avian migration routes. By integrating both satellite tracking and community-contributed data, we have explored how the uplift of the QTP could shape avian migration across the area. We believe our findings provide important insights of how birds balance their responses to large-scale climate change and geological barrier, which yields the most comprehensive picture to date of how the QTP uplift have shaped migratory patterns of birds. We have also discussed the study’s limitations – including the small number of tracking species (Lines 205218), the use of occurrence data as a proxy for breeding and wintering regions (Lines 200-202), the uneven sampling coverage in the QTP (Lines 202-205) and the assumptions behind the counterfactual scenario (Lines 91-98). This ensures that readers understand the context and constraints of our findings.

My modest suggestion for you is: go into the field. Ideally use bird radars along the plateau to document whether the birds shift the directions when facing the barrier.

We thank the reviewer for this suggestion. We agree that radar holds promise for understanding certain aspects of bird migration, particularly for detecting flight intensity, altitudes, and timing. However, the radar systems are currently challenging to resolve migration at the level of species, populations, or individuals, which are central to questions of migratory connectivity and route selection. Most radar signals cannot distinguish between species in mixed flocks, nor can they link breeding and wintering sites for tracked individuals. In addition, the spatial coverage of radar installations remains limited, especially across remote and high-elevation regions like the Qinghai-Tibet Plateau, where infrastructure and continuous power supply are still logistically prohibitive. 

The eBird dataset used in our study is itself a form of field-based observation, contributed by tens of thousands of birdwatchers across continents, including the QTP region (Figure S11). While eBird cannot provide individual-level tracking, it captures spatiotemporal patterns of occurrence at broad scales, making it a valuable complement to satellite tracking data. We would also emphasis that our team has extensive field experience in the Qinghai-Tibet Plateau (about twenty years), including multi-year expeditions to deploy satellite tags and observe migration at stopover sites. 

We agree that more direct tracking (e.g. GPS tagging) would be an ideal way to validate migration pathways and population connectivity. Using the satellite-tracking data, we have showed that most tracking species shifted their migration direction when facing the QTP (Figure S6). In this revision, as stated we managed to add a number of 12 more species with satellite tracking routes. We have also noted that future studies should build on our findings by using dedicated tracking of more individual birds and monitoring of migration over the QTP. We have cited recent advances in these techniques and suggested that incorporating more tracking data could further test the hypotheses generated by our work (Lines 205-218).

Reviewer #2 (Recommendations for the authors):

L55 "an important animal movement behaviour is.." Is there any unimportant animal movement? I mean this sentence is floppy, empty.

We used this sentence to introduce migration. We have removed “important” to reduce ambiguous phrasing.

L 152-154 This sentence is full of nonsense or you misinterpretation. First of all, the issue of inflexible initiation of migration was related to long-distance migrants only! The way you present it mixes apples and oranges (long- and short-distance migrants). It is not "owing to insufficient responses" but due to inherited patterns of when to take off, photoperiod and local conditions.

We stated that this claim is invoked for long-distance migrants before this sentence and have rewritten the sentence to highlight that this interpretation is for long-distance migrants. 

L 158 what is a migration circle? I do not know such a term.

We have amended it as “annual migration cycle”, which is a more common way to describe the yearly round-trip journey between breeding and wintering grounds of birds.

L 193 The way you present and mix capital and income breeding theory with your simulation study is quite tricky and super speculative.

We thank the reviewer for raising this important concern. We have presented this idea as an inference rather than a conclusion: “This pattern could be consistent with a ‘capital breeding’ strategy — where birds rely on endogenous reserved energy gained prior to reproduction — rather than an ‘income’ strategy where birds ingest nutrients mainly collected during the period of reproductive activity. This collaborates with studies on breeding strategies of migratory birds in Asian flyways. However, we note that this interpretation would require further study.” By adding this caution, we made it clear that we are not asserting this link as proven fact, only suggesting it as one possible explanation. We have also doublechecked that the rest of the discussion around this point is framed appropriately. Moreover, to help illustrate why we raised this ecological interpretation, we would also draw attention to examples of satellite tracking points from several species (e.g., Beijing Swift, Demoiselle Crane) in the following, which show obvious shifts in migratory direction near the QTP region. These turning points suggest potential behavioral responses to environmental constraints, such as climatic corridors or energy availability, which could help motivate our discussion of possible capital breeding strategies in these species.

For a sample, they calculate the number of total genomes using QCTs. Then, they calculate the fetal fraction using polymorphic loci. Based on the fetal fraction and the number of total genomes, they estimate the expected number of fetal antigen molecules (AEM) if the fetus is positive. If the AEM is lower than the threshold, they discard.

Then, they calculate the number of antigen molecules detected (ADM) using the read counts and QCTs. Then, they calculate the detected fetal antigen fraction (CFAF) as the number of detected molecules over the number of expected molecules. Across the 5 RhD loci, they take the second highest CFAF value and they compare it to the detection ranges to make the "antigen detected" call.

for - language - Wordbank - children's vocabulary - from - article - Atlantic - The Mystery of Babies’ First Words - https://hyp.is/OKsHnqU-EfCLelsZRWUhxw/www.theatlantic.com/family/archive/2019/04/babies-first-words-babbling-or-actual-language/588289/

for - from - search - Google - how new words divide the world in new ways - https://hyp.is/55MHUKUxEfC-TAfy9q1VjA/www.google.com/search?q=how+new+words+divide+the+world+in+new+ways&oq=how+new+words+divide+the+world+in+new+ways&gs_lcrp=EgZjaHJvbWUyBggAEEUYOTIHCAEQIRigATIHCAIQIRigATIHCAMQIRigAdIBCDgwODFqMGo0qAIAsAIB&sourceid=chrome&ie=UTF-8

• book review - The Language Animal
  • self awareness emerges out of intersubjectivity
  • like Melanie Klein
  • relationship is necessary to form self identity
  • culture and language are intertwingled
  • “The basic thesis of this book is that language can only be understood if we understand its constitutive role in human life.”

for - book review - The Language Animal

for - relationship defines the self - adjacency - relationship defines the self - Melanie Klein - Taylor

for - language - word meaning - adjacency - language - culture - adjacency - Taylor - Pearce

for - language philosophy - book - The Language Animal - Charles Taylor - language philosophy - book - Sources of the Self

Love this formulation of the classic "actions speak louder than words" wisdowm.

😢

for - key insight - language - wanting stability - adjacency - wanting stability - interpretant - Charles Saunders Pearce

for - language development - depends on social interaction

Royal KMM FPE HH KH 10 T1 B64 Typewriter Ribbon Install Rewind Respool Replace by [[Phoenix Typewriter]]

The spools for the standard Royal typewriters (Ten, H, KH, KHM, KMM, KMG, RP, HH, FP, Empress, 440, 660, etc.) have a custom metal mechanism for their auto-reverse. The spools are known as the T1 (which is the same as General Ribbon part # T1-77B , T1-77BR, and Nu-Kote B64.) If winding on universal ribbon onto them, remove the eyelette which isn't needed and may interfere with the auto reverse.

The function of the mechanism is fairly similar to that of the Remington, but the mechanism is on the spool itself rather than on the spindle.

If necessary, Ribbons Unlimited carries these metal spools: https://www.ribbonsunlimited.com/6N064-Royal-Standard-Electric-Ribbon-64-p/6n064.htm

I think the two roads symbolize the choice we all face in life - one may be destiny and the other fate, but no matter which we choose , it leads us to a certain way of living and becoming ourselves.

I wonder how much this represents a mandate from the higher ups

They should be given some leniency because they are minors but instead they are taken advantage of

Not surprising still wondering why

Where do they go after?

Why, whats the incentive

Correct on the first part to a great extent. Not so much on the second sentence.

Society once saw creative women as "mad", but their so-called madness is actually passion, vision and art.

for - search - Google - how new words divide the world in new ways - https://www.google.com/search?q=how+new+words+divide+the+world+in+new+ways&oq=how+new+words+divide+the+world+in+new+ways&gs_lcrp=EgZjaHJvbWUyBggAEEUYOTIHCAEQIRigATIHCAIQIRigATIHCAMQIRigAdIBCDgwODFqMGo0qAIAsAIB&sourceid=chrome&ie=UTF-8 interesting results returned - How words shape our world - https://hyp.is/v03HxqUxEfCM7h8cfH031w/www.prospectmagazine.co.uk/culture/48612/how-words-shape-our-world

Time is important in the things we've read so far. Here, time for the characters is now going to be the same as time for the audience. Remember that John had two intermissions over three days. And footfalls, despite being seven pages, took half an hour to perform.

All we know about the characters are their ages. We don't even know if they have glasses!

Remember how haunted it was to put an empty chair in the room? Now what if the chair had wheels!! and it could... MOVE on its OWN!

https://www.youtube.com/watch?v=bhgpY5K3s10 http://www.tatarowicz.com/the-humans/

this is the last thing that happens in the play

We can be pretty confident going into this play that these quotes are Forwards. Since there's so little context though (in fact, they are the context), we'll likely need to read backwards to make good sense of them

Your home familiar to you! But I've never been there...

Tools of the typewriter trade by [[Retrotype]]

Excellent overview of many of the basic tools for typewriter repair. Didn't have the strongest grasp of all the tools' specific names, but good enough for describing their general use cases.

Example of a typewriter toolset including a case made for telephone company repair, but which works with typewriters.

• Shore A durometer gauge 2:22
• nylon fishing/picture hanging wire spec to 25kg (for drawband replacement)
• thick waxed string/yarn for repairing fishing nets (for drawbands)
• nitrile gloves (to prevent staining, issues with mineral spirits, and other caustic chemicals)
• XPower pressure blower for blowing out dust/dirt and mineral spirits. (smaller than an air compressor)
• nail grooming set with tweezers, picks, etc. (not technically necessary, but sometimes useful)
• dental tools (for use as spring hooks)
• Renaissance micro-crystalline wax (non-corrosive, made for British Museum, good on marble, wood, leather, etc. Good on bare metal for treating previously rusted metal. (It's recommended to use an abrasive polish for improving the shine of glossy paint however)
• Pouch and set of precision screwdrivers (he only uses the flatheads though the set includes other) Prefer hollow ground tips which are squared off rather than wedges.
• Chapman bit set of screwdrivers (with hollow ground tips) He prefers these for hard to remove screws. Issue that it's a bit thicker at the tip.
• Liquid wrench penetrating oil for helping to loosen screws (he likes this better than WD-40)
• brash wire brushes
• steel wire brushes (uses less frequently as they're more abrasive)
• pouch of precision wrenches (imperial and metric) his are bladed, Moody tools wrenches (mfg.) prefer the thinnest tips
• microfiber cloths
• jig for soldering typeslugs on typearms
• pouch with various typewriter specific pliers:
  • 3 prong pliers (total of 9 prongs) for making bends/forming typebars (especially making bends in the middle of bars rather than the end.;
  • peening bend pliers;
  • bending pliers for sideways bends esp. with thinner typebars;
  • vertical adjustment pliers (with rollers) not good for making adjustments of 3mm or more;
  • forming pliers with screws on the end to rotate heads for bending, peening and cutting;
  • peening pliers (bending by metal displacement)
• Magnetized screwdrivers
• forceps
• screw grabber (active capture)
• spring hooks (push/pull)
• nylon brushes for dusting
• needle nose pliers
• t-bender with slotted head for forming metal
• small bottles for mineral spirits and sewing machine oil. They have small metal tips for precision application.

Observation: They met 3 men in a canoe who had hunted for a year and made $900 from furs. The men ran out of food and powder. It rained that night.

Interpretation: This shows how hunters worked for a long time in the wilderness and lived off trading furs.

Connection: It connects to the importance of hunting and trading in the early West.

I learned that fur trading was one of the main ways people survived and made money in the early West. This adds to my connection because it shows how important nature and animals were to the economy before cities and factories grew. It’s important because it helps me see how life and work changed over time from depending on hunting and trading to building towns and using new jobs and trades.

Change over time: Back then, selling furs was one of the main ways to earn money. Later, hunting slowed down because there were fewer animals, new towns were built, and people started finding other jobs instead of trading animal skins.

Observation: They left early, went past Big Monitu Creek, saw buffalo tracks, and Lewis checked out a salt spring with some men.

Interpretation: This shows they were looking at animals and natural things like salt while they traveled.

Connection: It links to how the trip was about learning what the land had, not just moving through it.

I learned that Lewis and his team studied everything around them, like animals and natural resources such as salt. This adds to my connection because it shows the expedition was about discovery and learning what the new land could offer. It’s important because their findings helped the U.S. understand the land’s value, resources, and how people could live there. It shows how exploration helped the country grow and use its new land wisely.

Context: In 1804, the U.S. had just bought this land in the Louisiana Purchase. People didn’t know what was there, so the expedition was sent to study the land, animals, and resources.

Observation: Lewis measured the sun, noticed traces of buffalo, said the land was pretty good, and wrote that he was sick with a sore throat and headache.

Interpretation: This shows they were still studying the land and sky, even when they felt sick.

Connection: It ties to how the trip was about learning and exploring, not just traveling.

I learned that Lewis kept studying the land and animals even when he was sick. This adds to my connection because it shows how hard he worked to help the U.S. learn about new places. It’s important because his notes and measurements taught people what the land was like and what could be found there.

Context: In 1804, the U.S. had just bought this land. The trip was meant to find out what was there like animals, land, and resources that people back east didn’t know about yet.

Observation: Lewis used a tool to measure the sun in the sky. He wrote down that the sun was 37° high.

Interpretation: This shows the group was using science and math to know where they were. They looked at the sun and moon instead of maps, because no maps were there.

Connection: This connects to how the trip was not just about travel. They were also studying the land and collecting facts to help the U.S. learn more.

I learned that Lewis and Clark’s trip was about science and discovery, not just travel. Their measurements helped the U.S. understand new lands after the Louisiana Purchase. This is important because it shows how their work helped make better maps and supported the country’s westward growth.

Causality: Because Lewis and Clark measured things like this, the U.S. could make better maps later. This helped people move west and use the new land

ideas don't just develop by themselves, they must be created and molded by societal and economic conditions

1. What Marx is criticizing

Marx is talking about a mistake in how people understand history:

Some thinkers separate dominant ideas from the people and social conditions that created them.

In other words, they treat ideas as if they exist independently, not as a product of society and its material conditions.

1. What happens when you do that

People start to believe that history is driven by ideas themselves, rather than by the economic and social realities behind them.

Philosophers like Hegel did this: they treated ideas as if they were self-developing forces shaping history.

1. Consequence of this way of thinking

If you focus only on “thought” or “the Idea,” you can end up thinking that all human relations and society come from ideas — like “Man” or “the essence of Man” — rather than from material conditions like class, work, or property.

not practice on idea, but idea on practice (more 'real')

1. Estrangement (alienation) — people become alienated from their work, from what they produce, and from each other under capitalism.

2. To get rid of this alienation (through revolution), two conditions must exist:

First condition:

There has to be:

a large group of people who own nothing (the working class, the “proletariat”),

and, in contrast, a world where wealth and culture are concentrated in the hands of a few.

This situation only appears when society’s productive power (technology, industry, organization of labor, etc.) has become very advanced — capitalism has created huge wealth but distributed it very unevenly.

The development of productive forces (technology, industry, science) must reach such a high level that there’s enough to go around for everyone.

If that’s not the case — if we try to have a revolution before society can produce enough for all — then:

everyone would still be struggling over scarce goods,

people would fight for survival again (“the need-driven struggle over necessities”),

and all the “old crap” (poverty, inequality, exploitation) would come back.

create objects necessary for being - first stage of history

this is the

semantic web approach uh all of them have their own Knowledge Graph their rdf um and Sparkle compliant and they can be

also um a various number of different standards can be applied and we do apply

them as well

so each knowledge as it has a certain amount of

metadata and indexing data that you would require to put them as public information but that's also very

flexible but everything else around it can be made private depending on the on the use case that we have and we work

with some very sensitive sensitive data has proven to work quite well

so each knowledge

and asset has a verifiable issuer and date of date on the blockchain uh meaning that you can

always be certain that some particular entity particular organization particular individual issued the body of

knowledge that you are actually looking at and also uh that it what you're looking at also um is uh represented uh

with the digital fingerprint on the blockchain meaning that you very can verify the data state that nobody

tampered with the data and so on

so we introduced the New Concept the akin to the URL in the web 2 World

um called the unique asset locator so each asset will have its own ual to be discoverable and found so it can be

found on on the decentralized knowledge graph

Timmy turner

urination

arbitrariness

labor, opening

interesting. maybe read

Reviewer #1 (Public review):

Summary:

In this article, Mirza et al developed a continuum active gel model of actomyosin cytoskeleton that account for nematic order and density variations in actomyosin. Using this model, they identify the requirements for the formation of dense nematic structures. In particular, they show that self-organization into nematic bundles requires both flow-induced alignment and active tension anisotropy in the system. By varying model parameters that control active tension and nematic alignment, the authors show that their model reproduces a rich variety of actomyosin structures, including tactoids, fibres, asters as well as crystalline networks. Additionally, discrete simulations are employed to calculate the activity parameters in the continuum model, providing a microscopic perspective on the conditions driving the formation of fibrillar patterns.

Strengths:

The strength of the work lies in its delineation of the parameter ranges that generate distinct types of nematic organization within actomyosin networks. The authors pinpoint the physical mechanisms behind the formation of fibrillar patterns, which may offer valuable insights into stress fiber assembly. Another strength of the work is connecting activity parameters in the continuum theory with microscopic simulations.

Weaknesses:

This paper is a very difficult read for nonspecialists, especially if you are not well-versed in continuum hydrodynamic theories. Efforts should be made to connect various elements of theory with biological mechanisms, which is mostly lacking in this paper. The comparison with experiments is predominantly qualitative. It is unclear if the theory is suited for in vitro or in vivo actomyosin systems. The justification for various model assumptions, especially concerning their applicability to actomyosin networks, requires a more thorough examination. The classification of different structures demands further justification. For example, the rationale behind categorizing structures as sarcomeric remains unclear when nematic order is perpendicular to the axis of the bands. Sarcomeres traditionally exhibit a specific ordering of actin filaments with alternating polarity patterns. Similarly, the criteria for distinguishing between contractile and extensile structures need clarification, as one would expect extensile structures to be under tension contrary to the authors' claim. Additionally, it's unclear if the model's predictions for fiber dynamics align with observations in cells, as stress fibers exhibit a high degree of dynamism and tend to coalesce with neighboring fibers during their assembly phase. Finally, it seems that the microscopic model is unable to recapitulate the density patterns predicted by the continuum theory, raising questions about the suitability of the simulation model.

Reviewer #2 (Public review):

Summary:

The article by Waleed et al discusses the self-organization of actin cytoskeleton using the theory of active nematics. Linear stability analysis of the governing equations and computer simulations show that the system is unstable to density fluctuations and self-organized structures can emerge.

Strengths:

(i) Analytical calculations complemented with simulations (ii) Theory for cytoskeletal network

Weaknesses:

Not placed in the context or literature on active nematics.

Comments on revised version:

The authors have satisfactorily responded to the comments

Reviewer #3 (Public review):

The manuscript "Theory of active self-organization of dense nematic structures in the actin cytoskeleton" analysis self-organized pattern formation within a two-dimensional nematic liquid crystal theory and uses microscopic simulations to test the plausibility of some of the conclusions drawn from that analysis. After performing an analytic linear stability analysis that indicates the possibility of patterning instabilities, the authors perform fully non-linear numerical simulations and identify the emergence of stripe-like patterning when anisotropic active stresses are present. Following a range of qualitative numerical observations on how parameter changes affect these patterns, the authors identify, besides isotropic and nematic stress, also active self-alignment as an important ingredient to form the observed patterns. Finally, microscopic simulations are used to test the plausibility of some of the most crucial assumptions underlying continuum simulations.

The paper is well written, figures are mostly clear, and the theoretical analysis presented in both, main text and supplement, is rigorous. Mechano-chemical coupling has emerged in recent years as a crucial element of cell cortex and tissue organization and it is plausible to think that both, isotropic and anisotropic active stresses, are present within such effectively compressible structures. Even though not explicitly stated this way by the authors, I would argue that combining these two is one of the key ingredients that distinguishes this theoretical paper from similar ones.

The diversity of patterning processes experimentally observed and theoretically described is nicely elaborated on in the introduction of the paper. The theory development and discussion of the continuum model itself is also well-embedded in a review of the relevant broad literature on active liquid crystals and active nematics, which includes plenty of previous results by the authors themselves. Interestingly, several of the patterns identified in the present work, such as 2D hexagonal and pulsatory patterns (Kumar et al, PRL, 2014), as well as contractile patches (Mietke et al, PRL 2019) have been observed previously in different, but related, active isotropic fluid models. In light of this crowded literature, the authors do good job in delineating key results obtained in the present manuscript from existing work.

The results of numerical simulations are well-presented. The discussion of numerical observations is comprehensive, but also at many times qualitative. Some of the observations resonate with recent discussions in the field, for example the observation of effectively extensile dynamics in a contractile system, which is interesting and reminiscent of ambiguities about extensile/contractile properties discussed in recent preprints (Nejad et al, Nat Comm 2024). It is convincingly concluded that, besides nematic stress on top of isotropic one, active self-alignment is a key ingredient to produce the observed patterns.

The authors must be complimented for trying to gain further mechanistic insights into their conclusions using microscopic filament simulations that were diligently performed. It is rightfully stated that these simulations only provide plausibility tests about key assumptions underlying the hydrodynamic theory. Within this scope, I would say the authors are successful. At the same time, it leaves open questions that could have been discussed more carefully. For example, I wonder what can be said about the regime \kappa>0 microscopically, in which the continuum theory does also predict the formation of stripe patterns? How does the spatial inhomogeneous organization the continuum theory predicts fit in the presented, microscopic picture and vice versa? The authors clearly explain the scope and limitations of the microscopic model, which suggests that questions like these will be interesting directions of future investigations.

Overall, the paper represents a valuable contribution to the field of active matter that should provide a fruitful basis to develop new hypothesis about the dynamic self-organisation and mechanics of dense filamentous bundles in biological systems.

Author response:

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

eLife assessment

In this study, the authors offer a theoretical explanation for the emergence of nematic bundles in the actin cortex, carrying implications for the assembly of actomyosin stress fibers. As such, the study is a valuable contribution to the field actomyosin organization in the actin cortex. While the theoretical work is solid, experimental evidence in support of the model assumptions remains incomplete. The presentation could be improved to enhance accessibility for readers without a strong background in hydrodynamic and nematic theories.

To address the weaknesses identified in this assessment, we have expanded the motivation and description of the theoretical model, specifically insisting on the experimental evidence supporting its rationale and assumptions. These changes in the revised manuscript are implemented in the two first paragraphs of Section “Theoretical model” and in a more detailed description and justification of the different mathematical terms that appear in that section. We have made an effort to map in our narrative different terms to mechanistic processes in the actomyosin network. Even if the nature of the manuscript is inevitably theoretical, we think that the revised manuscript will be more accessible to a broader spectrum of readers.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this article, Mirza et al developed a continuum active gel model of actomyosin cytoskeleton that account for nematic order and density variations in actomyosin. Using this model, they identify the requirements for the formation of dense nematic structures. In particular, they show that self-organization into nematic bundles requires both flow-induced alignment and active tension anisotropy in the system. By varying model parameters that control active tension and nematic alignment, the authors show that their model reproduces a rich variety of actomyosin structures, including tactoids, fibres, asters as well as crystalline networks. Additionally, discrete simulations are employed to calculate the activity parameters in the continuum model, providing a microscopic perspective on the conditions driving the formation of fibrillar patterns.

Strengths:

The strength of the work lies in its delineation of the parameter ranges that generate distinct types of nematic organization within actomyosin networks. The authors pinpoint the physical mechanisms behind the formation of fibrillar patterns, which may offer valuable insights into stress fiber assembly. Another strength of the work is connecting activity parameters in the continuum theory with microscopic simulations.

We thank the referee for these comments.

Weaknesses:

(A) This paper is a very difficult read for nonspecialists, especially if you are not well-versed in continuum hydrodynamic theories. Efforts should be made to connect various elements of theory with biological mechanisms, which is mostly lacking in this paper. The comparison with experiments is predominantly qualitative.

We understand the point of the referee. While it is unavoidable to present the continuum hydrodynamic theory behind our results, we have made an effort in the revised manuscript to (1) motivate the essential features required from a theoretical model of the actomyosin cytoskeleton capable of describing its nematic self organization (two first paragraphs of Section “Theoretical model”), and to (2) explicitly explain the physical meaning of each of the mathematical terms in the theory, and when appropriate, relate them to molecular mechanisms in the cytoskeleton. We hope that the revised manuscript addresses the concern of the referee.

Regarding the comparison with experiments, they are indeed qualitative because the main point of the paper is to establish a physical basis for the self-organization of dense nematic structures in actomyosin gels. Somewhat surprisingly, we argue that a compelling mechanism explaining the tendency of actomyosin gels to form patterns of dense nematic bundles has been lacking. As we review in the introduction, these patterns are qualitatively diverse across cell types and organisms in terms of geometry and dynamics, and for this reason, our goal is to show that the same material in different parameter regimes can exhibit such qualitative diversity. A quantitative comparison is difficult for several reasons. First, many of the parameters in our theory have not been measured and are expected to vary wildly between cell types. In fact, estimates in the literature often rely on comparison with hydrodynamic models such as ours. For this reason, we chose to delineate regimes leading to qualitatively different emerging architectures and dynamics. Second, the patterns of nematic bundles found across cell types depend on the interaction between (1) the intrinsic tendency of actomyosin gels to form such structures studied here and (2) other elements of the cellular context. For instance, polymerization and retrograde flow from the lamellipodium, the physical barrier of the nucleus, and the interaction with the focal adhesion machinery are essential to understand the emergence of stress fibers in adherent cells. Cell shape and curvature anisotropy control the orientation of actin bundles in parallel patterns in the wings and trachea of insects. Nuclear positions guide the actin bundles organizing the cellularization of Sphaeroforma arctica [11]. Here, we focus on establishing that actomyosin gels have an intrinsic ability to self organize into dense nematic bundles, and leave how this property enables the morphogenesis of specific structures for future work. We have emphasized this point in the revised section of conclusions.

(B) It is unclear if the theory is suited for in vitro or in vivo actomyosin systems. The justification for various model assumptions, especially concerning their applicability to actomyosin networks, requires a more thorough examination.

We thank the referee for this comment. Our theory is applicable to actomyosin gels originating from living cells. To our knowledge, the ability of reconstituted actomyosin gels from purified proteins to sustain the kind of contractile dynamical steady-states observed in living cells is very limited. In the revised manuscript, we cite a very recent preprint presenting very exciting but partial results in this direction [49]. Instead, reconstituted in vitro systems encapsulating actomyosin cell extracts robustly recapitulate contractile steady-states. This point has been clarified in the first paragraph of Section “Theoretical model”.

(C) The classification of different structures demands further justification. For example, the rationale behind categorizing structures as sarcomeric remains unclear when nematic order is perpendicular to the axis of the bands. Sarcomeres traditionally exhibit a specific ordering of actin filaments with alternating polarity patterns.

We agree with the referee and in the revised manuscript we have avoided the term “sarcomeric” because it refers to very specific organizations in cells. What we previously called “sarcomeric patterns”, where bands of high density exhibit nematic order perpendicular to the axis of the bands, is not a structure observed to our knowledge in cells. It is introduced to delimit the relevant region in parameter space. In the revised manuscript, we refer to this pattern as “banded pattern with perpendicular nematic organization” or “banded pattern” in short.

(D) Similarly, the criteria for distinguishing between contractile and extensile structures need clarification, as one would expect extensile structures to be under tension contrary to the authors' claim.

We thank the referee for raising this point, which was not sufficiently clarified in the original manuscript. We first note that in incompressible active nematic models, active tension is deviatoric (traceless and anisotropic) because an isotropic component would simply get absorbed by the pressure field enforcing incompressibility. Being compressible, our model admits an active tension tensor with deviatoric and isotropic components. We consider always a contractile (positive) isotropic component of active tension, but the deviatoric component can be either contractile (𝜅 > 0) or extensile (𝜅 < 0), where we follow the common terminology according to which in contractile/extensile active nematics the active stress is proportional to q with a positive/negative proportionality constant [see e.g. https://doi.org/10.1038/s41467018-05666-8]. Furthermore, as clarified in the revised manuscript, total active stresses accounting for the deviatoric and isotropic components are always contractile (positive) in all directions, as enforced by the condition |𝜅| < 1.

For fibrillar patterns, we need 𝜅 < 0, and therefore active stresses are larger perpendicular to the nematic direction. This means that the anisotropic component of the active tension is extensile, although, accounting for the isotropic component, total active tension is contractile (see Fig. 1c). This is now clarified in the text following Eq. 7 and in Fig. 1.

However, following fibrillar pattern formation and as a result of the interplay between active and viscous stresses, the total stress can be larger along the emergent dense nematic structures (“contractile structures”) or perpendicular to them (“extensile structures”). To clarify this point, in the revised Fig. 4 and the text referring to it, we have expanded our explanation and plotted the difference between the total stress component parallel to the nematic direction (𝜎∥) and the component perpendicular to the nematic direction (𝜎⊥), with contractile structures satisfying 𝜎∥ − 𝜎⊥ > 0 and extensile structures satisfying 𝜎∥ − 𝜎⊥ < 0. See lines 280 to 303. This is consistent with the common notion of contractile/extensile systems in incompressible nematic systems [see e.g. https://doi.org/10.1038/s41467-018-05666-8].

(E) Additionally, its unclear if the model's predictions for fiber dynamics align with observations in cells, as stress fibers exhibit a high degree of dynamism and tend to coalesce with neighboring fibers during their assembly phase.

In the present work, we focus on the self-organization of a periodic patch of actomyosin gel. However, in adherent cells boundary conditions play an essential role, as discussed in our response to comment (A) by this referee. In ongoing work, we are studying with the present model the dynamics of assembly and reconfiguration of dense nematic structures in domains with boundary conditions mimicking in adherent cells, possibly interacting with the adhesion machinery, finding dynamical interactions as those suggested by the referee. As an example, we show a video of a simulation where at the edge of the circular domain, there is an actin influx modeling the lamellipodium, and in four small regions friction is higher simulating focal adhesions. Under these boundary conditions, the model presented in the paper exhibits the kind of dynamical reorganizations alluded by the referee.

Author response video 1.

We would like to note, however, that the prominent stress fibers in cells adhered to stiff substrates, so abundantly reported in the literature, are not the only instance of dense nematic actin bundles. In the present manuscript, we emphasize the relation of the predicted organizations with those found in different in vivo contexts not related to stress fibers, such as the aligned patterns of bundles in insects (trachea, scales in butterfly wings), in hydra, or in reproductive organs of C elegans; the highly dynamical network of bundles observed in C elegans early embryos; or the labyrinth patters of micro-ridges in the apical surface of epidermal cells in fish.

(F) Finally, it seems that the microscopic model is unable to recapitulate the density patterns predicted by the continuum theory, raising questions about the suitability of the simulation model.

We thank the referee for raising this question, which needs further clarification. The goal of the microscopic model is not to reproduce the self-organized patterns predicted by the active gel theory. The microscopic model lacks essential ingredients, notably a realistic description of hydrodynamics and turnover. Our goal with the agent-based simulations is to extract the relation between nematic order and active stresses for a small homogeneous sample of the network. This small domain is meant to represent the homogeneous active gel prior to pattern formation, and it allows us to substantiate key assumptions of the continuum model leading to pattern formation, notably the dependence of isotropic and deviatoric components of the active stress on density and nematic order (Eq. 7) and the active generalized stress promoting ordering.

We should mention that reproducing the range of out-of-equilibrium mesoscale architectures predicted by our active gel model with agent-based simulations seems at present not possible, or at least significantly beyond the state-of-the-art. To our knowledge, these models have not been able to reproduce the heterogeneous nonequilibrium contractile states involving sustained self-reinforcing flows underlying the pattern formation mechanism studied in our work. The scope of the discrete network simulations has been clarified in lines 340 to 349 in the revised manuscript.

While agent-based cytoskeletal simulations are very attractive because they directly connect with molecular mechanisms, active gel continuum models are better suited to describe out-of-equilibrium emergent hydrodynamics at a mesoscale. We believe that these two complementary modeling frameworks are rather disconnected in the literature, and for this reason, we have attempted substantiate some aspects of our continuum modeling with discrete simulations. We have emphasized the complementarity of the two approaches in the conclusions.

Reviewer #1 (Recommendations For The Authors):

Questions on the theory:

Does rho describe the density of actin or myosin? The authors say that they are modeling actomyosin material as a whole, but the actin and myosin should be modeled separately. Along, similar lines, does Q define the ordering of actin or myosin?

Active gel models of the actomyosin cytoskeleton have been formulated with independent densities for actin and for myosin or using a single density field, implicitly assuming a fixed stoichiometry. Super-resolution imaging of the actomyosin cytoskeleton also suggest that in principle it makes sense to consider different nematic fields for actin and for myosin filaments. In the revised manuscript, we now explicitly mention that our density and nematic field are effective descriptions of the entire actomyosin gel (lines 82-84).

A more detailed model would entail additional material parameters, not available experimentally, which may help reproduce specific experiments but that would make the systematic study of the different behaviors much more difficult. Our approach has been to keep the model minimal meeting the fundamental requirements outlined in the first paragraphs of Section “Theoretical model”.

Should the active stress depend on material density? It seems strange (from Eq. 3) that active stress could be non-zero even where density is zero, since sigma_act does not depend on rho.

Yes, active stress is assumed to be proportional to density. Eq. 3 in the original manuscript was misleading (it was multiplied by rho in Eq. 2). In the revised manuscript, we have explained with a bit more detail the theoretical model, clarifying this point.

The authors should clearly explain their rationale for retaining certain types of nonlinear terms while ignoring others in theory. For instance, the nonlinearities in the equations of motion are sometimes quadratic in the fields, while there are also some cubic terms. Please remark up to what order in the fields the various interactions are modeled.

We thank the referee for raising this point. The nonlinearities in the theory are easily explained on the basis of a small number of choices. We have added a new paragraph towards the end of Section “Theoretical model” (lines 145 to 152) providing a rationale for the origin and underlying assumptions leading to different nonlinearities.

To connect with experiments and the biological context, please explain the biological origin of various terms in the model: (1) L-dependent terms in Eq. 2 and 4, (2) Flowalignment of nematic order and experimental evidence in support of it, (3) densitydependent susceptibility terms in Eq. 4

(1) Unfortunately, the L-dependent terms are very bulky, but are very standard in nematic theories. The best way to understand their physical significance is through the expression of the nematic free-energy, which is now given and explained in the revised manuscript (Eq. 3). The resulting complicated expression for the molecular field and the nematic stress (Eqs. 4 and 5) are mathematical consequences of the choice of nematic free energy. In the revised manuscript, we also attempt to provide a basis for these terms in the context of the actin cytoskeleton. (2) To our knowledge, the best reference supporting this term from experiments is Reymann et al, eLife (2016). In the revised manuscript, we have provided a physical interpretation. (3) We have expanded the motivation and plausible microscopic justification of this term.

There are different 'activity' terms in the model. Their biophysical origin is not made clear. For example, the authors should make clear if these activities arise from filament or motor activity. Relatedly, the authors should provide a comprehensive discussion of the signs of the different active parameters and their physical interpretations.

In an active gel model, activity parameters are phenomenological and how they map to molecular mechanisms is not precisely known, although conventionally contractile active tension is ascribed to the mechanical transduction of chemical power by myosin motors. The fact is that, besides myosin activity, there are many nonequilibrium processes in the actomyosin cytoskeleton that may lead to active stresses including (de)polymerization of filaments or (un)binding of crosslinkers. In the revised manuscript, we have added sentences illustrating how different terms may result from microscopic mechanisms, but providing a precise mapping between our model and nonequilibrium dynamics of proteins is beyond the scope of our work, although our discrete network simulations address this issue to a certain degree.

Following the suggestion of the referee, our description of the theory now discusses much more extensively the signs of activity parameters and their physical interpretations, e.g. the text following Eq. 7.

Throughout the paper, various activity terms are varied independently of each other. Is that a reasonable assumption given that activities should depend on ATP and are thus not independent of one another?

We agree that, ultimately, all active process depend on the conversion of chemical energy into mechanical energy. However, recent work has highlighted how active tension also depends on the microscopic architecture of the network controlled by multiple regulators of the actomyosin cytoskeleton (e.g. Chug et al, Nat Cell Biol, 2017). It is reasonable to expect that, for a given rate of ATP consumption, chemical power will be converted into mechanical power in different ways depending on the micro-architecture of the cytoskeleton, e.g. the stoichiometry of filaments, crosslinkers, myosins, or the length distribution of filaments (very long filaments crosslinked by myosins may be difficult to reorient but may contract efficiently).

We have added a paragraph in Section “Theoretical model” with a discussion, lines 153 to 156.

Sarcomeres are muscle fibers that exhibit alternating polarity pattern. Such patterning is not evident in what the authors call 'sarcomeres' in Fig. 2. I believe the authors should revise their terminology and not loosely interpret existing classifications in the field.

We thank the referee for raising this point. We have changed the terminology.

Fig 2a: Is the cartoon for filament alignment incorrect for kappa>0?

The cartoon is correct. In the revised manuscript we have explained more clearly the physical meaning of kappa in the text following Eq. 7. In the caption of Fig. 1 and of Fig. 2a, we have also clarified that when the absolute value of kappa is <1, then active tension is positive in all directions.

Within the section "Requirements for fibrillar and banded patterns", it will be useful to show the figures for varying the different active parameters in the main figures.

We have followed the referee’s suggestion and moved Supp. Fig. 1 of the original manuscript to the main figures.

How do the authors decide if bundles are contractile or extensile? Why are contractile bundles under tension while extensile bundles are under compression? I would expect the opposite.

We agree that this point deserves a more detailed explanation. In the revised manuscript and in the new Figure 4, we further develop this point. The fibrillar pattern forms when kappa<0. We further assume that -1<kappa<0, so that active tension is positive in all directions. In this regime, the deviatoric (anisotropic) part of active tension is extensile. However, following pattern formation and because of the interplay between active and viscous stresses, the total stress in the emerging bundles may become extensile or contractile, depending on whether the largest component of stress is perpendicular or along the bundle axis. This is now presented in the updated figure, with new panels presenting maps of the total tension. The text discussing this point has been rewritten and we hope that the new version is much clearer (lines 280 to 303).

A contractile bundle tends to shorten, but it cannot do it because of boundary conditions or the interaction with other bundles. As a result they are in tension. Conversely, an extensile bundle tries to elongate, but being constrained, it becomes compressed. As an analogy, consider the cortex of a suspended cell. The cortex is contractile, but it cannot contract because of volume regulation in th cell, which is typically pressurized. As a result, tension in the cortex is positive, as shown by Laplace’s law [10.1016/j.tcb.2020.03.005]. We have tried to clarify this point in the revised manuscript.

Can the authors reproduce alternating density patterns using the cytosim simulations? This is an important step in establishing the correspondence between the continuum theory and the agent-based model.

We have addressed this point in our response to public comment (F) of this referee.

The authors do not provide code or data.

The finite element code with an input file require to run a representative simulation in the paper is now made available, see Ref. [74].

The customizations of Cytosim needed to account for nematic order in our discrete network simulations are available, see Ref. [98].

Reviewer #2 (Public Review):

Summary:

The article by Waleed et al discusses the self organization of actin cytoskeleton using the theory of active nematics. Linear stability analysis of the governing equations and computer simulations show that the system is unstable to density fluctuations and self organized structures can emerge. While the context is interesting, I am not sure whether the physics is new. Hence I have reservations about recommending this article.

We thank the referee for these comments. In the revised manuscript, we have highlighted the novelty, particularly in the last paragraph of the introduction, the first two paragraphs of Section “Theoretical model”, and in the conclusions. Despite a very large literature on theoretical models of stress fibers, actin rings, and active nematics, we argue that the active self-organization of dense nematic structures from an isotropic and low-density gel has not been compellingly explained so far. Many models assume from the outset the presence of actin bundles, or explain their formation using localized activity gradients. The literature of active nematics has extensively studied symmetry breaking and the self-organization. However, most of the works assume initial orientational order. Only a few works study the emergence of nematic order from a uniform isotropic state, but consider dry systems lacking hydrodynamic interactions or incompressible and density-independent systems [37,38]. Yet, pattern formation in actomyosin gels is characterized by large density variations, and by highly compressible flows, which coordinate in a mechanism relying on an advective instability and self-reinforcing flows.

Our theoretical model is not particularly novel, and as we mention in the manuscript, it can be particularized to different models used in the literature. However, we argue that it has the right minimal features to capture nematic self-organization in actomyosin gels. To our knowledge, no previous study explains the emergence of dense and nematic structures from a low-density isotropic gel as a result of activity and involving the advective instability typical of symmetry-breaking and patterning in the actomyosin cytoskeleton. These are important qualitative features of our results that resonate with a large experimental record, and as such, we believe that our work provides a new and compelling mechanism relying on self-organization to explain the prominence and diversity of patterns involving dense nematic bundles in the actomyosin cytoskeleton across species.

Strengths:

(i) Analytical calculations complemented with simulations (ii) Theory for cytoskeletal network

Weaknesses:

Not placed in the context or literature on active nematics.

We agree with the referee that this was a weakness of the original manuscript. In the revised manuscript, within reasonable space constraints given the size and dynamism of the field of active nematics, we have placed our work in the context of this field (end of introduction and first two paragraphs of Section “Theoretical model”). The published version of our companion manuscript [45] also contributes to providing a clear context to our theoretical model within the field.

Reviewer #2 (Recommendations For The Authors):

The article by Waleed et al discusses the self organization of actin cytoskeleton using the theory of active nematics. Linear stability analysis of the governing equations and computer simulations show that the system is unstable to density fluctuations and self organized structures can emerge. While the context is interesting, I am not sure whether the physics is new. Hence I have reservations about recommending this article. I explain my questions comments below.

We have responded to this comment above.

(i) Active nematics including density variations have been dealt quite extensively in the literature. For example, the works of Sriram Ramaswami have dealt with this system including linear stability analysis, simulations etc. In what way is the present work different from the system that they have considered?

(ii) Active flows leading to self organization has been a topic of discussion in many works. For example: (i) Annual Review of Fluid Mechanics, Vol. 43:637-659, 2010, https://doi.org/10.1146/annurev-fluid-121108-145434 (ii) S Santhosh, MR Nejad, A Doostmohammadi, JM Yeomans, SP Thampi, Journal of Statistical Physics 180, 699-709 (iii) M. G. Giordano1, F. Bonelli2, L. N. Carenza1,3, G. Gonnella1 and G. Negro1, Europhysics Letters, Volume 133, Number 5. In what way this work is different from any of these?

(iii) I am confused about the models used in the paper. There is significant literature from Prof. Mike Cates group, Prof. Julia Yeomans group, Prof. Marchetti's group who all use similar governing equations. In the present paper, I find it hard to understand whether the model used is similar to the existing ones in literature or are there significant differences. It should be clarified.

Response to (i), (ii) and (iii).

We completely agree with this referee (and also the previous referee), that the contextualization of our work in the field of active nematics was very insufficient. In the revised manuscript, the last paragraph of the introduction and the first two paragraphs of Section “Theoretical model” now address this point. In short, previous active nematic models predicting patterns with density variations have been either for dry active matter (disregarding hydrodynamic interactions), or for suspensions of active particles moving in an incompressible flow. None of these previous works predict nematic pattern formation as a result of activity relying on the advective instability and self-reinforcing compressible flows, leading to high density and high order bundles surrounded by an isotropic low density phase. Yet, these are fundamental features observed in actomyosin gels. Many works deal with symmetry-breaking of a system with pre-existing order, but very few address how order emerges actively from an isotropic state. We thank the referee for pointing at the paper by Santhosh et al, who nicely make this argument and is now cited. Our mechanism is fundamentally different from that in Santhosh, whose model is incompressible and ignores density variations.

We hope that the revised manuscript addresses this important concern.

(i) >(iv) Below Eqn 6, it starts by saying that the “...origin..is clear...” Its not. I don't understand the physical origin of the instability, and this should be clarified, may be with some illustrations.

We apologize for this unfortunate sentence, which we have rewritten in the revised manuscript (lines 181 to 185).

Reviewer #3 (Public Review):

The manuscript "Theory of active self-organization of dense nematic structures in the actin cytoskeleton" analysis self-organized pattern formation within a two-dimensional nematic liquid crystal theory and uses microscopic simulations to test the plausibility of some of the conclusions drawn from that analysis. After performing an analytic linear stability analysis that indicates the possibility of patterning instabilities, the authors perform fully non-linear numerical simulations and identify the emergence of stripelike patterning when anisotropic active stresses are present. Following a range of qualitative numerical observations on how parameter changes affect these patterns, the authors identify, besides isotropic and nematic stress, also active self-alignment as an important ingredient to form the observed patterns. Finally, microscopic simulations are used to test the plausibility of some of the conclusions drawn from continuum simulations.

The paper is well written, figures are mostly clear and the theoretical analysis presented in both, main text and supplement, is rigorous. Mechano-chemical coupling has emerged in recent years as a crucial element of cell cortex and tissue organization and it is plausible to think that both, isotropic and anisotropic active stresses, are present within such effectively compressible structures. Even though not yet stated this way by the authors, I would argue that combining these two is of the key ingredients that distinguishes this theoretical paper from similar ones. The diversity of patterning processes experimentally observed is nicely elaborated on in the introduction of the paper, though other closely related previous work could also have been included in these references (see below for examples).

We thank the referee for these comments and for the suggestion to emphasize the interplay of isotropic and anisotropic active tension, which is possible only in a compressible gel, as mentioned in the revised manuscript. We have emphasized this point in different places in the revised manuscript. We thank the suggestions of the referee to better connect with existing literature.

To introduce the continuum model, the authors exclusively cite their own, unpublished pre-print, even though the final equations take the same form as previously derived and used by other groups working in the field of active hydrodynamics (a certainly incomplete list: Marenduzzo et al (PRL, 2007), Salbreux et al (PRL, 2009, cited elsewhere in the paper), Jülicher et al (Rep Prog Phys, 2018), Giomi (PRX, 2015),...). To make better contact with the broad active liquid crystal community and to delineate the present work more compellingly from existing results, it would be helpful to include a more comprehensive discussion of the background of the existing theoretical understanding on active nematics. In fact, I found it often agrees nicely with the observations made in the present work, an opportunity to consolidate the results that is sometimes currently missed out on. For example, it is known that self-organised active isotropic fluids form in 2D hexagonal and pulsatory patterns (Kumar et al, PRL, 2014), as well as contractile patches (Mietke et al, PRL 2019), just as shown and discussed in Fig. 2. It is also known that extensile nematics, \kappa<0 here, draw in material laterally of the nematic axis and expel it along the nematic axis (the other way around for \kappa>0, see e.g. Doostmohammadi et al, Nat Comm, 2018 "Active Nematics" for a review that makes this point), consistent with all relative nematic director/flow orientations shown in Figs. 2 and 3 of the present work.

We thank the referee for these suggestions. Indeed, in the original submission we had outsourced much of the justification of the model and the relevant literature to a related pre-print, but this is not reasonable. The companion publication has now been accepted in the New Journal of Physics, with significant changes to better connect the work to the field of active nematics. A preprint reflecting those changes is available in Ref. [64], but we hope to reference the published paper that will come out soon.

In the revised manuscript, we have significantly rewritten the Section “Theoretical model” to frame the continuum model in the context of the field of active nematics. While our model and results have commonalities with previous work, there are also important differences. We have highlighted the novelty of the present work along with the relation with previous studies and theoretical models in the last paragraph of the introduction and the first two paragraphs of Section “Theoretical model”. Furthermore, as suggested by the referee, we have made an effort to connect our results with previous work by Kumar, Mietke, Doostmohammadi and others.

Regarding the last point alluded by the referee (“extensile nematics, \kappa<0 here, draw in material laterally of the nematic axis and expel it along the nematic axis”), the picture raised by the referee would be nuanced for our compressible system as compared to the incompressible systems discussed in that reference. As we have elaborated in our response to point (D) of Referee #1, our systems are overall contractile (with positive active tension in all directions), but the deviatoric component of the active tension can be either extensile or contractile. In our “extensile” models (left in Fig. 2c), material is drawn to laterally to the nematic axis but it is not expelled along this axis. Instead, it is “expelled” by turnover. In the revised manuscript, we have added a comment about this.

The results of numerical simulations are well-presented. Large parts of the discussion of numerical observations - specifically around Fig. 3 - are qualitative and it is not clear why the analysis is restricted to \kappa<0. Some of the observations resonate with recent discussions in the field, for example the observation of effectively extensile dynamics in a contractile system is interesting and reminiscent of ambiguities about extensile/contractile properties discussed in recent preprints (https://arxiv.org/abs/2309.04224). It is convincingly concluded that, besides nematic stress on top of isotropic one, active self-alignment is a key ingredient to produce the observed patterns.

We thank the referee for these comments. We are reluctant to extend the detailed analysis of emergent architectures and dynamics to the case \kappa > 0 as it leads to architectures not observed, to our knowledge, in actin networks. In the revised manuscript, we have expanded and clarified the characterization of emergent contractile/extensile networks by reporting the relative magnitude of stress along and perpendicular to the nematic direction. Our revised manuscript clearly shows that even though all of our simulations describe locally contractile systems with extensile anisotropic active tension, the emergent meso-structures can be either extensile or contractile, with the extensile ones exhibiting the usual bend-type instability (a secondary instability in our system) described classically for extensile active nematic systems. We have rewritten the text discussing this (lines 280 to 303), where we have placed these results in the context of recent work reporting the nontrivial relation between the contractility/extensibility of the local units vs the nematic pattern.

I compliment the authors for trying to gain further mechanistic insights into this conclusion with microscopic filament simulations that are diligently performed. It is rightfully stated that these simulations only provide plausibility tests and, within this scope, I would say the authors are successful. At the same time, it leaves open questions that could have been discussed more carefully. For example, I wonder what can be said about the regime \kappa>0 (which is dropped ad-hoc from Fig. 3 onward) microscopically, in which the continuum theory does also predict the formation of stripe patterns - besides the short comment at the very end? How does the spatial inhomogeneous organization the continuum theory predicts fit in the presented, microscopic picture and vice versa?

We thank the referee for this compliment. We think that the point raised by the referee is very interesting. It is reasonable to expect that the sign of \kappa may not be a constant but rather depend on S and \rho. Indeed, for a sparse network with low order, the progressive bundling by crosslinkers acting on nearby filaments is likely to produce a large active stress perpendicular to the nematic direction, whereas in a dense and highly ordered region, myosin motors are more likely to effectively contract along the nematic direction whereas there is little room for additional lateral contraction by additional bundling. As discussed in our response to referee #1, we believe that studying the formation of patterns using the discrete network simulations is far beyond the scope of our work. We discuss in lines 332 to 341, as well as in the last paragraph of the conclusions, the scope and limitations of our discrete network simulations.

Overall, the paper represents a valuable contribution to the field of active matter and, if strengthened further, might provide a fruitful basis to develop new hypothesis about the dynamic self-organisation of dense filamentous bundles in biological systems.

Reviewer #3 (Recommendations For The Authors):

• The statement "the porous actin cytoskeleton is not a nematic liquid-crystal because it can adopt extended isotropic/low-order phases" is difficult to understand and should be clarified, as the next paragraph starts formulating a nematic active liquid crystal theory. Do the authors mean a crystal that "Tends to be in a disordered phase?", according to its equilibrium properties? It would still be a "nematic liquid crystal", only its ground state is not a nematic phase.

We agree with the referee, and we hope that changes in the introduction and in Section “Theoretical model” address this comment.

• I could not find what Frank energy is precisely used, that would be helpful information.

In the revised manuscript, we have provided the expression for the nematic free energy in Eq. 3.

• The Significance of green/purple arrows in Fig 2a sketch unclear, green arrows also in b,c, do they represent the same quantity? From the simulations images it is overall it is very difficult to see how the flows are oriented near the high-density regions (i.e. if they are towards / away from the strip).

We thank the referee for bringing this up. The colorcodings of the sketches were confusing. The modified figures (Fig. 1(c) and Fig. 2(a)) present now a clearer and unified representation of anisotropic tension. The green arrows in Fig. 2(c) represent the out-of-equilibrium flows in the steady state. We agree that the zoom is insufficient to resolve the flow structure. For this reason, in the revised Fig. 2, we have added additional panels showing the flow with higher resolution.

• It is currently unclear how the linear stability results - beyond identification of the parameter \delta - inform any of the remaining manuscript. Quantitative comparisons of the various length scales seen in simulated patterns (e.g. Fig. 2b, 3c etc) with linear predictions and known characteristic length scales would be instructive mechanistically, would make the overall presentation more compelling and probes limitations of linear results.

In the revised manuscript, we have provided further information so that the readers can appreciate the predictions and limitations of the linear stability results. We have added a sentence and a Figure to show that, in addition to the critical activity, the linear theory provides a good prediction of the wavelengh of the pattern. See lines 199 to 201.

• It is not clear what is meant by "[bundle-formation] requires that active tension perpendicular to nematic orientation is larger than along this direction", and therefore also not why that would be "counter-intuitive". If interpreted naively, I would say that a large tension brings in more filaments into the bundle, so that may well be an obviously helpful feature for bundle formation and maintenance. In any case, it would be helpful if clarity is improved throughout when arguments about "directions of tensions" are made.

We have significantly rewritten the first paragraphs of section “Microscopic origin…” to clarify this point (lines 330 to 339). This paragraph, along with other changes in the manuscript such as the explanation of Eq. 7 or the discussion about the stress anisotropy in the new version of Fig. 4 (see lines 280 to 303), provide a better explanation of this important point.

• All density color bars: Shouldn't they rather be labelled \rho/\rho_0?

Yes! We have corrected this typo.

• Scalar product missing in caption definition of order parameter Fig. 2

We have corrected this typo.

• Fig. 3a: I suggest to put the expression for q0 in the caption

We have changed q_0 by S_0 and clarified its meaning in the caption of what now is Fig 4.

• Paragraph on bottom right of page 6 should several times probably refer to Fig. 3c(...), instead of Fig. 3b

We have corrected this typo.

This paragraph identifies two key factors that have important roles in shaping the use of healthcare and the practice of preventive activities: system trust and family finances. Trust is determined to be foundational in achieving health outcomes and foretells important behaviors such as vaccination and uptake of medical advice. The pandemic of COVID-19 further highlighted the problem, specifically with respect to professional advice follow-through. Independently, the paper records that changes in family finances (from lost job, inflation, or healthcare expenses) prompt an affordability crisis that also extends to affect health behavior.

This paper discusses the manner in which the COVID-19 pandemic produced large shocks to individual lives and global systems, including derived changes in society values, family budgets, healthcare usage, and health behavior.

Clara and Francisco’s story is a reminder that opportunity can be gained but also lost. Success here feels less like a permanent destination, where generational progress depends on constant effort, access, and luck.

What Isabella takes as a normal part of her daily life is something her parents, Clara and Ricardo, once had to seek out through hard work and relocation. Safety is tied to class, geography, and access, showing how privilege can quietly shape how people move through the world.

Equal funding doesn’t guarantee equal opportunity. Even when schools appear balanced on paper, deeper social and economic forces shape what students actually experience. Troy High School and Santa Ana High School might have similar resources, but students enter those classrooms with vastly different support systems, expectations, and barriers, showing how structural inequality extends far beyond school walls.

This shows how quickly a place’s identity can evolve beneath the surface of long-held stereotypes. Orange County now reflects the layered histories of migration, language, and cultural blending. What was once portrayed as a single, uniform image is now an area of diverse communities which challenges outsiders to rethink what the region represents.

Economic stratification leads to residential segregation, which in turn creates class divisions within schools. Wealthy families select high-quality educational resources through housing in school districts or private schools, while children from disadvantaged groups are concentrated in under-resourced schools. Research in China also shows that the combination of the key school system, proximity-based enrollment policies, and the real estate market exacerbates educational inequality.

Sofia's case not only exposes the education system's structural exclusion of disadvantaged students, but also demonstrates that personalized, flexible educational interventions can break down class stratification. Its core message is that educational equity requires not only resource investment but also a restructuring of institutional logic—a shift from "selection and elimination" to "support for growth."

Lola's confusion—"Why can't I get into honors classes even though I'm smart?"—essentially reveals how the American education system reinforces class reproduction. As family background, rather than individual ability, increasingly determines academic outcomes, so-called "equal opportunity" has become a mere rhetoric. Reforms must directly address underlying issues like residential segregation and tax policies, rather than simply blaming schools or individual effort.

The Head Start program, a federal government early childhood education intervention for low-income families, demonstrated short-term success in these children—cultivating their interest in learning and enabling Sofia to demonstrate her talents (e.g., being selected for gifted programs). This confirms research showing the positive effects of early intervention on the cognitive and social-emotional development of disadvantaged children. However, the lack of continuity in this intervention and the absence of a follow-up support system made it difficult to maintain these early advantages.

While this educational model may be a rational choice at the individual level, it exacerbates social stratification at the collective level. Public policy should consider how to mitigate the "Matthew effect" in extracurricular education by subsidizing activities for low-income families and extending after-school programs. At the same time, the education evaluation system should reduce the weighting of paid extracurricular activities, allowing children from all walks of life to more equitably demonstrate their potential.

This passage reveals the complex sources of competitive pressure at Troy High School, reflecting the tension between individual families and the collective culture within the educational environment. Although Isabella emphasizes that her parents did not exert direct pressure but supported her with a "just do your best" attitude, she also acknowledges that the competitive pressure in the school's overall atmosphere permeates each student through the "spillover effect" of other families.

Students at Troy High School focus more on grades and academic achievement than on outward appearances. This atmosphere has made the word "nerd" no longer a derogatory term, but rather a recognition of academic effort, reflecting a difference in values. This environment is consistent with the widespread academic involution among "super high schools," where students pursue admission to prestigious schools through intense competition (such as perfect SAT scores and AP courses).

Kira's English teacher proactively provided emotional support upon learning of her family's tragedy. Not only did she intervene promptly ("If you ever need anyone"), but she also established a long-term, trusting relationship ("I still talk to her"). This support aligns with the core characteristics of "emotional support" in social support theory, effectively alleviating the student's psychological crisis and demonstrating the role of teachers, as "significant others," in shaping students' psychological capital—including fostering resilience and hope.

This passage demonstrates Clara's deep investment and strategic planning in her children's education, highlighting the close connection between educational resources and family decision-making. Clara's choice to relocate near a university was not only for geographical convenience, but also to give her children early access to higher-level educational resources (such as university-provided courses), thereby gaining an academic advantage. This "educational migration" behavior reflects the competitive pursuit of high-quality educational resources among contemporary parents, as well as the widespread desire to compensate for or enhance deficiencies in schooling through environmental interventions.

This phenomenon of educational migration is both a legitimate pursuit of equal opportunity for families and a reflection of the structural flaws of the American public education system. The government needs to mitigate disparities in school districts through measures like fiscal balance and teacher support. Otherwise, the "school selection based on housing" model will only perpetuate intergenerational class disparity. This statement also serves as a reminder that educational equity cannot rely solely on the economic capital of individual families; it requires systemic reform.

Francisco's words—"In just one generation, you can leap forward, but in just one generation, you can go backward"—deeply reveal the bidirectional nature and fragility of social mobility. His warning serves as both an affirmation of individual striving and an implicit critique of structural inequality.

This description contrasts sharply with the subsequent description of the living environment of impoverished families, highlighting the theme of unequal educational opportunities explored in the book. This scene not only depicts the physical space but also symbolizes the differences in social class, setting the stage for the subsequent discussion of the different growth environments for children from different socioeconomic backgrounds.

The widening economic gap between Latino and African American communities is closely linked to long-standing unjust public policies, including housing discrimination (such as redlining, which restricts mortgage lending for minorities), unequal distribution of educational resources, and racial segregation in the job market. For example, the median wealth of Latino families is only 19.1% of that of white families, and they are increasingly excluded from high-quality school districts.

The sharp contrast between the "poor areas" and "rich areas" described in the article reflects the class segregation in geographical space. This segregation is not only reflected in income differences, but also reinforces intergenerational inequality through dimensions such as educational resources and community safety.

The author compares Orange County, California, to the "Ellis Island of the 21st century," which profoundly reveals the central role of immigration in the demographic and social transformation of the United States. Ellis Island was the primary gateway for European immigrants entering the United States from the late 19th century to the early 20th century, symbolizing the population mobility and cultural integration of an era.

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Hypermapped coevolution

Multiplicity of views Shared and coteferenve in converdations debates

I found this page 6 years ago because I did a search for the phrase "metaphysics of adjacency" as the meaning/intent-full hash/name for what I've been out-tuiting towards at the time. That one post was the only one web page out of petazillions that had this three word combination on it I would call it today metanexialitic intenional tacit awareness

From the ones adjacents its the ones that are portals to the longest trails well worth blazing and expolring

all driven from future orientated awareness of possibilities

or even impossibiities that desrve to be rendered inevitable and present

How ridiculous LLMs look from this perspective?

not to mention bogus notions like the manyverse?

all its apparent plausibikity is lost if we consider new trails spanninn to the future starting at every moment

https://via.hypothes.is/https://hyperpost.peergos.me/%F0%9F%8E%AD/gyuri/do/web/snarf/%F0%9F%93%85/2025/10/2/Metaphysics.of.Adjacency.html

We will tackle this calculus problem using a spreadsheet method, as we have before!

The future-facing, imaginative, collaborative nature of Schomburg’s collecting and collection were powerful to me. Imagination may carry an unserious? Whimsical? connotation but in the context of Black archive building it is integral and deeply serious. The combination of thinking to the future and imagining a myriad of forms/uses/etc for the archives feels like a precursor to Afrofuturism. Schomburg and his cohort sought to legitimize Blackness by placing Black people firmly in history and documenting it, thus making it possible for Black people to seed themselves in the future. Not to sentimentalize, but the collaboration that was the foundation of this collecting and archive building is beautiful. In many ways the work of Schomburg and his cohort would not have been possible individually. It relied on social ties, and imagination and intent expanded because the thinking was collective. It reminds me of our class readings’ emphasis on collaboration for effective and deep public history.

The relationship between form and message is an interesting one. I’m very interested in the “unpretending” list, and the idea that meaning comes from connecting the dots, rather than a single narrative. I do wonder if it is possible for a narrative to be entirely absent though. I suppose the implication is that narrative comes from commentary (the “thousand comments and flourishes”), whereas a list simply invites lines of inquiry. I’m curious if another implication is that lists are neutral—I don’t think they are, necessarily, but I may be misinterpreting the text. I think again of Trouillot, and his assertion that there is no “perfect” history, that it is not possible to have a history with no silences. I think this extends to neutrality—there no such thing as a perfectly neutral historical narrative or historical source. I think this probably relates to Constructivism and Positivism, but I’m a bit at sea in understanding those concepts.

My mind jumped to Trouillot upon reading this. Invisibility seems to align with silencing at first glance, but as I was thinking about it, in the particular field of bibliography, an author may not be so easily silenced. Invisible, yes, as their names may not appear in records. However, their words continue on. Though they may not have as much reach, they are not as effectively silenced. On the other hand, this invisibility does align with the concept of leaving traces in history, and Trouillot’s ideas about uneven power and uneven traces. Author’s whose names were omitted from auction and sale records of their work may not leave traces in that field equal to their “power” or importance. For example, Frederick Douglas’s work garnering only one entry in 26 years of sales does not match his significance as a historical figure.

P. 17: The maiden's servants, left, and flew...they were her servants before becoming her servants.

eLife Assessment

This important work has the potential to expand the repertoire of transgenic animals for systems neuroscience investigations across multiple fields. The generation of new reagents has the potential to open new directions in experimental design, and the Cas9-based approach for generating mice may provide additional benefits compared to existing BAC transgenic mouse lines. However, whereas some of the imaging data are compelling, quantitative analysis of transgene fidelity is incomplete, as it relies on a qualitative description of reporter XFP expression at low magnification, with some electrophysiological characterization.

Reviewer #1 (Public review):

Summary:

I read with much attention the manuscript titled "Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons" in which the authors reveal five transgenic lines to target diverse neuronal populations of the basal ganglia. In addition, the authors also provide some assessments of the functionality of the lines.

Strengths:

Knockin lines made readily available through Jackson. Lines show specific expression.

Weaknesses:

Although I have no doubt these knocking lines will be broadly used by researchers in the field, I find the scientific advances of the study and the breadth of the resource provided quite limited. This is partly because 4 of these lines have been generated by other laboratories. For instance, there are already two other Dat-FlpO lines generated (JAX#: 033673 and 035436), with one of them already characterized (PMID: 33979604). Similarly, Drd1-Cre and Adora2a-Cre have been used abundantly since they were generated over a decade ago, and a novel Drd1-FlpO line has been characterized thoroughly recently (PMID: 38965445). Indeed, some of these lines were BAC transgenic, and I agree with the authors that there is a sound rationale for generating knock-in mice; however, the authors should then demonstrate if/how their new drivers are superior. Overall, the valuable resource generated by the authors would benefit from additional quantification and validation.

Reviewer #2 (Public review):

Summary:

The authors report the generation and validation of new knock-in mouse lines enabling precise targeting of basal ganglia projection neurons and midbrain dopamine neurons. By inserting recombinase sequences at endogenous loci, they provide tools that improve on older BAC-based models, with the additional benefit that all lines are openly available through Jackson Laboratories. This work is timely, fills a longstanding gap for the community, and will support both basic circuit mapping and disease-related research.

Strengths:

The major strength of this study is the provision of new genetic resources that will be widely used by the basal ganglia and dopamine research communities. Anatomical and electrophysiological data indicate appropriate expression and preserved intrinsic properties. The Flp lines, in particular, show labeling largely confined to basal ganglia circuits, making them especially attractive for circuit-based studies. A further strength is the use of a T2A-recombinase insertion at the native gene stop codon, which preserves endogenous regulation and maintains near-physiological expression of Adora2a, Drd1a, and DAT. The availability of both Cre and Flp versions enables powerful intersectional strategies, and open distribution through Jackson Laboratories ensures broad accessibility and long-term value.

Weaknesses:

The major limitation is the discrepancy between Cre and Flp lines, with Cre generally driving broader expression than Flp. This raises concerns about anatomical fidelity that require validation at the cellular level. For the DAT-FlpO line, efficiency remains insufficiently quantified, and higher-resolution co-labeling with TH immunostaining is needed. Electrophysiological comparisons between Cre and Flp versions are also incomplete; current data suggest potential physiological differences, which warrant additional statistical testing and, at a minimum, explicit discussion in the manuscript.

Reviewer #3 (Public review):

Summary:

Using latest knock-in technology, the authors generated a set of five mouse lines with expression of recombinases in striatal projection neurons and dopaminergic neurons for public use. They rigorously characterize the expression of the recombinases by intersectional crossing with reporter lines to demonstrate that these lines are faithful, and they perform electrophysiological experiments in slices to provide evidence that the respective neurons show the expected features in these assays.

Strengths:

The characterization of the new mouse lines is exceptional, and these will be widely used by the community. The mouse lines are openly available for the community to use.

Weaknesses:

No weaknesses were identified by this Reviewer.

Author response:

We thank all three reviewers for their thoughtful and constructive evaluations of our manuscript, “Generation of knock-in Cre and FlpO mouse lines for precise targeting of striatal projection neurons and dopaminergic neurons.” We are encouraged that the reviewers recognize the value, specificity, and utility of these new lines for the basal ganglia and dopamine research communities. Below, we summarize our planned revisions and clarifications in response to the reviewers’ comments.

(1) Novelty and comparison with existing lines

We appreciate Reviewer 1’s point regarding the existence of previously generated Cre and Flp lines targeting similar neuronal populations. Our project was initiated six years ago, and during the course of generating and characterizing all five lines, we became aware that similar individual lines have since been developed by other groups. Nevertheless, our study provides a coordinated and independently validated set of lines created using a standardized knock-in (KI) strategy and distributed through Jackson Laboratories for unrestricted community use. Importantly, whereas previous BAC transgenic approaches rely on random insertion, which can lead to position effects and ectopic expression, our design places the recombinase coding sequence immediately downstream of the endogenous stop codon using a self-cleaving T2A peptide. This ensures expression under native promoter and regulatory control, preserving physiological gene regulation.

To address the Reviewers’ points, we will (i) expand the Introduction and Discussion to clarify the rationale and advantages of endogenous promoter–driven recombinase expression over BAC-based systems, emphasizing that our lines provide a uniform, promoter-controlled, and publicly accessible toolkit for the community, (ii) and explore including a comparative table summarizing differences in construct design, expression fidelity, and recombination efficiency across published lines (e.g., PMID 33979604, 38965445).

(2) Quantification, validation, and comparison of Cre vs FlpO

We agree with Reviewers 1 and 2 that further quantification and discussion of Cre versus FlpO fidelity will strengthen the manuscript. The observed difference in expression breadth between Cre and FlpO lines likely reflects a fundamental property of the recombinases themselves rather than a discrepancy in targeting. Cre recombinase is significantly more enzymatically efficient than FlpO, meaning that even very low endogenous levels of gene expression (e.g., Drd1a or Adora2a) can drive Cre-dependent recombination, whereas FlpO requires higher expression thresholds. Consequently, reporter-based readouts will inherently appear broader for Cre lines, despite both being driven by the same endogenous promoters.

To address these points, we will (i) provide quantitative co-labeling analyses for the DAT-FlpO line with TH immunostaining to assess efficiency and specificity, (ii) clarify in the Results and Discussion that differences between Cre and FlpO expression patterns largely stem from differences in recombinase kinetics and sensitivity, not mismatched promoter activity, (iii) and include representative high-resolution images and relevant statistics in the revised figures. Importantly, we would like to note that RNAscope may not be an ideal validation approach in this context, as in situ transcript detection cannot capture the enzymatic threshold differences that determine reporter recombination and thus will not help address observed differences between Cre and FlpO lines. Finally, we are actively performing electrophysiological comparisons between Cre and FlpO lines to rigorously quantify potential physiological differences between them. Updated analyses will be incorporated as available or described as ongoing future work.

(3) Discussion of scope and interpretation

We appreciate the reviewers’ suggestions to better contextualize the scope of this resource. We will revise the Discussion to (i) highlight that the Cre–FlpO pairings enable powerful intersectional and cross-line strategies for dissecting basal ganglia and midbrain circuitry, (ii) and clarify that our goal was to generate a rigorously validated foundational resource, with detailed functional comparisons and manipulation studies to be explored in subsequent work.

In summary, we thank the reviewers for their insightful feedback. The planned revisions and clarifications will underscore the unique strengths of our knock-in design, explore potential Cre–FlpO differences, and highlight the value of this standardized and accessible toolkit for the neuroscience community.

eLife Assessment

This important study reports on the redundant roles of the decapping activators Edc3 and Scd6 in orchestrating post-transcriptional programs to modulate metabolic responses to nutrients in yeast. The authors employed mutagenesis studies in conjunction with a battery of transcriptome-wide analyses to provide convincing evidence supporting their conclusions. Considering the broad implications of post-transcriptional regulation of gene expression, this study will be of interest across a variety of biomedical disciplines ranging from biochemistry and molecular and cellular biology to those specializing in studying various pathologies.

Reviewer #1 (Public review):

Summary:

mRNA decapping and decay factors play critical roles in post-transcriptionally regulating gene expression. Here, Kumar and colleagues investigate how deleting two yeast decapping enhancer proteins (Edc3 and Scd6), either alone or in tandem, affects the transcriptome. Using RNA-Seq, CAGE-Seq and ribosome profiling, they conclude that these factors generally act in a redundant fashion, with a mutant lacking both proteins showing an increased abundance of select mRNAs. As these upregulated transcripts are also upregulated in mutants lacking the decapping enzyme, Dcp2, and show no increases in transcription of their cognate genes, the authors conclude that this is at the level of mRNA decapping and decay. This was further supported by CAGE-Seq analyses carried out in WT cells and the scd∆6edc3∆ double mutant. Their ribosome profiling data also lead them to conclude that Scd6 and Edc3 display functional redundancy and cooperativity with Dhh1/Pat1 in repressing the translation of specific transcripts. Finally, as their data suggest that Scd6 and Edc3 repress mRNAs coding for proteins involved in cellular respiration, as well as proteins involved in the catabolism of alternative carbon sources, they go on to show that these decapping activators play a role in repressing oxidative phosphorylation.

Strengths:

Overall, this manuscript is well-written and contains a large amount of compelling high-quality data and analyses. At its core, it helps to shed light on the overlapping roles Edc3 and Scd6 have in sculpting the yeast transcriptome.

Weaknesses:

While not essential, it would be interesting if the authors carried out add-back experiments to determine which domain within Scd6/Edce3 plays a critical role for enforcing the regulation that they see? Their double mutant now puts them in a perfect position to carry out such experiments.

Reviewer #2 (Public review):

Summary:

This manuscript by Kumar and Zhang presents compelling evidence that Edc3 and Scd6 decapping activators, present a high degree of redundancy that can only be overcome by double mutants of both. In addition, the authors provide strong evidence for their role in regulating starvation-induced pathways as evidenced by measurements of mitochondrial membrane potential, metabolomics and analysis of the flux of Krebs cycle intermediates.

Strengths:

Kumar, Zhang et al provide multiple source of evidence of the direct mechanism of Edc3 and Scd6, by using and comparing different approaches such as mRNA-seq, ribosome occupancies and translational efficiencies. By extensive analysis the authors show that this complex can also regulate genes outside the Environmental Stress Response (non-iESR) that are significantly up-regulated in all three mutants. Remarkably, the gene ontology analysis of these non-iESR genes identify enrichment for mitochondrial proteins that are implicated in the Krebs cycle. Overall, this study adds novel mechanistic insight into how nutrients control gene expression by modulating decapping and translational repression.

Weaknesses:

The authors show very nicely that growth phenotypes from scd6Δedc3∆ can be rescued by transformation of EDC3 (pLfz614-7) or SCD6 (pLfz615-5). Future work could make use of these rescue strategies, for example as a platform to further characterise protein-protein interactions between Edc3, Scd6 and Dhh1.

Reviewer #3 (Public review):

Summary:

In this paper, Kumar et al investigated the role of two decapping activators, Edc3 and Scd6, in regulating mRNA decay and translation in yeast. Using a variety of approaches including RNA-seq, ribosome profiling, proteomics, polysome analysis, and metabolomics the authors demonstrate that whereas single deletions of Edc3 or Scd6 have modest effects, the double mutant leads to increased abundance of mRNAs, many of which overlap with those targeted by the decapping activators Dhh1 and Pat1. The data suggest that Edc3 and Scd6 function redundantly to recruit Dhh1 to the Dcp2 decapping complex, thereby promoting mRNA turnover and translational repression. The authors show that these factors cooperate with Dhh1/Pat1 to repress transcripts involved in respiration, mitochondrial function, and alternative carbon source utilization, linking post-transcriptional regulation to nutrient responses. The study establishes Edc3 and Scd6 as important, but redundant regulators that fine-tune gene expression and metabolic adaptation in response to nutrient availability.

Strengths:

The paper has several strengths, including the comprehensive approach taken by the authors using multiple experimental techniques (RNA-seq, ribosome profiling, Western blotting, TMT-MS, polysome profiling, and metabolomics) to provide multiple lines of evidence to support their conclusions. The authors demonstrate clear redundancy of the factors by using single and double mutants for Edc3 and Scd6 and their global approach enables an understanding of these factors' roles across the yeast transcriptome. The work connects post-transcriptional processes to nutrient-dependent gene regulation, providing insights into how cells adapt to changes in their environment. The authors demonstrate the redundant roles of Edc3 and Scd6 in mRNA decapping and translation repression. Their RNA-seq and ribosome profiling results convincingly show that many mRNAs are derepressed only in the double mutants, confirming their hypothesis of redundancy. Furthermore, the functional cooperation between Edc3/Scd6 and Dhh1/Pat1 in regulating specific metabolic pathways, including mitochondrial function and carbon source utilization, is supported by the metabolomic data.

Weaknesses:

The study uses indirect evidence to support claims about the effect on mRNA stability rather than directly measuring mRNA stability. However, the combination of Pol II occupancy and RNA abundance measurements is consistent with the claims regarding mRNA stability. The addition of new experiments in the revision co-IPing Dhh1 and Dcp2 strengthens the argument that Edc3 and Scd6 recruit these factors.

Author response:

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

Reviewer #1 (Public review): 

Strengths: 

Overall, this manuscript is well-written and contains a large amount of high-quality data and analyses. At its core, it helps to shed light on the overlapping roles of Edc3 and Scd6 in sculpting the yeast transcriptome. 

Weaknesses: 

(1) While the data presented makes conclusions about mRNA stability based on corresponding ChIP-Seq analyses and analyzing other mutants (e.g. Dcp2 knockout), at no point is mRNA stability actually ever directly assessed. This direct assessment, even for select transcripts, would further strengthen their conclusions. 

We appreciate the reviewer’s concern but wish to emphasize that we conducted ChIP-Seq analysis of RNA Polymerase II occupancies in the CDSs of all genes, known to be a reliable indicator of transcription rate, and found only small increases in Pol II occupancies that cannot account for the increased transcript levels of the cohort of mRNAs up-regulated in the scd∆6edc3∆ double mutant (Fig. 3E). This provides strong evidence that increased transcription is not the main driver of increased mRNA abundance in this mutant.  Bolstering this conclusion, we showed that the Hap2/Hap3/Hap4/Hap5 complex of transcription factors responsible for induction of Ox. Phos. genes was not activated in scd6Δedc3Δ cells in glucose medium (Fig. 6F(ii)); nor was the Adr1 activator of CCR genes activated (Fig. S9C(i)), ruling out transcriptional induction of their target genes in glucose-replete scd6Δ/edc3Δ cells and instead favoring reduced degradation as the mechanism underlying derepression of Ox. Phos. and CCR gene transcripts in this mutant. In Fig. 3B, we further showed that the majority of mRNAs up-regulated in the scd6Δedc3Δ double mutant are also derepressed by dcp2Δ, and in Fig. 3D that the mRNAs up-regulated in scd∆6edc3∆ cells exhibit a higher than average codon protection index (CPI) indicating a heightened involvement of decapping and co-translational degradation by Xrn1 in their decay. To provide additional support for our conclusion, we have conducted new experiments to measure the abundance of capped mRNAs genome-wide by CAGE sequencing of total mRNA in both WT and scd∆6edc3∆ cells.  As established previously, normalizing CAGE TPMs to total mRNA TPMs determined by RNA-Seq, dubbed the C/T ratio, provides a reliable measure of the capped proportion of each transcript.  The new data presented in Fig. 3C indicate that the mRNAs up-regulated in the scd∆6edc3∆ mutant have significantly lower than average C/T ratios in WT cells, whereas the C/T ratios for the down-regulated transcripts are higher than average, and that these differences between the two groups and all expressed mRNAs are diminished in the scd∆6edc3∆ double mutant. These are the results expected if the up-regulated mRNAs are selectively targeted for decapping in WT cells dependent on Edc3/Scd6, whereas the downregulated mRNAs are targeted by Edc3/Scd6 less than the average transcript. In the original version of the paper, we came to the same conclusion by analyzing our previous CAGE data for the dhh1∆ mutant for the same transcripts dysregulated scd∆6edc3∆ cells, now presented as supportive data in Fig. S3F. Finally, we added the fact that among all four Dhh1 target mRNAs examined in the previous study of He et al. (2022) and found here to be up-regulated selectively in the scd6∆edc3∆ double mutant (Fig. S10), two of them (SDS23 and HXT6) were shown directly to have longer half-lives in dhh1∆ vs. WT cells by He et al. (2018). Hence, the combined evidence is compelling that selective up-regulation of particular mRNAs in the scd∆6edc3∆ mutant results from diminished decapping/decay rather than enhanced transcription; and we feel that the additional supporting evidence that would be provided by measuring half-lives of a small group of up-regulated transcripts would not justify the considerable effort required to do so.  Moreover, the standard approach for such experiments of impairing transcription with an inhibitor of Pol II or a Pol II Ts^- mutation has been criticized because of the known buffering (suppression) of mRNA decay rates in response to impaired transcription.

(2) Scd6 and Edc3 show a high level of functional redundancy, as demonstrated by the double mutant. As these proteins form complexes with other decapping factors/activators, I'm curious if depleting both proteins in the double mutant destabilizes any of these other factors. Have the authors ever assessed the levels of other key decapping factors in the double mutants (i.e. Dhh1, Pat1, Dcp2...etc)? I wonder if depleting both proteins leads to a general destabilization of key complexes. It would also be interesting to see if depleting Edc3 or Scd6 leads to a concomitant increase in the other protein as a compensatory mechanism. 

We thank the reviewer for this insight.  Examining our Ribo-Seq and TMT-MS data revealed that Dhh1 expression and steady-state abundance are increased ~2-fold in the scd6∆edc3∆ strain, indicating that the up-regulation of many of the same mRNAs by scd6∆edc3∆ and dhh1∆ does not result indirectly from reduced levels of Dhh1 in the scd6∆edc3∆ mutant. The predicted increased in Dhh1 expression might signify a compensatory response to the absence of Scd6/Edc3.  We also observed an ~40% reduction in Dcp2 translation (RPFs) and mRNA abundance in the scd6∆edc3∆ strain, which might contribute to the up-regulation of mRNAs dysregulated in this mutant. However, our new immunoblot analyses revealed no significant reduction in steady-state Dcp2 levels in scd6∆edc3∆ cells (Input lanes in Figs. 3F and S4C(i)-(ii)). Moreover, our previous finding that the majority of mRNAs subject to NMD, up-regulated by both upf1∆ and dcp2∆, are not upregulated by scd6∆edc3∆ implies that Dcp2 abundance in scd6∆edc3∆ cells is adequate for normal levels of NMD and favors a direct role for Scd6/Edc3 in accelerating degradation of most transcripts up-regulated in this mutant. We have added these points to the DISCUSSION.

(3) While not essential, it would be interesting if the authors carried out add-back experiments to determine which domain within Scd6/Edce3 plays a critical role in enforcing the regulation that they see. Their double mutant now puts them in a perfect position to carry out such experiments. 

We agree with the reviewer that our scd6∆edc3∆ strain provides an opportunity to dissect the Scd6 and Edc3 proteins to determine which domains and motifs of each protein are most critically required for their functions in activating mRNA decay. However, if conducted thoroughly, this would entail an extensive analysis requiring a combination of genetics, biochemistry and genomics.  Considering the large amount of data already presented in 43 and 34 panels of main and supplementary figures, respectively, we feel that these additional experiments would be conducted more appropriately as a stand-alone follow-up study.

Reviewer #2 (Public review): 

Weaknesses: 

The authors show very nicely in Figure S1A that growth phenotypes from scd6Δedc3∆ can be rescued by transformation of EDC3 (pLfz614-7) or SCD6 (pLfz615-5). The manuscript might benefit from using these rescue strategies in the analysis performed (e.g. RNA-seq, ribosome occupancies, and translational efficiencies). Also, these rescue assays could provide a good platform to further characterise the protein-protein interactions between Edc3, Scd6, and Dhh1. 

We responded to this point immediately above in responding to Rev. #1.

Reviewer #3 (Public review): 

Weaknesses: 

The limitations of the study include the use of indirect evidence to support claims that Edc3 and Scd6 recruit Dhh1 to the Dcp2 complex, which is inferred from correlations in mRNA abundance and ribosome profiling data rather than direct biochemical evidence. 

While the reviewer makes a valid point, it is important to note that the greater correlations between effects of scd6∆edc3∆ with those conferred by dhh1∆ vs. pat1∆ also extended to changes in metabolites (Fig. 7A-C). To provide more direct evidence that Edc3 and Scd6 recruit Dhh1 to the Dcp2 complex, we have now conducted co-immunoprecipitation experiments (presented in new Figs. 3F and S5) demonstrating that association of Dhh1 with Dcp2 is diminished in the scd6∆edc3∆ double mutant but not in either scd6∆ or edc3∆ single mutant, thus providing biochemical support for our proposal.

Also, there is limited exploration of other signals as the study is focused on glucose availability, and it is unclear whether the findings would apply broadly across different environmental stresses or metabolic pathways. Nonetheless, the study provides new insights into how mRNA decapping and degradation are tightly linked to metabolic regulation and nutrient responses in yeast. The RNA-seq and ribosome profiling datasets are valuable resources for the scientific community, providing quantitative information on the role of decapping activators in mRNA stability and translation control. 

While not disputing the facts of this comment, we think it is unjustified to label as a weakness that our study focused on glucose-grown cells considering the large amount of new data and insights made possible by our multi-omics approach, presented in >70 separate figure panels and nine supplementary datafiles, which the reviewer has characterized as being valuable to the scientific community.  Parallel studies in non-preferred carbon or nitrogen sources are underway and represent large-scale investigations in their own right, for which the current dataset in glucose-replete cells provides the critical reference condition.

Reviewer #1 (Recommendations for the authors): 

The authors made a note that a set of 37 mRNAs is repressed exclusively by Edc3 with little contribution by Scd6, a list that includes the RPS28B mRNA. Edc3 has been previously reported to promote the decay of this mRNA in a deadenylation-independent fashion by binding to an element in its 3'UTR (PMIDs 15225544, 24492965). Can the authors comment on whether Edc3 may be binding to similar elements in the 3'UTRs of these transcripts in their shortlist? This could be an interesting topic matter for discussion as well. 

While an interesting idea, this seems unlikely because the 3’UTR sequence in RPS28B mRNA was shown to bind Rps28 protein itself to confer heightened decapping and decay dependent on Edc3 in a negative autoregulatory loop that exerts tight control over Rps28 protein levels.  It would be surprising if Edc3mediated repression of the other 36 mRNAs would involve Rps28 as none of them encode cytoplasmic ribosomal proteins. Nevertheless, we searched for a conserved motif among the 3’UTRs of the 37 mRNAs using the MEME suite and found enrichment for motifs identified for RNA binding proteins Hrp1 and Nab2 and two novel motifs, but none of these motifs could be recognized within in the Rps28 autoregulatory loop.  We have chosen not to comment on these findings in the revised manuscript to avoid lengthening it unnecessarily with inconclusive observations.

Reviewer #2 (Recommendations for the authors): 

The authors show very nicely in Figure S1A that growth phenotypes from scd6Δedc3∆ can be rescued by the transformation of EDC3 (pLfz614-7) or SCD6 (pLfz615-5). The manuscript might benefit from using these rescue strategies on the analysis performed (e.g. RNA-seq, ribosome occupancies, and translational efficiencies); or expressing truncated mutants of EDC3 (pLfz614-7) or SCD6 (pLfz615-5), to show that they can act as dominant negative competitors, either on the binding to Dhh1 and Dcp2. 

We addressed this comment above in our response to this Reviewer.

Reviewer #3 (Recommendations for the authors): 

(1) Labels such as "mRNA_up_s6,e3" are not defined in figures or the text. I suggest clearer sample labeling throughout. 

The labels had been defined at first mention in the RESULTS but are now indicated there more explicitly, as well as in the legend to Fig. 1.

(2) In Figure 1D it is surprising that the mRNA profile has a peak in the 5' UTR. I would expect to see such a peak in ribosome footprinting data. Is it possible these are incorrectly labeled?

The figure is correctly labeled. Generally, one does not expect to see RPFs in the 5’UTR region unless there is an efficiently translated uORF, which appears not to be the case for MDH2.

In general, the information in this panel and C is inadequate. None of the numbers are clearly explained in the figure legend or in the figure. 

We had cited the legend to Fig. S3C for details of all such gene browser images but have now inserted this information into the Fig. 1D legend, at the first occurrence of such data in the regular figures. 

(3) Figures 1C and 1D are in the wrong order.

Corrected.

(4) Figure 2D is a very complicated Venn Diagram. I suggest using UpSet plots as an alternative to Venn diagrams to more clearly convey overlaps between sets.  

We provided additional explanatory text in the Fig. 2D legend to facilitate understanding.

(5) The use of the same color scheme to represent different sets in panels of the same figure is a source of confusion. E.g. the cyan in Figures 2A, 2D, and 2E indicates unrelated categories, but one would think they are related.

The use of the same cyan color in these three figure panels actually does designate results for the same set of 591 mRNAs up-regulated in the three mutants.  The application of the color schemes is now mentioned explicitly in Figs. 1, 2, and S3.

(6) Reporting of p-values = 0 in figures is not useful.

Corrected.

(7) The whole manuscript is extremely long which reduces the overall impact. For example, the introduction is six pages long. I suggest reducing redundant text and being more concise to enhance readability. 

We tried to streamline the text wherever possible, in particular shortening the Introduction by two pages.

(8) Many abbreviations are used throughout the text that are not introduced the first time they are used. 

Corrected throughout.

(9) The ERCC normalization is unclear. Were the spike-ins added before cell lysis to allow estimation of per-cell RNA counts or to the extracted RNA? If added to extracted RNA rather than cells it is not clear to me how the claim can be made regarding increased mRNA abundance in the mutants. 

We thank the reviewer for this comment. As we explained in the Methods, 2.4 µl of 1:100 diluted ERCC RNA Spike-In Control Mix 1 was added to 1.2 µg of each total RNA sample prior to cDNA library preparation.  Because the majority of total mRNA is comprised of rRNA, this normalization yields the abundance of each mRNA relative to rRNA. Owing to repression of rESR mRNAs encoding ribosomal proteins and biogenesis factors in the scd6∆edc3∆ strain (Fig. S3D), the ribosome content per cell is expected to be reduced in this mutant vs. WT. We showed previously that the isogenic dcp2∆ mutant that elicits an ESR response of similar magnitude, showed a 30% reduction in bulk ribosomal subunits per cell compared to same WT strain examined here {Vijjamarri, 2023 #7866}.  Assuming a similar reduction in ribosome abundance in the scd6∆edc3∆ mutant, the changes in mRNA per cell conferred by the scd6∆edc3∆ mutation are expected to be 0.7-fold of the ERCCnormalized values given in Fig. 3E, yielding fold-changes of 2.00 and 0.62 for the mRNA_up and mRNA_dn, groups, respectively, which still differ substantially from the corresponding changes in normalized Rpb1 occupancies of 1.2 and 0.93, respectively.  We have added this new analysis to the text of RESULTS.

(10) The use of the terms "up-regulated" and "derepressed" throughout is confusing. Both refer to observed increased abundance of mRNAs, but they imply different causes which are never clearly defined. 

We changed all occurrences of “derepressed” to “up-regulated”.

eLife Assessment

This manuscript revisits the well-studied KdpFABC potassium transport system from bacteria with a convincing set of new higher resolution structures, a protein expression strategy that permits purification of the active wildtype protein, and solid insight obtained from mutagenesis and activity assays. The thorough and thoughtful mechanistic analyses makes this a valuable contribution to the membrane transport field.

Reviewer #2 (Public review):

Summary:

The paper describes the high-resolution structure of KdpFABC, a bacterial pump regulating intracellular potassium concentrations. The pump consists of a subunit with an overall structure similar to that of a canonical potassium channel and a subunit with a structure similar to a canonical ATP-driven ion pump. The ions enter through the channel subunit and then traverse the subunit interface via a long channel that lies parallel to the membrane to enter the pump, followed by their release into the cytoplasm.

The work builds on the previous structural and mechanistic studies from the authors' and other labs. While the overall architecture and mechanism have already been established, a detailed understanding was lacking. The study provides a 2.1 Å resolution structure of the E1-P state of the transport cycle, which precedes the transition to the E2 state, assumed to be the rate-limiting step. It clearly shows a single K+ ion in the selectivity filter of the channel and in the canonical ion binding site in the pump, resolving how ions bind to these key regions of the transporter. It also resolves the details of water molecules filling the tunnel that connects the subunits, suggesting that K+ ions move through the tunnel transiently without occupying well-defined binding sites. The authors further propose how the ions are released into the cytoplasm in the E2 state. The authors support the structural findings through mutagenesis and measurements of ATPase activity and ion transport by surface-supported membrane (SSM) electrophysiology.

Reviewer #3 (Public review):

Summary:

By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wildtype protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway.

Strengths:

The high resolution (2.1 Å) of the current structure is impressive, and allows many new densities in the potassium transport pathway to be resolved. The authors are judicious about assigning these as potassium ions or water molecules, and explain their structural interpretations clearly. In addition to the nice structural work, the mechanistic work is thorough. A series of thoughtful experiments involving ATP hydrolysis/transport coupling under various pH and potassium concentrations bolsters the structural interpretations and lends convincing support to the mechanistic proposal. The SSME experiments are generally rigorous.

Weaknesses:

The present SSME experiments do not support quantitative comparisons of different mutants, as in Figures 4D and 5E. Only qualitative inferences can be drawn among different mutant constructs.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This study on potassium ion transport by the protein complex KdpFABC from E. coli reveals a 2.1 Å cryo-EM structure of the nanodisc-embedded transporter under turnover conditions. The results confirm that K+ ions pass through a previously identified tunnel that connects the channel-like subunit with the P-type ATPase-type subunit. 

Strengths: 

The excellent resolution of the structure and the thorough analysis of mutants using ATPase and ion transport measurements help to strengthen new and previous interpretations. The evidence supporting the conclusions is solid, including biochemical assays and analysis of mutants. The work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis. 

Weaknesses: 

There is insufficient credit and citation of previous work. 

The manuscript has been thoroughly revised with special attention to acknowledging all past work relevant to the study.

Reviewer #2 (Public review): 

Summary: 

The paper describes the high-resolution structure of KdpFABC, a bacterial pump regulating intracellular potassium concentrations. The pump consists of a subunit with an overall structure similar to that of a canonical potassium channel and a subunit with a structure similar to a canonical ATP-driven ion pump. The ions enter through the channel subunit and then traverse the subunit interface via a long channel that lies parallel to the membrane to enter the pump, followed by their release into the cytoplasm. 

Strengths: 

The work builds on the previous structural and mechanistic studies from the authors' and other labs. While the overall architecture and mechanism have already been established, a detailed understanding was lacking. The study provides a 2.1 Å resolution structure of the E1-P state of the transport cycle, which precedes the transition to the E2 state, assumed to be the ratelimiting step. It clearly shows a single K+ ion in the selectivity filter of the channel and in the canonical ion binding site in the pump, resolving how ions bind to these key regions of the transporter. It also resolves the details of water molecules filling the tunnel that connects the subunits, suggesting that K+ ions move through the tunnel transiently without occupying welldefined binding sites. The authors further propose how the ions are released into the cytoplasm in the E2 state. The authors support the structural findings through mutagenesis and measurements of ATPase activity and ion transport by surface-supported membrane (SSM) electrophysiology. 

Weaknesses: 

While the results are overall compelling, several aspects of the work raised questions. First, the authors determined the structure of the pump in nanodiscs under turnover conditions and observed several structural classes, including E1-P, which is detailed in the paper. Two other structural classes were identified, including one corresponding to E2. It is unclear why they are not described in the paper. Notably, the paper considers in some detail what might occur during the E1-P to E2 state transition, but does not describe the 3.1 Å resolution map for the E2 state that has already been obtained. Does the map support the proposed structural changes? 

As was seen in previous work by Silberberg et at. (2022), imaging KdpFABC under turnover conditions can produce multiple enzymatic states. We focus on the E1~P state and associated biophysical analyses to provide a clear and concise story that is focused on the conduction pathway for K⁺ ions. We continue to work with the cryo-EM data as well as other supporting methodologies and datasets with the goal of producing an additional manuscript that will describe other conformations. The class of particles producing the 3.1 Å structure shown in Fig. 1 – figure suppl. 2 is heterogeneous and thus requires further classification to elucidate conformational changes, as is apparent from the downstream processing of the E1 classes also shown in that figure. We cannot therefore derive any conclusions about the configuration of side chains at the CBS based on this structure. Nevertheless, two previous structures of the E2.Pi state - 7BGY and 7BH2 which were stabilized MgF₄ and BeF_x, respectively – show the structural change that is described in the paragraph discussing D583A. Given the consistency and relatively high resolution (2.9 and 3.0 Å, respectively) of these two independent structures, we believe that they provide strong support for our proposal for Lys586 acting as a built-in counter ion.

The paper relies on the quantitative activity comparisons between mutants measured using SSM electrophysiology. Such comparisons are notoriously tricky due to variability between SSM chips and reconstitution efficiencies. The authors should include raw traces for all experiments in the supplementary materials, explain how the replicates were performed, and describe the reproducibility of the results. Related to this point above, size exclusion chromatography profiles and reconstitution efficiencies for mutants should be shown to facilitate comparison between measured activities. For example, could it be that the inactive V496R mutant is misfolded and unstable? 

Similarly, are the reduced activities of V496W and V496H (and many other mutants) due to changes in the tunnel or poor biochemical properties of these variants? Without these data, the validity of the ion transport measurements is difficult to assess. 

To address this concern, we have generated a series of supplementary figures for Figs. 2, 4, 5, and 6, which show all of the raw traces underlying our SSME data (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1,Figure 5 - figure supplement 3, Figure 6 - figure supplement 2). We have also included further detail about the experimental protocols, including number and type of replicates, in an expanded "Activity Assays" section of Methods.

In addition, we have included SEC profiles for each of the V496 mutants, which show that they are all well behaved in detergent solution prior to reconstitution (Fig. 4 - figure supplement 1). We are not able to directly document reconstitution efficiencies as it is not practical to separate proteoliposomes from unincorporated protein prior to preparing the sensors used for SSME. Binding currents are seen for several of the inactive mutants (e.g., Q116R in Rb and NH₄ in Fig. 2 - figure supplement 3 and V496R in Fig. 4 - figure supplement 1), which demonstrate that protein is indeed present in the corresponding proteoliposomes even though no sustained transport current is observed.

The authors propose that the tunnel connecting the subunits is filled with water and lacks potassium ions. This is an important mechanistic point that has been debated in the field. It would be interesting to calculate the volume of the tunnel and estimate the number of ions that might be expected in it, given their concentration in bulk. It may also be helpful to provide additional discussion on whether some of the observed densities correspond to bound ions with low occupancy.  

As suggested, we calculated the internal volume of the tunnel within KdpA (from the S4 K⁺ site to the KdpA/KdpB subunit interface) based on the profile derived from Caver. Based on this volume (4.9 x 10^-25 L), a single K⁺ ion within this cavity would correspond to 3.4 M, which is near saturation for a solution of KCl. We added this information together with an acknowledgment of low-occupancy K⁺ to the fourth paragraph of the Discussion:

" Fourth, based on the volume of the cavity in KdpA, a single K⁺ ion would correspond to a concentration of 3.4 M, suggesting that multiple ions would exceed the solubility limit especially in the absence of counterions. Finally, map densities within the tunnel were either of comparable strength or weaker than surrounding side chain atoms, unlike at S3 and canonical binding sites. Although it is possible that weaker density could represent low occupancy K⁺ ions, we favor a mechanism whereby individual K⁺ ions occupy the tunnel transiently as they transit between the selectivity filter and the canonical binding site."

In order to make this analysis, we developed a python script to calculate the volume of the tunnel as defined by the Caver software (this software is available via github.com/dls4n/tunnel). In turn, this enabled us to distinguish water molecules that were actually in the tunnel rather than bound more deeply within the structure of KdpA. As a result, we updated the water distribution plot in Fig. 4b. Notably, the 17 water molecules within this cavity would correspond to 57.8 M, which is reasonably near the expected 55 M for an aqueous solution.

Reviewer #3 (Public review): 

Summary: 

By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wild-type protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway. 

Strengths:  

Although this structure is not so different from previous structures, its high resolution (2.1 Å) is impressive and allows the resolution of many new densities in the potassium transport pathway. The authors are judicious about assigning these as potassium ions or water molecules, and explain their structural interpretations clearly. In addition to the nice structural work, the mechanistic work is thorough. A series of thoughtful experiments involving ATP hydrolysis/transport coupling under various pH and potassium concentrations bolsters the structural interpretations and lends convincing support to the mechanistic proposal. 

Weaknesses: 

The structures are supported by solid membrane electrophysiology. These data exhibit some weaknesses, including a lack of information to assess the rigor and reproducibility (i.e., the number of replicates, the number of sensors used, controls to assess proteoliposome reconstitution efficiency, and the stability of proteoliposome absorption to the sensor). 

To address this concern, we have generated a series of supplementary figures for Figs. 2, 4, 5, and 6, which show all of the raw traces underlying our SSME data (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1,Figure 5 - figure supplement 3, Figure 6 - figure supplement 2). We have also included further detail about the experimental protocols, including number and type of replicates, in the "Activity Assays" section of Methods.

Reviewing Editor Comments

After discussing the evaluations, the Reviewers and Reviewing Editor have identified the following essential revisions that would need to be addressed to improve the eLife assessment:

(1) Work from others in the field should be adequately described and acknowledged: 

(a) Page 2: " A series of X-ray and cryo-EM structures of KdpFABC from E. coli have led to proposals of a novel transport mechanism befitting the unprecedented partnership of these two superfamilies within a single protein complex." 

The authors must give credit where credit is due (namely, the Haenelt/Paulino groups having discovered the transport pathway). Why don't they cite Stock et al., where this pathway was described first? The Stokes group proposed an entirely different pathway initially. 

Explicit reference to this work has been added to as follows:

“A series of X-ray and cryo-EM structures of KdpFABC from E. coli (Huang et al., 2017; Silberberg et al., 2022, 2021; Stock et al., 2018; Sweet et al., 2021) indicate a novel transport mechanism befitting the unprecedented partnership of these two superfamilies within a single protein complex. As first proposed by Stock et al. (Stock et al., 2018), there is now a consensus that K⁺ enters the complex from the extracellular side of the membrane through the selectivity filter of KdpA, but is blocked from crossing the membrane.”

(b) Page 4 " As a result, many previous structures (Huang et al., 2017; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021) feature the S162A mutation to avoid inhibition rather than the fully WT protein used for the current work." 

This is not correct. At least the work by Huang et al 2017 and Stock et al 2021 was done without the mutation. This is why the structures also captured the off-cycle state when no E2 inhibitor was used. But in Silberberg et al 2022 the mutant was used, but this is not mentioned 

The Q116R mutant was used by Huang et al., but indeed not used for the Stock et al paper. We have replaced the sentence in the manuscript with the following:

“Use of the KdpD knockout strain allowed us to produce WT and mutant protein free from Ser162 phosphorylation.”

(c) Page 4: " In the paper, we report on the most highly populated state (44% of particles)". Exactly the same was also seen in detergent solution, which should be mentioned. 

Reference to the Silberberg 2022 paper, where E1~P was the most highly populated state, has been added. The percentage of particles was removed as we are still processing data from the other states, which will we hope will be described in a future manuscript.

(d) Page 7 "Asp583 and Lys586 are two conserved residues on M5 that have previously been shown......indicating that this particular mutation interfered with energy coupling."  The lack of discussion of the Haenelt/Paulino 2021 paper, where they have analyzed the coupling in detail and described a proximal binding site where K+ is coordinated by D583 and the neighbouring Phe is very concerning. 

To correct this oversight, we made the following changes to the text: 

On pg. 7 in the Results section, we refer to the 2005 paper from Bramkamp & Altendorf:

“Consistent with earlier work on this mutant (Bramkamp and Altendorf, 2005), the D583A mutant displayed substantial ATPase activity (30% of WT) but no transport, indicating that this particular mutation interfered with energy coupling.”

At the end of pg. 10 in the Discussion, we revised the paragraph discussing D583 and Lys586 to explicitly refer to the mechanism of transport described in the 2021 paper from Silberberg et al, including proximal and distal binding sites as well as uncoupling due to the D583A mutation.

“Similar to the Glu370/Arg493 charge pair in KdpA, Asp583 and Lys586 are the only charged residues in the membrane core of KdpB. Although they are not seen to interact directly in our structure, they coordinate accessory waters associated with the canonical binding site. Previous molecular dynamics simulations (Silberberg et al., 2021) indicate that Asp583 couples with Phe232 to form a “proximal binding site” for K⁺ ions. Based on these simulations, these authors proposed a mechanism whereby neutralization of this site either by ion binding or by D583A substitution served to stimulate ATPase activity. Indeed, earlier work on D583A (Bramkamp and Altendorf, 2005) as well as current data demonstrate uncoupling, in which K⁺ independent ATPase activity was observed even though transport was abolished. A plausible explanation for this stimulation is seen in the behavior of Lys586 in previous structures of the E2·Pi state (7BGY and 7BH2) (Sweet et al., 2021). In these structures, M5 undergoes a conformational change that pushes the side chain of Lys586 into the CBS. As a consequence of the D583A mutation, this Lys could be freed to act as a built-in counter ion as in related P-type ATPases ZntA (Wang et al., 2014) and AHA2 (Pedersen et al., 2007). In regard to the proximal binding site and the partnering “distal binding site” on the KdpA-side of the subunit interface, our structure does not show densities at either site and thus does not provide any support for the related mechanism. In any case, in the WT complex it seems likely that Asp583 exerts allosteric control over Lys586 and ensures that its movement into the binding site is coordinated with the transition from E1~P to E2·Pi, thus leading to displacement of K⁺ from the CBS and release to the cytoplasm. “

(e) Page 8 " The intersubunit tunnel is arguably one of the most intriguing elements of the KdpFABC complex. Although it has been postulated to conduct K+, experimental evidence has been lacking. " 

Incorrect, see Silberberg 2021. 

On this point, we beg to differ. Although this 2021 paper shows densities in experimental cryo-EM maps and effects of mutations to residues at the KdpA and KdpB interface, the intra-tunnel transport mechanism is based on computational analysis (MD simulations) and not experimental evidence. We softened the statement to read as follows:

“Although it has been postulated to conduct K⁺, direct experimental evidence has been hard to come by.”

(f) In this context, also f232 is not mentioned anywhere in the text, although depicted in almost all figures. 

Phe232 is shown as a point of reference for the KdpA/KdpB subunit interface. We added a reference to Phe232 in the Results section labeled “Intersubunit tunnel” as well as the paragraph in the Discussion addressed in point d) above.

" These densities, which we have modeled as water, are most prevalent near the vestibule, which is the wider part of the tunnel, but then disappear completely at the subunit interface near Phe232, which is the narrowest part of the tunnel and also distinctly hydrophobic (Fig. 4)."

" Previous molecular dynamics simulations (Silberberg et al., 2021) indicate that Asp583 couples with Phe232 to form a “proximal binding site” for K⁺ ions."

(g) Page 2 "Later, it was recognized that KdpA belongs to the Superfamily of K+ Transporters (SKT superfamily), which also includes bona fide K+ channels such as KcsA, TrkH and KtrB (Durell et al., 2000). " 

KcsA is not a member of the SKT superfamily. 

Thanks. This is correct, although the SKT superfamily is believed to have evolved from KcsA. KcsA has been removed from the sentence and a reference added to a review of the SKT superfamily:

“which also includes bona fide K⁺ channels such as TrkH and KtrB (Diskowski et al., 2015; Durell et al., 2000).”

(2) Two other structural classes were identified, including one corresponding to E2. It is unclear why they are not described in the paper. Notably, the paper considers in some detail what might occur during the E1-P to E2 state transition, but does not describe the 3.1 Å resolution map for the E2 state that has already been obtained. Does the map support the proposed structural changes? 

As was seen in previous work by Silberberg et at. (2022), imaging KdpFABC under turnover conditions can produce multiple enzymatic states. We focus on the E1~P state and associated biophysical analyses to provide a clear and concise story. We continue to work with the cryo-EM data as well as other supporting methodologies and datasets with the goal of producing an additional manuscript that will describe other conformations. The class of particles producing the 3.1 Å structure shown in Fig. 1 – figure suppl. 2 is heterogeneous and thus requires further classification to elucidate conformational changes, as is apparent from the downstream processing of the E1 classes also shown in that figure. We cannot therefore derive any conclusions about the configuration of side chains at the CBS based on this structure. Nevertheless, two previous structures of the E2.Pi state - 7BGY and 7BH2 which were stabilized MgF₄ and BeF_x, respectively – show the structural change that is described in the paragraph discussing D583A. Given the consistency and relatively high resolution (2.9 and 3.0 Å, respectively) of these two independent structures, we believe that they provide strong support for our proposal for Lys586 acting as a built-in counter ion.

(3) The paper relies on the quantitative activity comparisons between mutants measured using SSM electrophysiology. Such comparisons are notoriously tricky due to variability between SSM chips and reconstitution efficiencies. The authors should include raw traces for all experiments in the supplementary materials, explain how the replicates were performed, and describe the reproducibility of the results. 

To address this concern, we have generated supplementary figures for Figs. 2, 4, 5, and 6, which show all of the raw traces underlying our SSME data (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1,Figure 5 - figure supplement 3, Figure 6 - figure supplement 2). We have also added a detailed description of replicates, sensor stability and the experimental protocols in the "Activity Assays" section of Methods. In addition, we have highlighted observations of pre-steady state binding currents that were seen for some mutants (e.g., Q116R assayed with Rb⁺, NH₄⁺ and Na⁺), in which an initial, transient current response was observed without an ensuing transport current. The depiction of this raw data has allowed us to explain our use of the current response at 1.25 s, after decay of this binding current, as a measure of transport rate. This approach is consistent with recommendations by the manufacturer, as documented in their 2023 publication (Bazzone et al. https://doi.org/10.3389/fphys.2023.1058583).

(4) Related to this point above, size exclusion chromatography profiles and reconstitution efficiencies for mutants should be shown to facilitate comparison between measured activities. For example, could it be that the inactive V496R mutant is misfolded and unstable? Similarly, are the reduced activities of V496W and V496H (and many other mutants) due to changes in the tunnel or poor biochemical properties of these variants? Without these data, the validity of the ion transport measurements is difficult to assess. 

We have included SEC profiles for each of the V496 mutants, which show that they are all well behaved in detergent solution prior to reconstitution (Fig. 4 - figure supplement 1). We are not able to directly document reconstitution efficiencies as it is not practical to separate proteoliposomes from unincorporated protein prior to preparing the sensors used for SSME. Binding currents are seen for several of the inactive mutants (e.g., Q116R in Rb and NH₄ in Fig. 2 - figure supplement 3 and V496R in Fig. 4 - figure supplement 1), which demonstrate that protein is indeed present in the corresponding proteoliposomes even though no sustained transport current is observed.

(5) What are the different lines in Figure 1 - Supplement 1, panel G? 

This panel depicted a series of SSME traces as an example of the raw data, but has been removed from the revised version given the inclusion of all the raw traces. These new figures include a legend explaining the conditions for each trace.

(6) How was the 44 % population of the single-occupancy E1 state estimated (it does not correspond to the number of particles in Figure 1 - Supplement 2. 

The calculation of 44% for the E1~P state was premature, given that we are still analyzing the data from the turnover conditions. The revised manuscript simply states that E1~P represented the largest population of particles, which is consistent with this state preceding the rate limiting step of the PostAlbers cycle. Reference is made to the Silberberg 2022 paper, which made a similar observation in a detergent-solubilized sample.

(7) The text states that Km for Q116E is "<10 uM". However, the fitted value is 90 µM in Figure 2e. 

This was a typographical error. The text now states that Km for Q116E is <100 M.

(8) The Km values for Rb, NH4, and Na in Figures 2g and h, and Na in Figure 2i do not make sense. They should be removed. 

The values for Km were determined by fitting the Michaelis-Menton equation to the data as detailed in the Methods section. Although the curves visually appear rather flat relative to other ions, the fitting generated respectable confidence limits and are therefore defensible in a statistical context. Furthermore, the curves that are shown are based on those values of Km and it would be inappropriate not to cite them.

(9) Figure 3 would benefit from a slice through the protein to orient the viewer. 

Thanks for the suggestion. We have added panels to Figs. 3, 5 and 6 in an effort to orient the reader to the site that is depicted.

(10) The differences between R493E, Q, and M do not appear to be significant. 

The y-axis is logarithmic which makes a visual comparison difficult. To alleviate this, P values were calculated based on one-way ANOVA analysis are results are indicated in Fig. 3c and 3d. They show that all of the Arg493 mutations have Km significantly higher than WT. Differences between R493E orR493Q and R493Q orR493M are not significant at the p<0.01 level, while the difference between R493E and R493M is highly significant (p<0.001).  The associated text on pg. 6 has been slightly modified as follows:

“Changes to Arg493 generally increase Km (lower apparent affinity) without affecting Vmax, with Met substitution having greater effect than charge reversal (R493E).”

(11) Page 5, paragraph 2. Q116R and G232D don't seem like the world's most intuitive mutations. It appears there is a historical reason for looking at these. Could the rationale be explained in the text? (Why R and D specifically?) 

These mutations have historical significance, having been generated by random mutagenesis during early characterization of the Kdp system by Epstein and colleagues. A sentence containing relevant references has been added to this paragraph to provide this context:

“Specifically, Q116R and G232D substitutions were initially discovered by random mutagenesis during early characterization of the Kdp system (Buurman et al., 1995; Epstein et al., 1978) and have featured in many follow-up studies (Dorus et al., 2001; Schrader et al., 2000; Silberberg et al., 2021; Sweet et al., 2020; van der Laan et al., 2002).”

Below are the recommendations from each of the reviewers, some of which were not included as essential revisions, but that can also be helpful to further strengthen the manuscript. 

Reviewer #1 (Recommendations for the authors): 

It is essential that the authors correct their selective, incomplete, and in places inappropriate references to work from others in the field. 

Specific points: 

(1) Page 2: " A series of X-ray and cryo-EM structures of KdpFABC from E. coli have led to proposals of a novel transport mechanism befitting the unprecedented partnership of these two superfamilies within a single protein complex." 

The authors must give credit where credit is due (namely, the Haenelt/Paulino groups having discovered the transport pathway). Why don't they cite Stock et al., where this pathway was described first? The Stokes group proposed an entirely different pathway initially. 

(2) Page 4 " As a result, many previous structures (Huang et al., 2017; Silberberg et al., 2021; Stock et al., 2018; Sweet et al., 2021) feature the S162A mutation to avoid inhibition rather than the fully WT protein used for the current work." 

This is not correct. At least the work by Huang et al 2017 and Stock et al 2021 was done without the mutation. This is why the structures also captured the off-cycle state when no E2 inhibitor was used. But in Silberberg et al 2022 the mutant was used, but this is not mentioned 

(3) Page 4: " In the paper, we report on the most highly populated state (44% of particles)". Exactly the same was also seen in detergent solution, which should be mentioned. 

(4) Page 7 "Asp583 and Lys586 are two conserved residues on M5 that have previously been shown......indicating that this particular mutation interfered with energy coupling."  The lack of discussion of the Haenelt/Paulino 2021 paper, where they have analyzed the coupling in detail and described a proximal binding site where K+ is coordinated by D583 and the neighbouring Phe is very concerning. 

(5) Page 8 " The intersubunit tunnel is arguably one of the most intriguing elements of the KdpFABC complex. Although it has been postulated to conduct K+, experimental evidence has been lacking. " 

Incorrect, see Silberberg 2021. 

(6) In this context, also f232 is not mentioned anywhere in the text, although depicted in almost all figures. 

References have been added to address all of these points. See item 1) under Reviewing Editor’s Comments above.

Other points: 

(7) Page 2 "Later, it was recognized that KdpA belongs to the Superfamily of K+ Transporters (SKT superfamily), which also includes bona fide K+ channels such as KcsA, TrkH and KtrB (Durell et al., 2000). " 

KcsA is not a member of the SKT superfamily. 

KcsA has been removed from the sentence and a reference added to a review of the SKT family:

“which also includes bona fide K⁺ channels such as TrkH and KtrB (Diskowski et al., 2015; Durell et al., 2000).”

(8) Page 9 " Our demonstration of coupled transport of NH4+ and Rb+ G232D not only confirms that the selectivity filter governs ion selection, but that the pump subunit, KdpB, is relatively promiscuous."  Check grammar. 

This sentence has been updated as follows:

“Our observation that G232D is capable of coupled transport for NH₄<sup>+</sup and Rb⁺ confirms not only that the selectivity filter governs ion selection, but that the pump subunit, KdpB, is relatively promiscuous.

Reviewer #2 (Recommendations for the authors): 

(1) From an editorial point of view, I suggest a few changes to enhance readability and clarity for non-specialists. A description of the overall transport cycle at the start of the paper (perhaps as a supplementary figure) could help put the work into perspective for general readers who may not be familiar with P-type ATPase mechanisms. It is unclear what "single" and "double" occupancy refer to in the structural classes description. Why is only one structural class described in detail? I would suggest moving the discussion of what is going on with the Nterminus of KdpB to the Results section, where it is described, and shortening the corresponding paragraph in the Discussion. I would furthermore suggest adding a figure that illustrates the proposed regulatory role of the terminus and how phosphorylation might affect it. Otherwise, this section of the results reads very hollow. 

A diagram showing the Post-Albers cycle is shown as part of Fig. 1 and is described at the end of the second paragraph. This sentence only mentioned KdpB, which may have caused confusion. We therefore changed the sentence to read as follows:

“Like other P-type ATPases, KdpFABC employs the Post-Albers reaction cycle (Fig. 1) involving two main conformations (E1 and E2) and their phosphorylated states (E1~P and E2-P) to drive transport (Albers, 1967; Post et al., 1969).”

Single and double occupancy was meant to refer to the number of KdpFABC complexes residing in a nanodisc. This can be seen in the class averages in Fig. 1 - figure supplement 2. The legends to Fig. 1 figure supplements 1 and 2 have been revised to explain this observation more explicitly:

"Slight asymmetry of the main peak is consistent with a subpopulation of nanodiscs containing two KdpFABC complexes (Fig. 1 - figure supplement 2)."

and

"A subset of these particles were further classified to generate four main classes representing nanodiscs with a single copy of KdpFABC in either E1 or E2 conformations, nanodiscs with two copies of KdpFABC which were mainly E1 conformation, and junk."

As stated above, the class of particles producing the 3.1 Å structure shown in Fig. 1 – figure suppl. 2 is heterogeneous and requires further classification to elucidate conformational changes, as is apparent from the downstream processing of the E1 classes also shown in that figure. We continue to analyze the cryo-EM data and aim to produce a second manuscript that will include descriptions of other conformations together with the additional biophysical analysis related to their function.

With regard to the N-terminus, we have gone on to generate a truncation of residues 2-9 in KdpB. After expression and purification, this construct remained coupled with ATPase and transport activities similar to WT, which makes proposals of a regulatory effect less compelling. Because of the novelty of observing the N-terminus and the possibility that it plays a subtle role in the kinetics of the cycle not revealed under the current assay conditions, we have retained a brief discussion of this structural observation, but moved it into the Results section as suggested.

"Given the regulatory roles played by N- and C-termini of a variety of other P-type ATPases (Bitter et al., 2022; Cali et al., 2017; Lev et al., 2023; Timcenko et al., 2019; Zhao et al., 2021), we generated a construct in which residues 2-9 of the N-terminus of KdpB were truncated. However, ATPase and transport activities remained coupled at levels similar to WT, indicating that any functional role of the N-terminus is relatively subtle and not manifested under current assay conditions."

(2) The wording "exceedingly strong densities" seems ambiguous. 

We have changed this to “strong” in the Abstract and "exceptionally strong" in the Discussion. The precise values for these densities are shown in density histograms in Fig. 2 – figure supplement 1 and Fig. 5 – figure supplement 2. In the text, the densities are described as follows:

Results sections describing the selectivity filter:

"In fact, this S3 site contains the strongest densities in the entire map, measuring 7.9x higher than the threshold used for Fig. 2a (Fig. 2 – figure suppl. 1a)."

Results section describing the CBS:

"Given that this is the strongest density in KdpB, measuring 5.6x higher than the map densities shown in Fig. 5 (Fig. 5 – figure suppl 2b), we have modeled it as K⁺."

(3) What are the different lines in Figure 1 - Supplement 1, panel G? 

This panel depicted a series of SSME traces as an example of the raw data, but has been removed from the revised version given the inclusion of all the raw traces. These new figures include a legend explaining the conditions for each trace.

(4) How was the 44 % population of the single-occupancy E1 state estimated (it does not correspond to the number of particles in Figure 1 - Supplement 2. 

The calculation of 44% for the E1~P state was premature, given that we are still analyzing the data from the turnover conditions. We will consider citing an updated value in a future publication once this analysis is complete. The revised manuscript simply states that E1~P represented the largest population of particles, which is consistent with this state preceding the rate limiting step of the Post-Albers cycle. Reference was made to the Silberberg 2022 paper, where a similar observation was made.

(5) Panel 1d is called out of order after panel 1e. Please label Ser 162 in the panel. 

The order of these panels have been switched and Ser162 has been labelled as suggested.

(6) Several panels in Figure 1- Supplement 1 are neither referenced nor described. 

This figure supplement is referred to multiple times in the Results and the Methods sections of the text as well as in the figure legends. Although each panel is not individually referenced, all of this information is relevant at different points in the manuscript and is explained in the legend.

(7) Is the coordinating geometry for the S3 site consistent with what was previously observed for KcsA and relatives? 

The general arrangement of carbonyl atoms in the S3 site is the same in KcsA and KdpA, described by the MacKinnon group as a square antiprism. However, KcsA has strict four-fold symmetry and KdpA does not. As a result, there are small discrepancies between the coordinating geometries in the two structures. This point was made graphically in our original report on the X-ray structure of KdpFABC (Huang et al. 2007, Extended Data Fig. 3), though the positions of the carbonyls are more accurately determined in the current structure due to increased resolution. We added a sentence to the Selectivity Filter section of the Results stating the following:

"This coordination geometry is also consistent with that seen in the K⁺ channel KcsA, though the strict four-fold symmetry of that homo-tetramer produces a more regular structure, as indicated by the smaller variance in liganding distance (2.77 Å with s.d. 0.075 Å in 1K4C) and as depicted by Huang et al. in Extended Data Fig. 3 (Huang et al., 2017)."

(8) Label G232D in Figure 2a. 

G232 is out of the plane shown in Fig. 2a. However, we have added a label for Cys344 to help identify the selectivity filter strands that are shown. Note, however, that G232 is visible and labeled in Fig. 2 - figure suppl. 1. This has now been noted in the legend for Fig. 2.

(9) The text states that Km for Q116E is "<10 uM". However, the fitted value is 90 uµ in Figure 2e. 

This was a typographical error. The text now states that Km for Q116E is <100 M.

(10) The Km values for Rb, NH4, and Na in Figures 2g and h, and Na in Figure 2i do not make sense. They should be removed. 

The values for Km were determined by fitting the Michaelis-Menton equation to the data as detailed in the Methods section. Although the curves visually appear rather flat relative to other ions, the fitting generated respectable confidence limits and are therefore defensible in a statistical context. Furthermore, the curves that are shown are based on those values of Km and it would be inappropriate not to cite them.

(11) Figure 3 would benefit from a slice through the protein to orient the viewer. 

Thank you for the suggestion. We have added panels to Figs. 3, 5 and 6 in an effort to orient the reader to the site that is depicted.

(12) The differences between R493E, Q, and M do not appear to be significant. 

The y-axis is logarithmic which makes a visual comparison difficult. To alleviate this, P values were calculated based on one-way ANOVA analysis are results are indicated in Fig. 3c and 3d. They show that all of the Arg493 mutations have Km significantly higher than WT. Differences between R493E orR493Q and R493Q orR493M are not significant at the p<0.01 level, while the difference between R493E and R493M is highly significant (p<0.001).  The associated text on pg. 6 has been slightly modified as follows:

“Changes to Arg493 generally increase Km (lower apparent affinity) without affecting Vmax, with Met substitution having greater effect than charge reversal (R493E).”

Reviewer #3 (Recommendations for the authors): 

Overall, the text was very clear, experiments were rationalized well, and conclusions were justified. A few small comments: 

(1) Page 5, paragraph 2. Q116R and G232D don't seem like the world's most intuitive mutations. It appears there is a historical reason for looking at these. Could the rationale be explained in the text? (Why R and D specifically?) 

These mutations are of historical importance, having been generated by random mutagenesis during early characterization of the Kdp system. A sentence containing relevant references has been added to this paragraph to provide this information as context:

“Specifically, Q116R and G232D substitutions were initially discovered by random mutagenesis during early characterization of the Kdp system (Buurman et al., 1995; Epstein et al., 1978) and have featured in many follow-up studies (Dorus et al., 2001; Schrader et al., 2000; Silberberg et al., 2021; Sweet et al., 2020; van der Laan et al., 2002).”

(2) Typo: page 14, "diluted" 

This typo has been corrected.

(3) The Methods section for SSM electrophysiology could use some additional description of how the data/statistics were collected. How many replicates? Were all replicates from a single sensor/ were multiple sensors examined? Were controls done to test whether the same number of liposomes remain absorbed by the sensor over the length of the experiment? 

We have extended our description of experimental protocols in the "Activity Assays" section of Methods. This includes the number and type of replicates as well as a discussion of binding currents that were seen for some mutants. Furthermore, a new series of supplementary figures for Figs. 2, 4, 5, and 6 show all of the raw traces for the SSME measurements (Figure 2 - figure supplements 2-4, Figure 4 - figure supplement 1, Figure 5 - figure supplement 3, Figure 6 - figure supplement 2).

We have included SEC profiles for each of the V496 mutants, which show that they are all well behaved in detergent solution prior to reconstitution (Fig. 4 - figure supplement 1). We are not able to directly document reconstitution efficiencies as it is not practical to separate proteoliposomes from unincorporated protein prior to preparing the sensors used for SSME. Binding currents are seen for several of the inactive mutants (e.g., Q116R in Rb and NH₄ in Fig. 2 - figure supplement 3 and V496R in Fig. 4 - figure supplement 1), which demonstrate that protein is indeed present in the corresponding proteoliposomes even though no sustained transport current is observed.

What they would gain from War

YES but also hirap maghighlight sa website na itoh

Taking nothing from his tribe- selfish

These are all questions that we should be asking as we are writing our papers. Depending on the type of paper we are writing, it is important to use correct and appropriate wording or phrasing.

This is important information to use when we are preparing to write a paper because it helps to build potential information that we can use in writing in the future.

This is important to the writing process because it teaches us a new method with how we should retain information, or potentially write information down with more emphasis on how we say certain things. This makes this very important for the writing process.

Returns the year of a date as an integer relative to 1900.

Please remove the entire line. This function is not implemented.

How did increased African American political representation in the 1990s reshape Southern politics and challenge traditional power structures?

How did the political and cultural climate of the Reagan era shape both the defense and the rejection of Confederate symbols in the South?

Why was the location of these Confederate symbols on city-owned property significant, and how did it shape debates about history, ownership, and representation?

This line shows the role of student protest and educational institutions in confronting racial injustice during the civil rights movement.

eLife Assessment

This is a methodologically rich manuscript that is important for revealing the center-surround inhibition profile of expectation in orientation space. The analyses are compelling in validating the critical role of predictive coding feedback. The findings provide novel insights into how expectation optimizes perception via enhancement and suppression.

Reviewer #1 (Public review):

Summary:

The authors tested two competing mechanisms of expectation (1) a sharpening model that suppresses unexpected information via center-surround inhibition; (2) a cancellation model that predicts a monotonic gradient response profile. Using two psychophysical experiments manipulating feature space distance between expected and unexpected stimuli, the results consistently supported the sharpening model. Computational modeling further showed that expectation effects were explained by either sharpened tuning curves or tuning shifts. Finally, convolutional neural network simulations revealed that feedback connections critically mediate the observed center-surround inhibition.

Strengths:

The manuscript provides compelling and convergent evidence from both psychophysical experiments and computational modeling to robustly support the sharpening model of expectation, demonstrating clear center-surround inhibition of unexpected information.

Comments on revisions:

I appreciate the authors' thoughtful revisions. I have no further comments.

Reviewer #2 (Public review):

Summary:

This is a compelling and methodologically rich manuscript. The authors used a variety of methods, including psychophysics, computational modeling, and artificial neural networks, to reveal a non-monotonic, center-surround "Mexican-hat" profile of expectation in orientation space. Their data convincingly extend analogous findings in attention and working memory, and the modeling nicely teases apart sharpening vs. shift mechanisms.

Strengths:

The findings are novel and important in elucidating the potential neural mechanisms by which expectation shapes perception. The authors conducted a series of well-designed psychophysical experiments to careful examination of the profile of expectation's modulation. Computational modeling also provides further insights, linking the neural mechanisms of expectation to behavioral results.

Comments on revisions:

I think the authors did a great job in addressing my previous comments. I have no further comments.

Author response:

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

Reviewer #2 (Public review):

(1)  The sharpening model of expectation can predict surround suppression. The authors could further clarify how the cancellation model predicts a monotonic profile of expectation (Figure 1C) with the highest response at the expected orientation, while the cancellation model suggests a suppression of neurons tuned toward the expected stimulus.

We thank the reviewer for the comment. We would like to emphasize that as the expected signal is suppressed, the relative weight or salience of unexpected inputs increases. We have clarified this interpretation in the manuscript as follows:

“Here, given these two mechanisms making opposite predictions about how expectation changes the neural responses of unexpected stimuli, thereby displaying different profiles of expectation, we speculated that if expectation operates by the sharpening model with suppressing unexpected information, we should observe an inhibitory zone surrounding the focus of expectation, and its profile then should display as a center-surround inhibition (Fig. 1c, left). If, however, expectation operates as suggested by the cancelation model with highlighting unexpected information, the inhibitory zone surrounding the focus of expectation should be eliminated, and the profile should instead display a monotonic gradient (Fig. 1c, right).”

(2) I'm a bit concerned about whether the profile solely arises from modulation of expectation. The two auditory cues are each associated with a fixed orientation, which may be confounded by other cognitive processes like visual working memory or attention (which I think the authors also discussed). Although the authors tried to use SFD task to render orientation task-irrelevant, luminance edges (i.e., orientation) and spatial frequency in gratings are highly intertwined and orientation of the gratings may help recall the first grating's SF (fixed at 0.9 c/{degree sign}), especially given the first and second grating's orientations are not very different (4.8{degree sign}).

We agree that dissociating expectation from attention and other top-down processes remains a key challenge in visual expectation research (see Summerfield & Egner, 2009; Summerfield & de Lange, 2014; de Lange et al., 2018). As is generally acknowledged, expectation reflects the probability of a sensory event, while selective attention relates to its behavioral relevance. To minimize attentional influences, our task design ensured that grating orientation was not taskrelevant: on each trial, participants discriminated either orientation or spatial frequency difference, such that orientation itself did not require attentional allocation, a point already discussed in the manuscript.

Regarding visual working memory, we argue that even if participants recalled the first grating’s spatial frequency in the SFD task, they were not required to retain its precise spatial frequency (or orientation), as their task was simply to judge whether the second grating appeared denser or sparser. In other words, orientation (or spatial frequency) itself was not task-relevant. Moreover, although not included in the manuscript, we conducted a post-experiment debriefing in which participants were asked whether they noticed any association between the auditory tone and the grating orientation. None of the participants reported this relationship correctly, suggesting that the tone-orientation mapping remained implicit and was unlikely to be driven by strategic attention or memory.

However, we acknowledge that certain confounding processes such as statistical learning or implicit mapping acquisition cannot be fully ruled out given the current paradigm. Future studies using methods with higher temporal resolution (e.g., EEG/MEG) may help to dissociate these mechanisms more precisely.

(3) For each of the expected orientations (20{degree sign} or 70{degree sign}), the unexpected ones are linearly separable (i.e., all unexpected ones lie on one side of the expected angle). This might further encourage people to shift their attended or expected orientation, according to the optimal tuning hypothesis. Would this provide an alternative explanation to the tuning shift that the authors found?

We thank the reviewer for pointing out the relevance of the optimal tuning hypothesis. We acknowledge that the optimal tuning theory (Navalpakkam & Itti, 2007) is an important framework, particularly in visual search paradigms, where attentional templates may shift away from non-target features to enhance discriminability.

In our task, this hypothesis would predict a shift of expectation toward <20° in E20° trials and >70° in E70° trials, given that all unexpected orientations lie on one side of the expected angle. Importantly, the optimal tuning hypothesis predicts such shifts not only in Δ20°, Δ25°, and Δ30° trials but also in the Δ0° trials. In this regard, the observed shift in Δ20° and Δ30° (Experiment 2) and Δ25° (Experiment 3) trials is broadly consistent with the predictions of the optimal tuning account. However, we did not observe a corresponding shift away from nontarget features in the Δ0° condition, suggesting limited behavioral evidence for optimal tuning effects under our current task settings.

It is important to note that most previous studies supporting optimal tuning (e.g., Navalpakkam & Itti, 2007; Scolari & Serences, 2009; Geng, DiQuattro, & Helm, 2017; Yu & Geng, 2019) have used visual search paradigms that differ from our design in several critical ways, including the number of stimuli presented, their spatial arrangement (eccentricity), task demands, and so on. Therefore, it is difficult to determine whether the optimal tuning hypothesis could serve as an alternative explanation within the context of our current study. We agree that future studies could further examine how such task parameters influence the presence or absence of optimal tuning.

(4) It is great that the authors conducted computational modeling to elucidate the potential neuronal mechanisms of expectation. But I think the sharpening hypothesis (e.g., reviewed in de Lange, Heilbron & Kok, 2018) focuses on the neural population level, i.e., narrowing of population tuning profile, while the authors conducted the sharpening at the neuronal tuning level. However, the sharpening of population does not necessarily rely on the sharpening of individual neuronal tuning. For example, neuronal gain modulation can also account for such population sharpening. I think similar logic applies to the orientation adjustment experiment. The behavioral level shift does not necessarily suggest a similar shift at the neuronal level. I would recommend that the authors comment on this.

We thank the reviewer for this to-the-point comment. As de Lange et al. (2018) noted, “there is not always a direct correspondence between neural-level and voxel-level selectivity patterns.” That is, neuronal tuning, population-level tuning, voxel-level selectivity, and behavioral adaptive outcomes may reflect different underlying mechanisms and do not necessarily align in a one-toone fashion. We fully acknowledge that population-level tuning effects may also result from various neuronal mechanisms such as gain modulation (for review, see Salinas & Thier, 2000), shifts in preferred orientation (Ringach, et al., 1997; Jeyabalaratnam et al., 2013), asymmetric broadening of tuning curves (Schumacher et al., 2022), or tuning curve sharpening (Ringach, et al., 1997; Schoups et al., 2001).  

In our modeling, we implemented sharpening and shifts of neuronal tuning curves as a conceptual model simplification, intended to explore potential mechanisms underlying expectation-related center-surround suppression effects. While sharpening-based accounts (e.g., Kok et al. 2012) have often been emphasized, we stress that other mechanisms, such as gain modulation or tuning shifts, may also contribute. Our goal is not to provide a definitive account, but to highlight such plausible mechanisms and encourage future investigation. We have revised the Discussion to emphasize that multiple mechanisms may underlie the observed effects.

“We note that our implementation of sharpening and shifts at the neuronal level serves as a conceptual model simplification, as population-level tuning, voxel-level selectivity, and behavioral adaptive outcomes may reflect different underlying neuronal mechanisms and do not necessarily align in a one-to-one fashion. Here, we stress that other potential mechanisms beyond sharpening, such as tuning shifts, may also contribute to visual expectation.” 

(5) If the orientation adjustment experiment suggests that both sharpening and shifting are present at the same time, have the authors tried combining both in their computational model?

We agree with the reviewer that it is necessary to consider the combined model. Accordingly, we implemented a computational model incorporating sharpening of the expected orientation channel together with shifting of the unexpected orientation channels. This model

successfully captured the sharpening of the expected-orientation channel and the shift of the unexpectedorientation channels (Supplementary Fig. 3). For the expected orientation (Δ0°) , results showed that the amplitude change was significantly higher than zero on both OD (t(23) = 2.582, p = 0.017, Cohen’s d = 0.527) and SFD (t(23) = 2.078, p = 0.049, Cohen’s d = 0.424) tasks (Supplementary Fig. 3e, vertical stripes); the width change was significantly lower than zero on both OD (t(23) = -2.438, p = 0.023, Cohen’s d = 0.498) and SFD (t(23) = -2.578, p = 0.017, Cohen’s d = 0.526) tasks (Supplementary Fig. 3e, diagonal stripes). For unexpected orientations (Δ10°-Δ40°), however, the amplitude and width changes were not significant with zero on either OD (amplitude change: t(23) = 0.443, p = 0.662, Cohen’s d = 0.091; width change: t(23) = -1.819, p = 0.082, Cohen’s d = 0.371) or SFD (amplitude change: t(23) = 1.130, p = 0.270, Cohen’s d = 0.231; width change: t(23) = -1.710, p = 0.101, Cohen’s d = 0.349) tasks (Supplementary Fig. 3f). In the meantime, the location shift was significantly different than zero for unexpected orientations (Δ10°-Δ40°, OD task: t(23) = 3.611, p = 0.001, Cohen’s d = 0.737; SFD task: t(23) = 2.418, p = 0.024, Cohen’s d = 0.493 (Supplementary Fig. 3g). These results provided further evidence that tuning sharpening and tuning shift jointly contribute to center– surround inhibition in expectation.  

Reviewer#1 (Recommendation for the Author):

(1) A direct comparison between tasks (baseline vs. expectation conditions) would have strengthened the findings. Specifically, contrasting performance in the orientation discrimination task with the spatial frequency discrimination task could have provided clearer evidence that participants actually used the auditory cues to attend to the expected orientation. This comparison would be particularly important for validating cue manipulation in the orientation discrimination task.

We agree that a direct comparison between the orientation discrimination (OD) and spatial frequency discrimination (SFD) tasks could further clarify how expectation (auditory cues) differentially modulates orientation relevance. However, the primary goal of the current study was to examine expectation effects within each task separately and to demonstrate that such effects are independent of attentional modulation driven by the task-relevance of orientation.

In addition, the OD and SFD tasks differ not only in the relevant task features (orientation vs. spatial frequency discrimination), but also in stimulus properties and difficulty, for example, the arbitrary use of 20–70° as the orientation range and ~0.9 cycles/° as the spatial frequency setting, a direct comparison could introduce confounding factors unrelated to expectation.

Importantly, Previous studies (e.g., Kok et al., 2012, 2017; Aitken et al., 2020) and our current results show that participants performed significantly better when the auditory cue matched the expected orientation, supporting the validity of our expectation manipulation.

(2) An interesting consideration is why the center-surround inhibition profile of expectation was independent of the task-relevance of orientation. Previous studies (e.g., Kok et al., 2012) have found that orientation discrimination patterns differ depending on whether orientation is taskrelevant or irrelevant. This could be useful to discuss the possible discrepancies.

We thank the reviewer for this inspiring comment. Kok et al. (2012) showed that both orientation and contrast tasks elicited similar fMRI decoding results, regardless of task relevance, suggesting neural mechanisms of expectation operate independently of whether orientation is task relevant. Behaviorally, they reported better performance for expected versus unexpected trials in the orientation task (3.4° vs. 3.8°, t(17) = 2.8, p = 0.013), and a marginal trend (although not significant) in the contrast task (4.3% vs. 5.0%, t(17) = 1.9, p = 0.075). If any differences between the two tasks exist, they may lie in the correlation between behavioral and fMRI effects, a question that goes beyond the scope of the current study. Therefore, it is hard to strongly conclude that orientation discrimination patterns differ depending on whether orientation is taskrelevant or irrelevant in their paper.

Our study differs from theirs in at least two important ways, which may account for the clearer expectation facilitatory effect we observed in the expectation (Δ0°) condition. First, in our study, the orientation-irrelevant task involved spatial frequency discrimination (SFD) rather than contrast discrimination. Compared to contrast, spatial frequency has been shown to exhibit a clear cueing effect, as reported in Fang & Liu (2019). Second, our design included a baseline condition, which was absent in their study. We computed discrimination sensitivity (DS) to quantify how much the discrimination threshold (DT) changed relative to baseline. By using this baseline-referenced approach, we observed a significant facilitatory expectation effect in the Δ0° condition, an effect that shifted from marginal significance in their orientation-irrelevant task to clear significance in our study.

(3) The authors might consider briefly explaining how the orientation adjustment paradigm used in this study is particularly effective for examining the potential co-existence of tuning sharpening and tuning shift computations, and how this approach complements traditional orientation discrimination tasks in characterizing expectation-related mechanisms.

We thank the reviewer for this valuable suggestion. We agree that further clarification is needed to better connect the two experiments. To explain this, we have elaborated further in the manuscript.

“To further explore the co-existence of both Tuning sharpening and Tuning shift computations in center-surround inhibition profile of expectation, participants were asked to perform a classic orientation adjustment experiment. Unlike profile experiment (discrimination tasks), the adjustment experiment provides a direct, trial-by-trial measure of participants’ perceived orientation, capturing the full distribution of responses. This enables the construction of orientation-specific tuning curves, allowing us to detect both tuning sharpening and tuning shifts, thereby offering a more nuanced understanding of the computational mechanisms underlying expectation.”

(4) These interesting findings raise important questions about their relationship to existing hybrid models of attentional modulation. Could the authors discuss how their results might align with or extend previous work demonstrating combined feature-similarity gain and surround suppression effects for orientation (e.g., Fang & Liu, 2019)? Could a hybrid model potentially provide a better account of these data than the pure surround suppression model?

We thank the reviewer for this valuable comment. We agree that hybrid model should be mentioned in the manuscript and we have elaborated further in the Discussion.

“For example, within the orientation space, the inhibitory zone was about 20°, 45°, and 54° for expectation evident here, feature-based attention[21], and visual perceptual learning[35], respectively; within the feature-based attention, it was about 30° and 45° in color [77] and motion direction [53] spaces, respectively These variations hint at the exciting possibility that the width of the inhibitory surround may flexibly adapt to stimulus context and task demands, ultimately facilitating our perception and behavior in a changing environment. This principle is consistent with the hybrid model of feature-based attention [53,54,75], where attention is deployed adaptively to prioritize task-relevant information through feature-similarity gain which filters out the most distinctive distractors, and surround suppression which inhibits similar and confusable ones, thereby jointly shaping the attentional tuning profile.”

(5) On page 19, there appears to be a missing symbol in the description of the Tuning Sharpening model. The text states: 'the tuning width of each channel's tuning function is parameterized by ??', where the question marks seem to indicate a missing parameter symbol.

We appreciate the reviewer’s careful attention. Yes, the "ơ" is missing, which was likely caused by a formatting issue. We have corrected it.

eLife Assessment

This important study reports the results of efforts to replicate two phenomena of significant interest to early-career scientists and scientific policymakers: the Matthew effect and the early-career setback effect. Several previous studies of these effects have focused on early-career researchers with grant proposals that fell just below or just above a funding threshold. Those just above the threshold were more likely to be successful when they applied for funding later in the career (an example of the well-known Matthew effect), while those just below were more likely to go on to have stronger publication records (the early-career setback effect). In this study the Matthew effect was found to be robust across funders, and to generalize from those close to the funding threshold to the whole population. The early-career setback effect was not robust across funders and did not generalize to the whole population. The evidence reported is convincing.

Reviewer #1 (Public review):

Summary:

The authors performed a multi-funder study to determine if the Matthew effect and early-career setback effect were reproducible across funding programs and processes. The authors extended the analysis of these effects to all applicants and compared the results to the prior studies that only looked at near-hit/near-miss applicants to determine if the effects were generalizable to the whole applicant pool. Further, the authors included new models that also account for researcher behavior and their overall likelihood to reapply for later funding and how this behavior may resolve what appears to be a paradox between the Matthew effect and the early-career setback effect.

Strengths:

Figure 4 shows that the "Post (late) MFCR" is the same for the funded and unfunded groups, indicating that the impact of early career funding (at least, in terms of citation metrics) is transient in researcher's overall careers. This finding should encourage researchers to persevere when needed and that long-term success is attainable.

The inclusion of the collider bias in the models to account for researcher behavioral responses is a key strength of the paper and enhance the analysis and nuanced discussion of the results.

Weaknesses:

The discussion of limitations is thorough and point to the need for additional studies. One limitation that is acknowledged is that the authors only looked at applicants who reapplied for funding at the same funder. Given that the authors had the names and affiliations of the applicants from all of the funders, it would be helpful to understand why they were not able to look at applicants across their full data set. Was the limitation technical or a result of the study design? What would have to change to enable this broader analysis?

In Section 4.1, the authors make a statement that the "between MFCR" difference was seen at 5 years, but not at 10 years, and so the authors chose to use the 5-year period for the presentation of their results. It would be helpful to also see the 10-year analysis and have further justification from the authors on why they selected to look at the 5-year period and how their conclusions might or might not change if they consider the longer time period.

The discussion could also include that many funders require novel research directions as a condition of receiving an early-career award. For those who receive these awards, they must establish the new research program, begin publishing, and they may initially see a lower citation rate until the impact of the research is more broadly recognized. Are there ways to explore how these time lags impact the "Between MFCR" on those who were funded more so than those who were not funded?

Reviewer #2 (Public review):

Summary:

The manuscript evaluates the generalizability of two phenomena of great interest to early-career scientists and scientific policymakers. These phenomena describe how early funding success can promote future funding success (the Matthew Effect) and how initially unsuccessful applicants may later succeed (the early-career setback effect). Given the often-normative aspirations of science-of-science studies, the manuscript represents a much-needed and highly significant effort, as it allows a broader audience to assess whether they should reconsider their behavior or policies.

Strengths:

The evidence provided by the authors for the generalizability of the Matthew Effect is very strong and convincing. The manuscripts addresses an important topic of practical concern to early-career scientists and scientific policymakers.

Weaknesses: If I am correctly interpreting S11 and S12, the statements on the early-career setback effect could be stronger and more direct. The argument in the main text relies on assumptions and simulations to suggest that observations of the early-career setback effect may depend on reapplications. In contrast, S11 and S12 appear to provide more direct evidence against its generalizability, showing that the effect seems to exist in, and be driven by, only one of the six funding agencies considered (FWF). This narrow replication may not be obvious to readers ("the early-career setback effect also replicates, but is not robust across funders").

I would also suggest that the authors provide a more nuanced discussion of the limitations of their Bayesian model. While the model seems appropriate for accounting for major factors, it appears to exclude others, such as the emergence of new scientific fields or the strategic reorientation of funders toward such fields.

Reviewer #3 (Public review):

Summary:

This paper investigates the Matthew effect, where early success in funding peer review can translate into potentially unwarranted later success. It also investigated the previously found "setback" effect for those who narrowly miss out on funding.

Strengths:

The study used data from six funding agencies, which increases the generalisability, and was able to link bibliographic data for around 95% of applicants. The authors nicely illustrate how the previously found "setback" effect for near-miss applicants could be a collider bias due to those who chose to apply sometime later. This is a good explanation for the counter-intuitive effect and is nicely shown in Figure 5.

Weaknesses:

Most of the methods were clearly presented, but I have a few questions and comments, as outlined below.

In Figure 4(a) why are the "post" means much lower than the "pre"? This contradicts the expected research trajectory of researchers. Or is this simply due to less follow-up time? But doesn't the field citation ratio control for follow-up time?

The choice of the log-normal distribution for latent quality was not entirely clear to me. This would create some skew, rather than a symmetric distribution, which may be reasonable but log-normal distributions can have a very long tail which might not mimic reality, as I would not expect a small number of researchers to be extremely above the crowd. However, then the skew was potentially dampened by using percentile scores. Some further reasoning and plots of the priors would help.

Can the authors confirm the results of Figure S9 which show no visible effect of altering the standard deviation for the review parameter or the mean citations? Is this just because the prior for quality is dominated by the data? Could it be that the width of the distribution for quality does not matter, as it's the relative difference/ranking that counts? So the beta in equation 6 changes to adjust to the different quality scale?

The contrary result for the FWF is not explained (Table S3). Does this funder have different rules around re-applicants or many other competing funders?

The outlined qualitative research sounds worthwhile. Another potential mechanism (based on anecdote) is that some researchers react irrationally to rejection or acceptance, tending to think that the whole agency likes or hates their work based on one experience. Many researchers do not appreciate that it was a somewhat random selection of reviewers who viewed their work, and it will unlikely be the same reviewers next time.

"A key implication is the importance of encouraging promising, but initially unsuccessful applicants to reapply." Yes, A policy implication is to give people multiple chances to be lucky, perhaps by giving fewer grants to more people, which could be achieved by shortening the funding period (e.g., 4 year fellowships instead of 5 years). Although this will have some costs as applicants would need to spend more time on applications and suffer increased stress of shorter-term contracts. The bridge grants is potentially an ideal half-way house between many short-term and few long-term awards. Giving more grants to fewer people is supported by this analysis showing a diminishing returns in research outputs with more funding, DOI: 10.1371/journal.pone.0065263.

Making more room for re-applicants also made me wonder if there should be an upper cap on funding, potentially for people who have been incredibly successful. Of course, funders generally want to award successful researchers, but people who've won over some limit, for example $50 million, could likely be expected to win funding from other sources such as philanthropy and business. Graded caps could occur by career stage.

eLife Assessment

This important research addresses the effects of subjective control and task difficulty on experienced stress using a novel behavioral task administered on the same day in two large online samples. Convincing evidence is provided, establishing the internal and external task validity of the task, as well as a relationship between the sense of control and task difficulty, with individual differences in relevant mental health constructs. Evidence for the specificity of the link between control and stress would be more substantial if the design had not conflated control and reward rate. This work will be of interest to psychologists and clinicians studying the concepts of controllability, stress, and psychopathology.

Reviewer #1 (Public review):

Summary:

This work investigated how the sense of control influences perceptions of stress. In a novel "Wheel Stopping" task, the authors used task variations in difficulty and controllability to measure and manipulate perceived control in two large cohorts of online participants. The authors first demonstrate that their behavioral task exhibits good internal consistency and external validity, indicating that perceived control during the task is linked to relevant measures of anxiety, depression, and locus of control. Most importantly, manipulating controllability in the task resulted in reduced subjective stress, demonstrating a direct impact of control on stress perception. However, this work has some minor limitations to this work due to the design of the stressor manipulations/measurements and the necessary logistics associated with online versus in-person stress studies.<br /> Nevertheless, this research adds to our understanding of when and how control can influence the effects of stress and has particular relevance for mental health interventions.

Strengths:

The primary strength of this research is the development of a unique and clever task design that can reliably and validly elicit variations in beliefs about control. Impressively, higher subjective control in the task was associated with decreased psychopathology measures such as anxiety and depression in a non-clinical sample of participants. In addition, the authors found that lower control and higher task difficulty led to higher perceived stress, suggesting that the task can reliably manipulate perceptions of stress. Prior tasks have not included both controllability and difficulty in this manner and have not directly tested the direct influence of these factors on incidental stress, making this work both novel and important for the field.

Weaknesses:

One minor weakness of this research is the validity of the online stress measurements and manipulations. In this study, the authors measure subjective stress via self-report both during the task and after either a Trier Social Stress Test (high-stress condition) or a memory test (low-stress condition). One concern is that these stress manipulations were really "threats" of stress, where participants never had to complete the stress tasks (i.e., recording a speech for judgment). While this is not unusual for an in-lab study and can reliably elicit substantial stress/anxiety, in an online study, there is a possibility for communication between participants (via online forums dedicated to such communication), which could weaken the stress effects. That said, the authors did find sensible increases and decreases in perceived stress between relevant time points; however, future work could improve upon this design by including more comprehensive stress manipulations and by measuring implicit physiological signs of stress.

Comments on revisions:

I appreciate the authors' responses to my comments and concerns. I have decided not to make changes to my public review, as I believe it remains relevant and fair after revisions.

Reviewer #2 (Public review):

Summary:

The authors have developed a behavioral paradigm to experimentally manipulate the sense of control experienced by participants by varying the level of difficulty in a wheel-stopping task. In the first study, this manipulation is tested by administering the task in a factorial design with two levels of controllability and two levels of stressor intensity to a large number of participants online, while simultaneously recording subjective ratings of perceived control, anxiety, and stress. In a second study, the authors employed the wheel stopping task to induce a high sense of controllability and investigate whether this manipulation buffers the response to a subsequent stress induction when compared to a neutral task, such as watching pleasant videos.

Strengths:

(1) The authors validate a method to manipulate stress.

(2) The authors use an experimental manipulation to induce an enhanced sense of controllability to test its impact on the response to stress induction.\

(3) The studies involved big sample sizes.

Weaknesses:

(1) The study was not preregistered.

(2) The control manipulation is conflated with task difficulty and, therefore, the reward rate. In the revised version of the manuscript, the authors perform statistical analysis to demonstrate that the relationship between perceived level of control and subjective stress remains robust after the inclusion of win rate in the model. This analysis strengthens the authors's claims, but the evidence would more substantial if the design did not conflate reward rate and control. The authors properly discuss this issue in the revised manuscript.

This study will be of interest to psychologists and cognitive scientists who are interested in understanding how controllability and its subjective perception influence how people respond to stress exposure. The demonstration that an increased sense of control buffers/protects against subsequent stress is important and may trigger further studies to characterize this phenomenon better. However, beyond the highlighted weaknesses, the current study only studied the effect of stress induction consequent to the performance of the WS task on the same day, and its generalizability is not warranted.

Reviewer #3 (Public review):

Summary:

This is an interesting investigation on the benefits of perceiving control and its impact on the subjective experience of stress. To assess the subjective sense of control, the authors introduce a novel wheel stopping (WS) task where control is manipulated via size and speed to induce conditions of low and high control. The authors demonstrate that the subjective sense of control is associated with experienced subjective stress and individual differences related to mental health measures. In a second experiment, they further demonstrate that an increased sense of control buffers subjective stress induced by a trier social stress manipulation, more so than a typical stress-buffering mechanism of watching neutral/calming videos.

Strengths:

Several strengths of the manuscript can be highlighted. For instance, the paper introduces a new paradigm and a clever manipulation to test a significant and important question. Additionally, it is a well-powered investigation that allows for confidence in replicability and demonstrate both high internal consistency and high external validity, along with an interesting set of individual difference analyses. Finally, the results are quite interesting and support prior literature, while also making a significant contribution to the field in understanding the benefits of perceiving control.

Weaknesses:

The authors have addressed all my queries, and I believe the revised paper has been improved and will make an important contribution to the literature.

Author response:

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

Reviewer #1 (Public review):

Summary:

This work investigated how the sense of control influences perceptions of stress. In a novel "Wheel Stopping" task, the authors used task variations in difficulty and controllability to measure and manipulate perceived control in two large cohorts of online participants. The authors first show that their behavioral task has good internal consistency and external validity, showing that perceived control during the task was linked to relevant measures of anxiety, depression, and locus of control. Most importantly, manipulating controllability in the task led to reduced subjective stress, showing a direct impact of control on stress perception. However, this work has minor limitations due to the design of the stressor manipulations/measurements and the necessary logistics associated with online versus in-person stress studies.

Nevertheless, this research adds to our understanding of when and how control can influence the effects of stress and is particularly relevant to mental health interventions.

We thank the reviewer for their clear and accurate summary of the findings. 

Strengths:

The primary strength of this research is the development of a unique and clever task design that can reliably and validly elicit variations in beliefs about control. Impressively, higher subjective control in the task was associated with decreased psychopathology measures such an anxiety and depression in a non-clinical sample of participants. In addition, the authors found that lower control and higher difficulty in the task led to higher perceived stress, suggesting that the task can reliably manipulate perceptions of stress. Prior tasks have not included both controllability and difficulty in this manner and have not directly tested the direct influence of these factors on incidental stress, making this work both novel and important for the field.

We thank the reviewer for their positive comments.

Weaknesses:

One minor weakness of this research is the validity of the online stress measurements and manipulations. In this study, the authors measure subjective stress via self-report both during the task and also after either a Trier Social Stress Test (high-stress condition) or a memory test (low-stress condition). One concern is that these stress manipulations were really "threats" of stress, where participants never had to complete the stress tasks (i.e., recording a speech for judgment). While this is not unusual for an in-lab study and can reliably elicit substantial stress/anxiety, in an online study, there is a possibility for communication between participants (via online forums dedicated to such communication), which could weaken the stress effects. That said, the authors did find sensible increases and decreases of perceived stress between relevant time points, but future work could improve upon this design by including more complete stress manipulations and measuring implicit physiological signs of stress.

We thank the reviewer for urging us to expand on this point. The reviewer is right that stress was merely anticipatory and is in that sense different to the canonical TSST. However, there are ample demonstrations that such anticipatory stress inductions are effective at reliably eliciting physiological and psychological stress responses (e.g. Nasso et al., 2019; Schlatter et al., 2021; Steinbeis et al., 2015). Further, there is evidence that online versions of the TSST are also effective (DuPont et al., 2022; Meier et al., 2022), including evidence that the speech preparation phase conducted online was related to increases in heart rate and blood pressure (DuPont et al., 2022). Importantly, and as the reviewer notes in relation to our study specifically, the anticipatory TSST had a significant impact on subjective stress in the expected direction demonstrating that it was effective at eliciting subjective stress. We have elaborated further on this in our manuscript (pages 8 and 9) as follows: 

“Prior research has found TSST anticipation to elicit both psychological and physiological stress responses [37-39], suggesting that the task anticipation would be a valid stress induction despite participants not performing the speech task. Moreover, prior research has validated the use of remote TSST in online settings [40, 41], including evidence that the speech preparation phase (online) was related to increased heart rate and blood pressure compared to controls [40].”

Reviewer #2 (Public review):

Summary:

The authors have developed a behavioral paradigm to experimentally manipulate the sense of control experienced by the participants by changing the level of difficulty of a wheel-stopping task. In the first study, this manipulation is tested by administering the task in a factorial design with two levels of controllability and two levels of stressor intensity to a large number of participants online while simultaneously recording subjective ratings on perceived control, anxiety, and stress. In the second study, the authors used the wheel-stopping task to induce a high sense of controllability and test whether this manipulation buffers the response to a subsequent stress induction when compared to a neutral task, like looking at pleasant videos.

We thank the reviewer for their accurate summary.

Strengths:

(1) The authors validate a method to manipulate stress.

(2) The authors use an experimental manipulation to induce an enhanced sense of controllability to test its impact on the response to stress induction.

(3) The studies involved big sample sizes.

We thank the reviewer for noting these positive aspects of our study. 

Weaknesses:

(1) The study was not preregistered.

This is correct.

(2) The control manipulation is conflated with task difficulty, and, therefore the reward rate. Although the authors acknowledge this limitation at the end of the discussion, it is a very important limitation, and its implications are not properly discussed. The discussion states that this is a common limitation with previous studies of control but omits that many studies have controlled for it using yoking.

We agree that these are very important issues to consider in the interpretation of our findings. It is important to note, that while our task design does not separate these constructs, we are able to do so in our statistical analyses. For example, our measure of perceived difficulty was included in analyses assessing the fluctuations in stress and control in which subjective control still had a unique effect on the experience of stress over and above perceived difficulty, suggesting that subjective control explains variance in stress beyond what is accounted for by perceived difficulty. Similarly, we have also included additional analyses in which we include the win rate (i.e. percentage of trials won) as a covariate when assessing the relationship between subjective control, perceived difficulty and subjective stress, in which subjective control and perceived difficulty still uniquely predict subjective stress when controlling for win rate. This suggests that there is unique variance in subjective control, separate from perceived task difficulty and win rate that is relevant to stress. We have included these analyses (page 16 of manuscript) as follows:

“To further isolate the relationship between subjective control and stress separate from perceived task difficulty or objective task performance, we also included the overall win rate (percentage of trials won during the WS task) in the models. In Study 1, lower feelings of control were related to higher levels of subjective stress (β= -0.12, p<.001) even when controlling for both  win rate (β= -0.06, p=.220) and perceived task difficulty (β= 0.37, p<.001, Table S10). This also replicated in Study 2, where lower subjective control was associated with higher feelings of stress (β= -0.32, p<.001) when controlling for perceived task difficulty (β= 0.31, p<.001) and win rate (β= -0.11, p=.428, Table S11). This suggests that there is unique variance in subjective feelings of control, separate from task performance, relevant to subjective stress.”

As well as expanding on this in the Discussion (pages 27 and 28) as follows:

“While our task design does not separate control from obtained reward, we are able to do so in the statistical analyses. Like with perceived difficulty, we statistically accounted for reward rate and showed that the relationship between subjective control and stress was not accounted for by reward rate, for example. Similarly, participants received feedback after every trial, and thus feedback valence may contribute to stress perception. However, given that overall win rate (which captures the feedback received during the task) did not predict stress over and above perceived difficulty or subjective control, it suggests that feedback is unlikely to relate to stress over and above difficulty. Future work will need to disentangle this further to rule out such potential confounds.”

Further, in terms of the wider literature on these issues, we have added more to this point in our discussion, especially in relation to previous literature that also varies control by reward rate (e.g. Dorfman & Gershman, 2019, who use a reward rate of 80% in high control conditions and 50% in low control conditions). This can be found in the manuscript on page 27 as follows: 

“Previous research typically accounts for different outcomes (e.g. punishment) by yoking controllable and uncontrollable conditions [3] though other work has manipulated the controllability of rewards by changing the reward rate [for example 30] where a decoy stimulus is rewarded 50% of the time in the low control condition but 80% in the high control condition).”

(3) The methods are not always clear enough, and it is difficult to know whether all the manipulations are done within-subjects or some key manipulations are done between subjects.

We have added more information in the methods section (page 8) clarifying withinsubject manipulations (WS task parameters) and between-subject manipulations (stressor intensity task, WS task version in Study 1, and WS task/video task in Study 2). Additionally, as recommended by Reviewer 1, we have provided more information in the methods section and Table S3 regarding the details of on-screen written feedback provided to participants after each trial of the WS Task.

(4) The analysis of internal consistency is based on splitting the data into odd/even sliders. This choice of data parcellation may cause missed drifts in task performance due to learning, practice effects, or tiredness, thus potentially inflating internal consistency.

We agree that this can indeed be an issue, though drift is likely to be present in any task including even in mood in resting-state (Jangraw et al., 2023). To respond to this specific point, we parcellated the timepoints into a 1^st/2^nd half split and report the ICC in the supplementary information. While values are lower, indeed likely due to systematic drifts in task performance as participants learn to perform the task (especially for Study 2 since the order of parameters were designed to get easier throughout the experiment), the ICC values are still high. Control sliders: Study 1 = 0.82, Study 2: = 0.68; Difficulty sliders: Study 1: = 0.84, Study 2 = 0.57; Stress sliders: Study 1 = 0.45, Study 2 = 0.71. As seen, the lowest ICC is for stress sliders in Study 1. This may be because the first 3 sliders (included in the 1^st half split) were all related to the stress task (initial, post-stress, task, post-debrief) and the final 4 sliders (in the 2^nd half split) were the three sliders during the WS task and shortly afterwards. 

(5) Study 2 manipulates the effect of domain (win versus loss WS task), but the interaction of this factor with stressor intensity is not included in the analysis.

We agree that this would be a valuable analysis to include. We have run additional analyses (section Sensitivity and Exploratory Analyses, pages 24 and 25), testing the interaction of Domain (win or loss) with stressor intensity (and time) when predicting the stress buffering and stress relief effects. This revealed no significant main effects of domain or interactions including domain, suggesting that domain did not impact the stress induction or relief differently depending on whether it was followed by the high or low stressor intensity condition. While the control by time interaction (our main effect of interest) still held for stress induction in this more complex model, the control by time interaction did not hold for the stress relief. However, this more complex model did not provide a better fit for the data, motivating us to continue to draw conclusions from the original model specification with domain as a covariate (rather than an interaction).

We outline these analyses on page 24 of the manuscript, as follows:

“Third, we included the interaction of domain with stressor intensity and with time, to test whether the win or loss domain in the WS task significantly impacted stress induction or stress relief differently depending on stressor intensity. There were no significant effects or interactions of domain (Table S14) for stress induction or stress relief, and the main effect of interest (the interaction between time and control) still held for the stress induction (β= 10.20, SE=4.99 p=.041, Table S14), though was no longer significant for the stress relief  (β= 6.72, SE=4.28, p=.117, Table S14). This more complex model did not significantly improve model fit (χ^²(3)= 1.46, p=.691) compared to our original specification (with domain as a covariate rather than an interaction) and had slightly worse fit (higher AIC and BIC) than the original model (AIC = 5477.2 versus 5472.7, BIC = 5538.5 versus 5520.8).”

This study will be of interest to psychologists and cognitive scientists interested in understanding how controllability and its subjective perception impact how people respond to stress exposure. Demonstrating that an increased sense of control buffers/protects against subsequent stress is important and may trigger further studies to characterize this phenomenon better. However, beyond the highlighted weaknesses, the current study only studied the effect of stress induction consecutive to the performance of the WS task on the same day and its generalizability is not warranted.

We thank the reviewer for this assessment and agree that we cannot assume these findings would generalise to more prolonged effects on stress responses.

Reviewer #3 (Public review):

Summary:

This is an interesting investigation of the benefits of perceiving control and its impact on the subjective experience of stress. To assess a subjective sense of control, the authors introduce a novel wheel-stopping (WS) task where control is manipulated via size and speed to induce low and high control conditions. The authors demonstrate that the subjective sense of control is associated with experienced subjective stress and individual differences related to mental health measures. In a second experiment, they further show that an increased sense of control buffers subjective stress induced by a trier social stress manipulation, more so than a more typical stress buffering mechanism of watching neutral/calming videos.

We agree with this accurate summary of our study. 

Strengths:

There are several strengths to the manuscript that can be highlighted. For instance, the paper introduces a new paradigm and a clever manipulation to test an important and significant question. Additionally, it is a well-powered investigation that allows for confidence in replicability and the ability to show both high internal consistency and high external validity with an interesting set of individual difference analyses. Finally, the results are quite interesting and support prior literature while also providing a significant contribution to the field with respect to understanding the benefits of perceiving control.

We thank the reviewer for this positive assessment. 

Weaknesses:

There are also some questions that, if addressed, could help our readership.

(1) A key manipulation was the high-intensity stressor (Anticipatory TSST signal), which was measured via subjective ratings recorded on a sliding scale at different intervals during testing. Typically, the TSST conducted in the lab is associated with increases in cortisol assessments and physiological responses (e.g., skin conductance and heart rate). The current study is limited to subjective measures of stress, given the online nature of the study. Since TSST online may also yield psychologically different results than in the lab (i.e., presumably in a comfortable environment, not facing a panel of judges), it would be helpful for the authors to briefly discuss how the subjective results compare with other examples from the literature (either online or in the lab). The question is whether the experienced stress was sufficiently stressful given that it was online and measured via subjective reports. The control condition (low intensity via reading recipes) is helpful, but the low-intensity stress does not seem to differ from baseline readings at the beginning of the experiment.

We agree that it would be helpful to expand on this further. Similar to the comment made by Reviewer 1, we wish to point out that there are ample demonstrations that such anticipatory stress inductions are effective at reliably eliciting physiological and psychological stress responses (e.g. Nasso et al., 2019; Schlatter et al., 2021; Steinbeis et al., 2015). Further, there is evidence that online versions of the TSST are also effective (DuPont et al., 2022; Meier et al., 2022), including evidence that the speech preparation phase conducted online was related to increases in heart rate and blood pressure (DuPont et al., 2022). We have elaborated further on this in our manuscript on pages 8 and 9 as follows:

“Prior research has found TSST anticipation to elicit both psychological and physiological stress responses [37-39], suggesting that the task anticipation would be a valid stress induction despite participants not performing the speech task. Moreover, prior research has validated the use of remote TSST in online settings [40, 41], including evidence that the speech preparation phase (online) was related to increased heart rate and blood pressure compared to controls [40].”

(2) The neutral videos represent an important condition to contrast with WS, but it raises two questions. First, the conditions are quite different in terms of experience, and it is interesting to consider what another more active (but not controlled per se) condition would be in comparison to the WS performance. That is, there is no instrumental action during the neutral video viewing (even passive ratings about the video), and the active demands could be an important component of the ability to mitigate stress. Second, the subjective ratings of the stress of the neutral video appear equivalent to the win condition. Would it have been useful to have a high arousal video (akin to the loss condition) to test the idea that experience of control will buffer against stress? That way, the subjective stress experience of stress would start at equivalent points after WS3.

We agree with the reviewer that this is an important issue to clarify. In our deliberations when designing this study, we considered that that any task with actionoutcome contingencies would have a degree of controllability. To better distinguish experiences of control (WS task) to an experience of no/neutral control (i.e., neither high nor low controllability), we decided to use a task in which no actions were required during the task itself. Importantly, however, there was an active demand and concentration was still required in order to perform the attention checks regarding the content of the videos and ratings of the videos. 

Thank you for the suggestion of having a high arousal video condition. This would indeed be interesting to test how experiencing ‘neutral’ control and high(er) stress levels preceding the stressor task influences stress buffering and stress relief, and we have included this suggestion for future research in the discussion section (page 28) as below:

“Another avenue for future research would be to test how control buffers against stress when compared to a neutral control scenario of higher stress levels, akin to the loss domain in the WS Task, given that participants found the video condition generally relaxing. However, given that we found no differences dependent on domain for the stress induction in the WS Task conditions, it is possible that different versions of a neutral control condition would not impact the stress induction.”

(3) For the stress relief analysis, the authors included time points 2 and 3 (after the stressor and debrief) but not a baseline reading before stress. Given the potential baseline differences across conditions, can this decision be justified in the manuscript?

We thank the reviewer for raising this. Regarding the stress relief analyses (timepoints 2 and 3) and not including timepoint 1 (after the WS/video task) stress in the model, we have added to the manuscript that there was no significant difference in stress ratings between the high control and neutral control (collapsed across stress and domain) at timepoint 1 (hence why we do not think it’s necessary to include in the stress relief model). Nevertheless, we have now included a sensitivity analysis to test the Timepoint*Control interaction of stress relief when including timepoint 1 stress as a covariate. The timepoint by control interaction still holds, suggesting that the initial stress level prior to the stress induction does not impact our results of interest. The details of this analysis are included in the Sensitivity and Exploratory Analyses section on page 24:

“Although there were no significant differences between control groups in subjective stress immediately after the WS/video task (t(175.6)=1.17, p=.244), we included participants’ stress level after the WS/video task as a covariate in the stress relief analyses (Table S12). The results revealed a main effect of initial stress (β= 0.643, SE=0.040, p<.001, Table S12) on the stress relief after the stressor debrief. Compared to excluding initial stress as in the original analyses (Table 4), there was now no longer a main effect of domain (β= 0.236, SE=2.60, p=.093, Table S12), but the inference of all other effects remained the same. Importantly, there was still a significant time by control interaction (β= 9.65, SE=3.74, p=.010, Table S12) showing that the decrease in stress after the debrief was greater in the highly controllable WS condition than the neutral control video condition, even when accounting for the initial stress level.”

(4) Is the increased control experience during the losses condition more valuable in mitigating experienced stress than the win condition?

We agree that this would be helpful to clarify. To test whether the loss domain was more valuable at mitigating experiences of stress than the win condition, we ran additional analyses with just the high control condition (WS task) to test for a Domain*Time interaction. This revealed no significant Domain*Time interaction, suggesting that the stress buffering or stress relief effect was not dependent on domain in the high control conditions. These analyses are outlined in the Sensitivity and Exploratory Analyses section on page 25:

“Finally, to test whether the loss domain was more valuable at mitigating experiences of stress than the win condition, we ran additional analyses with just the high control condition (WS task) for the stress induction and stress relief to test for an interaction of domain and time. For the stress induction, there was no significant two-way interaction of domain and time (β= -1.45, SE=4.80, p=.763), nor a significant three-way interaction of domain by time by stressor intensity (β= -3.96, SE=6.74, p=.557, Table S15), suggesting that there were no differences in the stress induction dependent on domain. Similarly for the stress relief, there was no significant two-way interaction of domain and time (β= -5.92, SE=4.42, p=.182), nor a significant three-way interaction of domain by time by stressor intensity interaction (β= 8.86, SE=6.21, p=.154, Table S15), suggesting that there were no differences in the stress relief dependent on the WS Task domain.

(5) The subjective measure of control ("how in control do you feel right now") tends to follow a successful or failed attempt at the WS task. How much is the experience of control mediated by the degree of experienced success/schedule of reinforcement? Is it an assessment of control or, an evaluation of how well they are doing and/or resolution of uncertainty? An interesting paper by Cockburn et al. 2014 highlights the potential for positive prediction errors to enhance the desire for control.

We thank the reviewer for this comment. Similar to comments regarding reward rate, our task does not allow us to fully separate control from success/reinforcement because of the manipulation of difficulty. However, we did undertake sensitivity analyses and the inclusion of overall win rate accounted for limited variance when predicting stress over and above subjective control and difficulty (page 16). 

“To further isolate the relationship between subjective control and stress separate from perceived task difficulty or objective task performance, we also included the overall win rate (percentage of trials won during the WS task) in the models. In Study 1, lower feelings of control were related to higher levels of subjective stress (β= -0.12, p<.001) even when controlling for both  win rate (β= -0.06, p=.220) and perceived task difficulty (β= 0.37, p<.001, Table S10). This also replicated in Study 2, where lower subjective control was associated with higher feelings of stress (β= -0.32, p<.001) when controlling for perceived task difficulty (β= 0.31, p<.001) and win rate (β= -0.11, p=.428, Table S11). This suggests that there is unique variance in subjective feelings of control, separate from task performance, relevant to subjective stress.” 

(6) While the authors do a very good job in their inclusion and synthesis of the relevant literature, they could also amplify some discussion in specific areas. For example, operationalizing task controllability via task difficulty is an interesting approach. It would be useful to discuss their approach (along with any others in the literature that have used it) and compare it to other typically used paradigms measuring control via presence or absence of choice, as mentioned by the authors briefly in the introduction.

We are delighted to expand on this particular point and have done so in the Discussion on page 27:

“Previous research typically accounts for different outcomes (e.g. punishment) by yoking controllable and uncontrollable conditions [3] though other work has manipulated the controllability of rewards by changing the reward rate [for example 30] where a decoy stimulus is rewarded 50% of the time in the low control condition but 80% in the high control condition). While our task design does not separate control from obtained reward, we are able to do so in the statistical analyses.” 

(7) The paper is well-written. However, it would be useful to expand on Figure 1 to include a) separate figures for study 1 (currently not included) and 2, and b) a timeline that includes the measurements of subjective stress (incorporated in Figure 1). It would also be helpful to include Figure S4 in the manuscript.

We have expanded Figure 1 to include both Studies 1 and 2 and a timeline of when subjective stress was assessed throughout the experiment as well as adding Figure S4 to the main manuscript (now top panel within Figure 4). 

Reviewer #1 (Recommendations for the authors):

(1) Study 2 shows a greater decrease in subjective stress after the high-control task manipulation than after the pleasant video. One possible confound is whether the amount of time to complete the WS task and the video differ. It could be helpful to look at the average completion time for the WS task and compare that to the length of the videos. Alternatively, in future studies, control for this by dynamically adjusting the video play length to each participant based on how long they took to complete the WS task.

This is an interesting suggestion. As a result, we have included the time taken as a covariate in the stress induction and stress relief analyses to ensure that any differences in time between the WS task and video task were not accounting for any of the stress induction or relief analyses. Controlling for the total time taken did not impact the stress induction or relief results. This is included in the Sensitivity and Exploratory Analyses section on page 24:

“Our second sensitivity analyses was conducted because the experiment took longer to complete for the video condition (mean = 54.3 minutes, SD = 12.4 minutes) than the WS task condition (mean = 39.7 minutes, SD = 12.8 minutes, t(186.19)=-9.32, p<.001). We therefore included the total time (in ms) as a covariate in the stress induction and stress relief analyses for Study 2. This showed that accounting for total time did not change the results of interest (Table S13), further highlighting that the time by control interactions were robust.”

(2) Because participants received feedback about their success/failure in the WS task, a confounding factor could be that they received positive feedback on highly controllable trials and negative feedback on low control trials (and/or highly difficult trials). This would suggest that it is not controllability per se that contributes to stress perception but rather feedback valence. The authors show that this is a likely factor in their results in Study 2, which shows significant effects of the loss domain on perceived control and stress. Was a similar analysis done in Study 1? Do participants receive feedback in Study 1? It would be helpful to include this information somewhere in the manuscript. I would be curious to know whether *any* feedback at all influences controllability/stress perceptions.

We thank the reviewer for this interesting suggestion. It is an interesting question as to whether feedback valence is related to stress in Study 1, and we have added this point to the Discussion on pages 27 and 28. To speak to this point, when we include the overall win rate (which captures the subsequent feedback received) when predicting subjective stress, win rate is not a significant predictor of stress over and above perceived difficulty and subjective control, suggesting that overall feedback valence may not be related to stress in Study 1. We take this as evidence that feedback may not be as important in terms of accounting for the relationship between stress and control. However, we unfortunately do not have any data in which there was no feedback provided to speak to this conclusively. This would be an interesting future study. The excerpt below is added to pages 27 and 28 of the discussion section:

“Like with perceived difficulty, we statistically accounted for reward rate and showed that the relationship between subjective control and stress was not accounted for by reward rate, for example. Similarly, participants received feedback after every trial, and thus feedback valence may contribute to stress perception. However, given that overall win rate (which captures the feedback received during the task) did not predict stress over and above perceived difficulty or subjective control, it suggests that feedback is unlikely to relate to stress over and above difficulty. Future work will need to disentangle this further to rule out such potential confounds.”

To respond specifically to the reviewer’s question about the feedback given to participants, written feedback was provided on screen to participants on a trial-bytrial basis also in Study 1 (i.e. for both studies), and we have provided more clarity about this in the manuscript on page 8 as well as providing additional details in Table S3:

“After each trial, participants were shown written feedback on screen as to whether the segment had successfully stopped on the red zone (or not), and the associated reward (or lack of). See Table S3 for details.”

(3) I'm not sure how to interpret the fact that in Figure S1, the BICs are all essentially the same. Does this mean that you don't really need all of these varying aspects of the task to achieve the same effects? Could the task be made simpler?

The similarity of BIC values suggests that a simpler WS task would have produced a worse account of the data approximately in keeping with the extent to which it is a simpler model. Here, the BIC scores for the models are similar, suggesting that adding these parameters adds explanatory power in keeping with what would have been expected from adding a parameter, but not more. We do note that the BIC is a relatively strict and conservative comparison. The fact that the most complex model overall narrowly improves parsimony; combined with the interpretable parameter values and the prior expectations given the task setup led us to focus on this most complex model.  

(4) A minor point, but the authors refer to their sample as "neurotypical." Were they assessed for prior/current psychopathology/medications? If not, I might use a different term here (perhaps "non-clinical sample"), since some prior work has shown that online samples actually have higher instances of psychopathology compared to community samples.

We have changed the phrasing of ‘neurotypical’ to a ‘non-clinical sample’ as recommended.

Reviewer #2 (Recommendations for the authors):

Figure 4S is very informative and could be presented in the main text.

We have expanded Figure 1 to include both Studies 1 and 2 and a timeline of when subjective stress was assessed throughout the experiment as well as adding Figure S4 to the main manuscript (top panel of Figure 4). 

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