Proposal is nearly identical to what  Anne Gorsuch did to EPA enforcement during the first two years of the Reagan Administration, as part of a far-reaching assault against the EPA's basic mission.  She and others in that administration also proposed cutting the agency's budget by one-third; though public outcry by 1983 drove her out of office and led the Reagan White House toward a more supportive approach.  Whereas the Trump administration was able to "achieve" a historic drop in EPA enforcement efforts without resorting to this measure, apparently a second Trump administration may draw from the early Reagan playbook.   https://pubmed.ncbi.nlm.nih.gov/29698097/ On the Trump admnistration's successes in cutting agency enforcement work without this measure, see https://envirodatagov.org/publication/a-sheep-in-the-closet-the-erosion-of-enforcement-at-the-epa/

Pollution and hazardous wastes have disproportionate negative health consequences in communities of color, on Tribal lands, and in low-income neighborhoods around the nation, from Flint, Michigan, to the Louisiana Gulf Coast. Beneficial environmental amenities, such as parks, green spaces, and recreation areas, which promote human health, are also unevenly distributed. In response to these documented disparities, the Office of Environmental Justice and External Civil Rights (OEJECR), which Project 2025 seeks to eliminate, was first established in 1992 as the Office of Environmental Equity by Republican President George H.W. Bush. The OEJECR now anchors the EPA’s efforts to remedy these longstanding inequalities. The OEJECR works to ensure that everyone has “equitable access to a healthy, sustainable, and resilient environment” and to protect people from adverse human health and environmental burdens, including those attributable to climate change, cumulative exposures, and “the legacy of racism or other structural or systemic barriers.” https://www.epa.gov/aboutepa/about-office-environmental-justice-and-external-civil-rights Since environmental racism was identified as a serious national problem in the 1980s, progress has been halting, but the OEJECR represents a significant commitment to address environmental inequalities. Politicizing federal agencies by reorganizing departments and by replacing experienced civil service experts with political appointees stands out as a key element of the Project 2025 plan. Eliminating the OEJECR would reduce environmental protections for all. For a history of this office and its fate during the Trump Administration, see https://www.liebertpub.com/doi/abs/10.1089/env.2021.0012

Already, all peer-reviewed findings can receive scrutiny and comment in the public record.  What this rhetoric of "transparency" targets for exclusion are studies of people who are actually exposed to environmental chemicals, which often draw on medical records whose privacy would be compromised by this "transparency." See comment and links at annotation #2 above of this chapter.

The EPA has long had a compliance program, as it should, but as many studies of EPA enforcement have shown, a lack of strong enforcement will lead to more violations that put public health and the environment at risk. https://www.journals.uchicago.edu/doi/abs/10.1093/reep/req017 https://envirodatagov.org/publication/a-sheep-in-the-closet-the-erosion-of-enforcement-at-the-epa/

Ordinarily "tangible" would be taken as meaning, among other things, scientifically demonstrable. Yet the aversion here to acknowledging the science of how intensifying storms or droughts have been worsening through climate change suggests otherwise. Instead, "tangible" seems to refer only to those problems that authors of this report and their allies--none of them scientists themselves--are willing to acknowledge as perceptible and real, without necessarily listening to what any scientists say.

States lack the budgets, enforcement authority, and breadth of expertise that the federal agency can provide.

They can also be susceptible to regulatory capture--a major reason why the state-based patchwork of pollution controls prior to the EPA was widely considered to have failed. For just one instance, see this analysis of the mining/smelting industry in Idaho. https://upittpress.org/books/9780822964483/

Rescusitates this slogan from Scott Pruitt, Trump's first appointee as EPA administrator. Before resigning under a cloud of multiple scandals, he claimed his vision for the agency to harken back to an earlier EPA, when its agenda was more "basic" and its relationships with states more harmonious. For a critical look by an actual historian at what Pruitt's vision missed, see this piece by Leif Fredrickson: https://www.washingtonpost.com/news/made-by-history/wp/2017/07/14/scott-pruitt-is-wrong-about-the-origins-of-the-epa/

False; the EPA's workforce size and budget trends do not support this claim. Employment at the EPA, which actually peaked in 1999, shrank during the Obama administration. https://federalnewsnetwork.com/unions/2023/02/epa-employees-voice-concerns-about-low-pay-understaffing-burnout/ Also see charts 4 and 11 here which actually show slow declines in EPA's budget and staff over the Obama years: https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

Project 2025’s goal of devolving environmental regulation to the states would undermine longstanding federal policies that enjoy bipartisan support and have proved remarkably effective in improving public health. For example, the 1987 Montreal Protocol on Substances that Deplete the Ozone Layer, negotiated and signed during the Reagan administration, required participating countries to curb the release of chlorofluorocarbons (CFCs) that were damaging the ozone layer. Rules implementing global treaties are of necessity federal regulations. The international agreement, signed by all of the world’s countries, worked because signatories agreed to reduce their output of CFCs by one-half within ten years. As a result, worldwide use of CFCs dropped by 80 percent in the first eight years. Thirty years later, the ozone hole was the smallest recorded. https://www.unep.org/news-and-stories/story/thirty-years-what-montreal-protocol-doing-protect-ozone The result: improvements in public health due to reductions in UV radiation, which can cause skin cancer and eye damage. https://www.epa.gov/sites/default/files/2015-07/documents/achievements_in_stratospheric_ozone_protection.pdf<br /> September 16, 2024, marks the International Day for the Preservation of the Ozone Layer.

A critical aspect of the origins of the EPA is not stated here: States and localities had anti-pollution laws in place prior to the EPA's creation, but those laws were either inadequate or weakly enforced. Anti-pollution laws also existed at the federal level, but these were primarily designed to fund and otherwise assist states and localities. This system, however, failed to protect public health and the environment. The EPA, and the much stronger federal laws that were created in conjunction with it, constituted a historic response to these regulatory as well as market failues. A Republican President, with bipartian support from Congress, created a national agency and laws that could control pollution far more effectively than the preexisting system. States had a role, and would be given more authority if they proved they would effectively implement environmental laws. This document argues for a return to "state leadership," precisely what by 1970s was widely agreed to have poorly controlled the nation's pollution problems. https://ajph.aphapublications.org/doi/full/10.2105/AJPH.2018.304396

"...perceived threat...": another side-step from the scientific consensus about threats from climate change.

The EPA's failures in Flint are exactly the failures that Project 2025's state devolutionary approach would compound. The Grist article cited here by this Project 2025 author explains the EPA's failure differently from what is here implied, in the following way:

"The [Office of Inspector General] report cites five possible oversight actions that the EPA could have taken under the Safe Drinking Water Act, including alerting Flint residents about possible harms and acting in the place of state authorities when there is 'substantial endangerment' to human health." https://grist.org/article/the-epa-failed-flint-now-we-know-exactly-how/

But in almost every case, officials deferred to their state counterparts, rather than using their legal authority to step in. As the report’s authors note, such oversight tools — like most tools out there — are “only effective when used.”

Additionally, the Flint water crisis was the consequence of decades of deliberate underfunding of Detroit's water system. During the Nixon and Ford administrations, the EPA failed to allocate sufficient funds, through its Municipal Wastewater Construction Grants Program, to the Detroit Water and Sewer Department. This refusal worsened a municipal debt spiral that would eventually contribute to the Flint water crisis. Moreover, the EPA failed to adequately ensure DWSD's compliance with agreed-upon treatment standards and deadlines. For more on Detroit and the EPA's Construction Grants Program, see:

https://uncpress.org/book/9781469665764/toxic-debt/

https://academic.oup.com/jah/article-abstract/111/1/71/7695574?redirectedFrom=fulltext

Across both liberal and conservative administrations, there have been tensions between Congress and the Executive Branch over the EPA. But the most notorious case happened during the conservative administration of Ronald Reagan, when a bi-partisan Congress held Reagan's appointees accountable for their malfeasance and attempts to undermine the mission of the agency. As a result, Reagan was forced to re-install the original EPA administrator, William Ruckelshaus, to revive the agency's legitimacy and fend off a growing backlash from Congress and the public. In 2017, Ruckelshaus (a Republican) criticized the Trump administration for again undermining the basic mission of the EPA. https://www.nytimes.com/2017/03/07/opinion/a-lesson-trump-and-the-epa-should-heed.html; https://ajph.aphapublications.org/doi/full/10.2105/AJPH.2018.304396

Needing context: EPA staff levels actually peaked in 1999. Despite declining budgets and staff over the Obama years, largely driven by conservatives in Congress rather than the Obama White House, the Trump administration (of which this author was a part) repeatedly proposed draconian budget cuts for the agency, as much as 32%. Though Congress restored much of this money, both the agency's budget and staff declined significantly over the Trump years. https://www.apeoplesepa.org/home/origins https://envirodatagov.org/embattled-landscape-series-part-2b-the-declining-capacity-of-federal-environmental-science/

The argument that EPA has been coopted by the Left is a long-standing canard of the Republican party. In reality, Republican presidents have been the leaders in appointing partisan operatives to head and staff the agency, especially in more recent times. While Reagan appointed Anne Gorsuch early in his presidency, the public outcry against her actions, led the Reagan White House to reverse course after two years. And from the return of William Ruckelshaus, the well-respected first leader of the agency, in 1983 through the appointment of former New Jersey Governor Christine Whitman by George W. Bush in 2001, Republican presidents did appoint EPA administrators who were committed the agency's basic mission. But the Republican Party turned more aggressively anti-environmental stances by the 2010s lead to Republican appointees during the Trump administration who actively opposed and sought to undermine much of this agency's mission and ongoing work. https://pubmed.ncbi.nlm.nih.gov/29698097/

"Open-source" science is code language for conservative efforts to undermine the work of scientists and the role of science in the regulatory process. It is the most recent variation on calls for "sound science," which was a talking point for EPA Administrator Scott Pruitt at the start of the Trump administration. Calls for "open source" or "sound science" seek to limit the kinds of scientific research that can be used in regulatory decisionmaking. In particular, they seek to eliminate any studies that draw upon sensitive healthcare records. https://fivethirtyeight.com/features/the-easiest-way-to-dismiss-good-science-demand-sound-science/

This "open-source science" agenda would exclude from regulatory considerations practically all the burgeoning science done over the past two decades centering on actual people who are exposed to toxic pollutants, including what are considered by scientists to be the best and most conclusive studies in epidemiology. That's because all these studies involve confidential medical records the laws like HIPA make impossible to fully release to the public without violating patients' privacy. https://nyuelj.org/2021/06/transparency-in-regulatory-science-for-whom/

I like this sentence and how the writers convey the idea that getting lost can be something positive. It helps people to think, become more creative, and explore new things.

• You need to pay attention to what is happening to the person
• You have to maintain that punishment in memory
• You have to repeat that behaviour when you're motivated to do so
• Reinforcement is affecting motivation the most

• Rewarded based on the amount of responses and it increases responses
• Immune to extinction

• Hinders drug-addiction recovery because the context is still fresh in the one's mind

• Are you able to access the reward even if you don't do the behaviour?
• Eg. We still need to feed dogs which is why food is not good for reinforcement

• How quickly after the behavior the reward can be received
• Eg. Obedient classes and treat

• Refers to the opportunity value of engaging in an activity
• The ability to do something is reinforcing behaviour, even when you don't actually plan on doing the behaviour

• We learn this reinforcer through conditioning Eg. Social status(eg. promotion), grades
• Used much more than secondary since the use of the former is inhumane

• Fulfill biological needs Eg. Fulfillment of food and sustenance, warmth(shelter, clothing, etc.), sleep, sex, etc.
• Praise is debatable -> We can live without it, but the absence of it has detrimental consequences in children's development

• Learn precursor behaviour from the deliverer of the punishment Eg. Avoid the person doing the punishment even when the behavior is committed which means the behavior continues

• Run away from the punishment; not effective in this case

• Negative reinforcement

• Taking something away to increase the probability of a behavior occurring Eg. Absolving a child the responsibility of doing chores if they're nice to their siblings

• Often confused with positive reinforcement

• • Negative punishment 2.

• Positive reinforcement
• Not synonymous with good and bad but refers to addition and subtraction in regards to the situation

• Increases the possibility of a behaviour continuing Eg. Rewarding a kid a piece of candy for being nice to their friends

• Positive punishment

• Decreasing the probability of a behaviour occurring

• Two stimuli become associated and we develop a conditioned response to that association

• Happens to people who regularly use psychotic drugs
• Individuals get a conditioned response to their environment and can contribute to drug tolerance. At the extreme, it contributes to overdose when individuals use drugs in different settings

• Cases of regular drug users
• Happens unintentionally around the use of the drugs
• Refers to the context which the drugs are used

https://ashby.info/journal/index.html

Note that the URL http://www.rossashby.info/index.html has moved.

https://forum.zettelkasten.de/discussion/2415/zettelkaesten-in-the-fields-of-science-and-history

I learned that some writers only plan the beginning and the ending, and the middle is changeable, allowing anything to come to the writer's mind.

reply to Signynt at https://discord.com/channels/686053708261228577/979886299785863178/1293207926013427733

Does Carl Linnaeus' incarnation work? Isabelle Charmantier and Staffan Müller-Wille have a number of journal articles on his "invention" and use of index cards in his research and writing work. If you dig around you'll find references to Gottfried Wilhelm von Leibniz' use of index cards and the Arca Studiorum (Krajewski, MIT, 2011); Computer scientist Gerald Weinberg wrote Gerald M. Weinberg on Writing: The Fieldstone Method. New York, N.Y: Dorset House, 2005, which might appeal; you'll also find examples in physicist Mario Bunge, and, although he had a mixed practice of notebooks and index cards, W. Ross Ashby's collection of notes on complexity can be found at https://ashby.info/. Hundreds of other scientists and mathematicians had practices, though theirs typically fall under the heading of commonplace books (Erasmus Darwin, Charles Darwin, et al.) or as in the case of Isaac Newton and others the heading of "waste books". While looking at others' examples or reading about it may feel like it's going to get you somewhere (better?), having some blind faith and proceeding with your own practice is really the better way to go. Others have certainly done it. Generally it's far rarer for mathematicians, engineers, or scientists to write about their note making/methods so you're unlikely to find direct treatises the way you would for historians, sociologists, anthropologists, humanists, etc.

syndication link: https://discord.com/channels/686053708261228577/979886299785863178/1293663556197417082

Uses "they" a considerable amount of the time to give us more context to her situation

Personification

Use of humor to mediate the pain

As the Mariner awakes from his trance he realizes that the ship is making its way toward his native land. While he is taking in the sights of all things familiar he appears to witness angelic beings rising from the bodies of fallen sailors before seeing a Hermit who he hopes will be able to wash away all his sins. (GJ)

Definition (n): a revolving pointer that shows the direction of the wind in the shape of a rooster (GJ)

A mysterious ghastly figure that wins the Mariner's soul in a gamble. Then he is faced with a consequence worth than death: life-in-death, he will have to wander the world meaninglessly and unable to fade away.

Definition (n): a specific time of day dedicated to prayer, the ninth canonical hour being dedicated to God the Son. Originated with the Catholic tradition. 'Vesper' means 'evening' in Latin and was used to describe the time of evening prayers. The specific phrase 'vesper's nine' is unique to Coleridge's poem and means that the Albatross perched for nine days (or through nine vespers). KF

Loewen likely intended this book to show us the raw, unfiltered truth of history. If we had been taught the full story from the beginning, we could use those lessons to improve our lives today. It's understandable why people often choose to ignore or downplay the negative aspects of history. They prefer to believe in heroic figures and a hopeful future rather than confront past mistakes. However, we can see how history is repeating itself because we were never taught the "dark side" of the past. This lack of knowledge prevents us from recognizing the warning signs and stopping harmful events from happening now.

The highlighted portion discusses the true nature of the early Virginians. When we are told the story of Columbus, Jamestown, and the Indians, we are told that everyone worked together. The Indians wanted to help the Virginians by teaching them how to plant crops, and in return, the Virginians educated the Natives in English culture. However, Loewen tells us that what happened was after Squanto volunteered to help them with fishing, other Virginians "took Indian prisoners and forced them to teach colonists how to farm."

These couple of paragraphs do not sit right with me. The reason is because I did not know that this happened. In my years of education, I was never told that A) the disease during this time was so bad and B) that the American Indians were used this way. Loewen demonstrated how the European Americans used the fear of the Natives to gain power over them. In the last paragraph highlighted, Loewen states, "many American Indians surrendered to alcohol, converted to Christianity, or simply killed themselves."

They were saying earlier in the text that legal research is a science. This section here truly explains why that is true. In science there are so many things that touch that specific topic, and, as a researcher, you must know where to start and how to look through these related items. The pooling of knowledge is imperative if we want growth and understanding as there are just too many things to look at. Pooling knowledge allows the field to come to an agreement of what rule they want to follow and any other approaches that people have tried that work or don't work. In the law we need to have a consensus on how we are interpreting and applying the law or we will not have the results that we are looking for when implementing them.

I had no idea what shepardizing was until a couple months ago; however, I think it is one of the most important steps in your research. At the OJ Simpson trial the prosecutor based one of her arguments off of a case that had not been shepardized and she was called out by the judge. The case she based her argument off of had been overturned. She could have saved herself a lot of embarrassment and time spent crafting that argument if she had just shepardized before she relied on the case.

One thing that I always struggle with is that legal problems usually arise from "laymen" but then legal terms of art and complicate concepts are brought in to complicate the problem. One major issue in the legal realm is that the law is not accessible to the average laymen person because they simply do not understand it. I understand that our job as lawyers is to write the legal questions and concepts in legal terms but the explain it to the clients clearly. However, if the legal issues are arising from the laymen, then why do we have to complicate the matter for the public and add in legal terms and concepts that makes the law more inaccessible? I think it's important to keep this in mind moving forward as we all start practicing law The part in this sentence that says "clearly and concisely" is going to be very important when we begin to interact with clients.

I actually like that he is advocating his system as being more effective than most systems of legal research used in his time. I think that one of the problems that I've noticed amongst lawyers, is that many are too adversarial. They often don't want to help each other, and sometimes actively work to undermine each other. The "cagey, old lawyer" hiding the book is an example of a common behavior amongst the lawyers who want to be sharks. They are jealous of their knowledge and experience, and rather than sharing that knowledge and experience with the inexperienced or ignorant, they use that knowledge and experience to eat their young. Even Gardner displays some of that attitude, making fun of the uneducated mountaineer, and not showing much empathy for the frustrated widow who was desperate to find some case that would help her cause.

They provide legal bases for people (NGOs, journalists, activists, etc.) to ask bureaucrats information about the government performance.

Definition. These laws are introduced in response to increasing dissatisfaction with secrecy around government policy.

DCD is an improvement on CD towards the very expensive EMD method.

need to do both directions because of the minimization

resisting gender essentialism

the absent patronymic

This just shows how difficult it is to climb the income ladder. Lampra, who went to school for a specific practice, still struggles to find work, despite the work, time, and money she put into gaining skills and knowledge about her practice. Having connections is important, but difficult when you may not have access to resources and opportunities to build those connections. For Michael, "network engineer" sounds like a job that also requires education and that he has to be equipped with the right skills to do well. However, it does not come with health insurance which makes it a lot more difficult to support him and his family.

When thinking back to Week 1 when we discussed the purpose of schools, a majority of the class voted for "social mobility" as the main purpose of schools now, and I agree with this. However, reading about the effects of where you live and seeing the map of different districts on Tuesday, makes me wonder if this can be possible for students and families who live in poverty.

but what is the input transformation?

I believe IRC and Discord are quite similar, despite being social media platforms from different decades. On Discord, like IRC, users can join groups focused on specific topics. Within these groups, there are designated channels for particular discussions. For example, the UW swim club has its own group, and within that group, separate channels like "workouts" or "swim times" help keep conversations organized and clear for everyone.

After everything Sonny has been through as well as the Narrater they both don't know how to communicate with each other. Sonny does not seem like himself because he has been through a trauma which can seem to the narrater there is no way to get through to him. Could open communication solve this issue?

This letter is a great representation of Sonny's side of the story and how grateful he is to hear from his brother. Having that sense of connection shows a lot about his character.

Why is he scared for Sonny? The narrater seems to be empathic towards Sonny due to the story he read in the paper.

This is a great opening paragraph in terms of catching the readers eye. I read this and was hooked wanting to know more. It sets up a mysterious story and has me wondering what he is reading.

I think that this is my favorite passage, just because of all of the details that are in it. How the narrator describes the band being held back, how Creole waits and watches Sonny to let him take control.

I just had an 'aha' moment readinf this sentence because of all I learned from last year's Ap US History class that I know about this Bill and the benefits to the men who served in the military

I thought this quote was significant because the narrator is saying that sonny is the same person he always was but with experiences that changes him. I also think that the narrator regrets not getting to know his baby brother but that he can still see bits of him buried underneath all of the prison time and drug addictions. There is also the symbol of darkness vs. light reappearing.

I thought this quote was really significant, not only because it's a really good piece of advice, but also because I kind of feel like I'm going through the same problem at home with my younger sister, who wants to do things, no matter the cost. I wanted to know from my classmates if anyone else has felt this way.

Their laughter isn't the childish laughter that you might expect, full of happiness, but instead it's mean and scathing. It emphasizes how this is the angry laughter of men who are already hardened against the world even though they shouldn't have to be.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

Welcome to Talking with machines! Please leave a comment here or below. You can post anonymously. If you sign up @ https://web.hypothes.is/ all your posts from across the interwebs are collected in your account. Click the little arrow next to the Hypothes.is logo above to close this panel.

why not be allies? bc if others see that you fear us, they will respect us more

negotiaion of Athens, be our slaves and we won't kill you

Athens motives

Athenians sens reps to speak to Melians, but Melians only allow them to speak infront of a few governing, Athenians claim they do this to prevetn the people from hearing their reason Melians reply that Athenians give them option of war or slavery, they see them as a threat

Brooklyn Engineers’ Club. Brooklyn Engineer’ Club Proceedings for 1906: Constitution and By-Laws and Catalogue of Reference Works Added to the Library During the Year. Brooklyn Engineers’ Club, 1907.

Winslow-Yost uses a lot of contrast and paradox to illustrate the complex emotional experiences offered by video games.

Technological systems lure people with apparent benefits while demanding something in return. The repeated listing of verbs ("oversee, track, quantify, guide...") highlights technological control, making the reader aware of how intrusive these systems are.

I've generally found that Olympia machines with a dedicated 1 key and a 4/$ key will usually have a script font. Additionally they don't have ribbon selectors (which are most often on the right hand side of the keyboard when they are present) or only have black and stencil settings.

The lack of bichrome settings on these machines is due to the taller/lower extenders on many script glyphs.

South Carolina shows itself why, the sheriff and the the governor and many other higher indiviuals have control of it.

Group is too insignificant to call it, at that point it should be called a political party, South Carolina proves it.

So lower class people didnt care what about the upper class that have power..? did it not appear to them as well to be involved?

why does every lawyer pass a few weeks after a brutal case..?

This shows that some oral or written artifacts could be intentionally created. Mostly, they created these artifacts to represent something already there but didn't have any evidence to prove it, so it could be made to describe that thing.

second kind of sources; testimonies

Definition of source

' the branch of psychology concerned with evaluation and use of psychological tests the application of statistical and mathematical techniques to psychological test

https://www.liberatingstructures.com/1-1-2-4-all/

I did not clue into this in my first read. Very interesting comparison between Gawain being hunted by the Lady Berdilak and the doe being hunted by Lord Berdilak.

I think the mind/body binary dovetails well with the man/nature one.

Gawain, at the end of the narrative has meted the chivalric/natural opposition in himself. Gawain was introduced as a personification of the human half of the binary. Gawain is chivalrous to his gleaming boots, noble and brave. He is polite, courteous, and socially adept. Devout in his religion and rational in his decision making. Gawain's journey results in a kind of transformation. He has not lost his chivalry, rather, he gains a deeper understanding of the natural. Gawain becomes more honest and emotionally mature through his experience, as the essay says more in touch with the 'inner man'.

ABCDE:

A. Asymmetry (of the moles) B. Border (Regular vs irregular border) C. Color (mult shades. etc) D. Diameter (<5-6 mm) E. Evolving (becoming bigger)

Cardiac biomarkers: Troponin, CK-MB CXR Echo CMP EKG

rupture of atherosclerotic plaque thrombus --> clot forms --> so now the clot is partially blocking the blood vessels

Low PO2 and HTN from reduced blood vessels and narrowed blood vessels

Yes it's bad - interferes with COX1 and COX2 stomach function

patients' lipid panel should be measured every 3 months to 1 yr if patients have statin

EKG shows elevated ST wave and depressed T wave:

General summary: In AMI, there is transmural injury - infarction damages the entire thickness of the heart across all layers, this is worse than subendocardial injury where only the subendocardium (inner layers of the heart) is damaged

EKG will show inverted T wave ST elevation for STEMI and ST depression for NSTEMI, prolonged Q wave

Normally, ventricles repolarize from epicardium to endocardium. During ischemia or MI, AP duration is reduced, and ventricles repolarizes from endocardium to epicardium instead. This reversed repolarization leads to an inverted T-wave on ECG (negative deflection).

Angina does not change the cardiac biomarkers

Acute MI do --> elevated troponin that peaks and dies faster than CK-MB that takes longer

Troponin I = sensitive and specific, rise in ~4hr and peak in 24hr, lasts 7-10 days

CK-MB: less sensitive and specific, rise in 4-8hr and lasts 48-72 hr (2-3 days), good for looking for reinfarction

Unstable angina, atherosclerotic plaque has ruptured forming a thrombi that partially blocked blood flow

His angina has worsened to an extent where taking nitroglycerin isn't relieving the symptoms

He may be experiencing a side effect of nitroglycerin overdose, ends up having reflex tachycardia and feeling worse

Chapman's reflexes at the bilateral second intercostal spaces - relates to viscerosomatic dysfunction on BETH - Bronchus, Thoracic, Heart, Esophagus

mic drop

so he's against relying on anything/anyone but yourself

I like this imagery

but if we're advocating for self-reliance shouldn't he get his own supply of corn rather than someone hand it to him?

ew nope

the crutches help but it all matters on him and his own well-being and support on the inside

so basically focus needs to be shifted towards self improvement and less towards societal improvement

create something new rather than take something old

I never really realized how much we imitate wow

and if he doesn't? is he not wise?

use of "you" interesting. who is this aimed at?

why must we classify the new minds? classification is no bueno

interesting

small things add to the progression of repairing evil

im glad others also thought of Farmer James

becoming more self-reliant can bring major change to all aspects of life

living in the present and has opportunities and the ability to take more risks and do more.

Love

society should not have the upper hand

Real, I couldn't agree more :)

This is very interesting.

I like this for some reason.

I understand we shouldn't dwell on the past and look forward but isn't it also important to look in the past cause how else do you learn?

???

I don't like this also who is the "great man"?

will it always bring you out safe though?

The devil's child is a common known thing but for it to be mentioned like this is really interesting to me.

Interesting

I like how this narrative switch from being oneself, to the universe, to god, to straight up politics.

Liar

Wow. Literature.

I fear it does advance there are just backs and differing opinions. All I know is that society has advanced to the point I can have a nose piercing and tattoo in peace.

one of the best things he's said so far

how self helping can one be if they rely on a higher power for everything?

If youre focused, faithful and joyus a man can make his own decisions

It is great that you want to be yourself, But I think you have loved yourself enough, Maybe focus a little bit on caring for other people.

im getting the feeling he doesn't love or care about any one.

Is this guy on drugs. yes or no?

Throwing own self out the window to implant his ideas into future spawn

live your truth.... unless you do not believe in god.

Everything is connected.

I like how he mentioned so many positive things about man, and the one thing he said about femininity is rage. Like excuse me?

This is giving too much main character complex. It does not seem like he has concern for others.

why would you want that? icky.

why does the rage have to be feminine? I feel like that is a little odd.

non popular opinions are seen as outcasts and separated from the rest

I feel like this is based on prospective. He is not fair to say that their truths are not quite true. They might not be, his might not be. It does not mean it makes it less true to the person at hand.

I feel like this represents a choice though??? Is that such a bad thing to choose? Obviously if one feels forced that's not a good thing, but if its a choice you make willingly?

thin line between love and hate or portraying one as the other????

believes in his argument so much that he would accept being a devil's child

Capitalism, Societal conform.

Infancy comes with a form of purity, untainted by the world and others opinions

who would be the absolutely trustworthy? god???

Take action and responsibility into your own hands. Be true to your being.

adapting others opinions to conform is the death of oneself

Taking ideas from other people to use as your own instead of creating one. The idea of being original to nothing being original

A reminder, almost like a conscious reminding the soul?

Strength in wildlife connecting back to reliance.

This is such a powerful statement, its very unique

Do not seek outside yourself, prevalent to the title at hand

Listen to the words you just said. "Her own internal monologue....but didn't recognize it". What exactly do you think hearing voices is, as defined clinically??

Exactly what would be expected in PD. When anxiety or anxiety triggers occur, this is when decoupling is happening. Reality processing > alogia and dissociation > full personality dissociative splitting

Meekness

Jacobs

so this is a difference to haskell, bc + is a defined function occurring in a pattern, not a constructor. Also, the pattern is non-linear. Aha, so this point is addressed later on.

for - Indyweb dev - dynamic graph networks

for - quote - climate crisis - behavioral change - system change - importance of showing impacts - example - climate departure project

quote - climate crisis - behavioral change - system change - importance of showing impacts - example - climate departure project - Eltahir - To get people to act, my hypothesis is, you need to reach them - not just by convincing them to be good citizens and saying it’s good for the world to keep below 1.5 degrees, but - showing how they individually will be impacted,”

the use of brackets make this revelation (and the future bracketed terms) feel secretive, hidden, and quiet

the revelations and contempt of the speaker grows and grows in power throughout the poem until it becomes vocalized at the end with this sentiment no longer whispered but instead declared

accusatory tone of the piece culminates here, where the speaker is telling readers that the land itself is not responsible and that America, the physical place, is not evil or committing the sins of its people

Call to action focuses on redeeming the physical land and on connecting with nature

African-Americans, just like nature, are the victims of White American racism and ought to be made America again - free, individual, beautiful, cultured, etc

meaning "going" - roots in Caribbean and U.S. Southern regional manners of speech, showing how the pains of the singer are likely responsive to the pains of the American Empire - both colonialism and segregationist pains and sufferings caused by America are resulting in African-American and global African sufferings and this general sense of exhaustion

personification of the piano itself as singing - not just an accompaniment which is subservient to the singer but the instruments itself are powerful, vocal, and centered

however, also implies that something is being done to the piano that forces it to sing (rather than singing, its moaning - involuntary and pained rather than joyous or enthusiastic)

connected with the sense of pathos throughout the poem - the forcing of the piano to play is reflective of the singer themselves who seems to be forced by something itself to play; not an excited performance but a 'weary' one

Author response:

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

Reviewer #1 (Public Review): 

Summary: 

Dr. Santamaria's group previously utilized antigen-specific nanomedicines to induce immune tolerance in treating autoimmune diseases. The success of this therapeutic strategy has been linked to expanded regulatory mechanisms, particularly the role of T-regulatory type-1 (TR1) cells. However, the differentiation program of TR1 cells remained largely unclear. Previous work from the authors suggested that TR1 cells originate from T follicular helper (TFH) cells. In the current study, the authors aimed to investigate the epigenetic mechanisms underlying the transdifferentiation of TFH cells into IL-10-producing TR1 cells. Specifically, they sought to determine whether this process involves extensive chromatin remodeling or is driven by preexisting epigenetic modifications. Their goal was to understand the transcriptional and epigenetic changes facilitating this transition and to explore the potential therapeutic implications of manipulating this pathway. 

The authors successfully demonstrated that the TFH-to-TR1 transdifferentiation process is driven by pre-existing epigenetic modifications rather than extensive new chromatin remodeling. The comprehensive transcriptional and epigenetic analyses provide robust evidence supporting their conclusions. 

Strengths: 

(1) The study employs a broad range of bulk and single-cell transcriptional and epigenetic tools, including RNA-seq, ATAC-seq, ChIP-seq, and DNA methylation analysis. This comprehensive approach provides a detailed examination of the epigenetic landscape during the TFH-to-TR1 transition. 

(2) The use of high-throughput sequencing technologies and sophisticated bioinformatics analyses strengthens the foundation for the conclusions drawn. 

(3) The data generated can serve as a valuable resource for the scientific community, offering insights into the epigenetic regulation of T-cell plasticity. 

(4) The findings have significant implications for developing new therapeutic strategies for autoimmune diseases, making the research highly relevant and impactful. 

We thank the reviewer for providing constructive feedback on the manuscript.

Weaknesses: 

(1) While the scope of this study lies in transcriptional and epigenetic analyses, the conclusions need to be validated by future functional analyses. 

We fully agree with the reviewer’s suggestion. We have added the following text to the Discussion to address this concern: “The current study provides a foundational understanding of how the epigenetic landscape of TFH cells evolves as they transdifferentiate into TR1 progeny in response to chronic ligation of cognate TCRs using pMHCII-NPs. Our current studies focus on functional validation of these observations, by carrying out extensive perturbation studies of the TFH-TR1 transdifferentiation pathway in conditional transcription factor gene knock-out mice. In these ongoing studies, genes coding for a series of transcription factors expressed along the TFH-TR1 pathway are selectively knocked out in T cells, to ascertain (i) the specific roles of key transcription factors in the various cell conversion events and transcriptional changes that take place along the TFH-TR1 cell axis; (ii) the roles that such transcription factors play in the chromatin re-modeling events that underpin the TFH-TR1 transdifferentiation process; and (iii) the effects of transcription factor gene deletion on phenotypic and functional readouts of TFH and regulatory T cell function.”

(2) This study successfully identified key transcription factors and epigenetic marks. How these factors mechanistically drive chromatin closure and gene expression changes during the TFH-to-TR1 transition requires further investigation. 

Agreed. Please see our response to point #1 above.  

(3) The study provides a snapshot of the epigenetic landscape. Future dynamic analysis may offer more insights into the progression and stability of the observed changes. 

We have previously shown that the first event in the pMHCII-NP-induced TFH-TR1 transdifferentiation process involves proliferation of cognate TFH cells in the splenic germinal centers. This event is followed by immediate transdifferentiation of the proliferated TFH cells into transitional and terminally differentiated TR1 subsets. Although the snapshot provided by our single cell studies reported herein documents the simultaneous presence of the different subsets composing the transdifferentiation pathway at any given time point, the transdifferentiation process itself is extremely fast, such that proliferated TFH cells already transdifferentiate into TR1 cells after a single pMHCII-NP dose (Sole et al., 2023a). This makes it extremely challenging to pursue dynamic experiments. Notwithstanding this caveat, ongoing studies of cognate T cells post treatment withdrawal, coupled to single cell studies of the TFHTR1 pathway in transcription factor gene knockout mice exhibiting perturbed transdifferentiation processes are likely to shed light into the progression and stability of the epigenetic changes reported herein. 

To address this limitation in the manuscript, we have added the following paragraph to the Discussion: “Although the snapshot provided by our single cell studies reported herein documents the simultaneous presence of the different subsets composing the TFH-TR1 cell pathway upon the termination of treatment, the transdifferentiation process itself is extremely fast, such that proliferated TFH cells already transdifferentiate into TR1 cells after a single pMHCII-NP dose (6). This makes it extremely challenging to pursue dynamic experiments. Notwithstanding this caveat, ongoing studies of cognate T cells post treatment withdrawal, coupled to single cell studies of the TFH-TR1 pathway in transcription factor gene knockout mice exhibiting perturbed transdifferentiation processes are likely to shed light into the progression and stability of the epigenetic changes reported herein”. 

Reviewer #1 (Recommendations for the authors): 

The authors may consider the following suggestions to improve this study: 

(1) The authors may include a brief background on type 1 diabetes and the model involving BDC2.5 T cells to provide context for readers who may not be familiar with these aspects. 

We have added this information to the first paragraph in the Results section: “BDC2.5mi/I-Ag7-specific CD4+ T cells comprise a population of autoreactive T cells that contribute to the progression of spontaneous autoimmune diabetes in NOD mice. The size of this type 1 diabetes-relevant T cell specificity is small and barely detectable in untreated NOD mice, but treatment with cognate pMHCII-NPs leads to the expansion and formation of antidiabetogenic TR1 cells that retain the antigenic specificity of their precursors (3). As a result, treatment of hyperglycemic NOD mice with these compounds results in the reversal of type 1 diabetes (3).”

(2) It is understandable that further biological and functional experiments are beyond the scope of this paper, but it would be of interest to know how the authors envision future studies based on the transcriptional and epigenetic information obtained thus far. 

We have added the following text to the Discussion section: “The current study provides a foundational understanding of how the epigenetic landscape of TFH cells evolves as they transdifferentiate into TR1 progeny in response to chronic ligation of cognate TCRs using pMHCII-NPs. Our current studies focus on functional validation of these observations, by carrying out extensive perturbation studies of the TFH-TR1 transdifferentiation pathway in conditional transcription factor gene knock-out mice. In these ongoing studies, genes coding for a series of transcription factors expressed along the TFH-TR1 pathway are selectively knocked out in T cells, to ascertain (i) the specific roles of key transcription factors in the various cell conversion events and transcriptional changes that take place along the TFH-TR1 cell axis; (ii) the roles that such transcription factors play in the chromatin re-modeling events that underpin the TFH-TR1 transdifferentiation process; and (iii) the effects of transcription factor gene deletion on phenotypic and functional readouts of TFH and regulatory T cell function.”

(3) The authors may consider adjusting figures where genes are crowded or difficult to read due to small font size. 

Figures with crowded text have been modified to facilitate reading.

Reviewer #2 (Public Review): 

Summary: 

This study, based on their previous findings that TFH cells can be converted into TR1 cells, conducted a highly detailed and comprehensive epigenetic investigation to answer whether TR1 differentiation from TFH is driven by epigenetic changes. Their evidence indicated that the downregulation of TFH-related genes during the TFH to TR1 transition depends on chromatin closure, while the upregulation of TR1-related genes does not depend on epigenetic changes. 

Strengths: 

(1) A significant advantage of their approach lies in its detailed and comprehensive assessment of epigenetics. Their analysis of epigenetics covers chromatin open regions, histone modifications, DNA methylation, and using both single-cell and bulk techniques to validate their findings. As for their results, observations from different epigenetic perspectives mutually supported each other, lending greater credibility to their conclusions. This study effectively demonstrates that (1) the TFH-to-TR1 differentiation process is associated with massive closure of OCRs, and (2) the TR1-poised epigenome of TFH cells is a key enabler of this transdifferentiation process. Considering the extensive changes in epigenetic patterns involved in other CD4+ T lineage commitment processes, the similarity between TFH and TR1 in their epigenetics is intriguing. 

(2) They performed correlation analysis to answer the association between "pMHC-NPinduced epigenetic change" and "gene expression change in TR1". Also, they have made their raw data publicly available, providing a comprehensive epigenomic database of pMHC-NPinduced TR1 cells. This will serve as a valuable reference for future research. 

We thank the reviewer for his/her constructive feedback and suggestions for improvement of the manuscript.

Weaknesses: 

(1) A major limitation is that this study heavily relies on a premise from the previous studies performed by the same group on pMHC-NP-induced T-cell responses. This significantly limits the relevance of their conclusion to a broader perspective. Specifically, differential OCRs between Tet+ and naïve T cells were limited to only 821, as compared to 10,919 differential OCRs between KLH-TFH and naïve T cells (Figure 2A), indicating that the precursors and T cell clonotypes that responded to pMHC-NP were extremely limited. This limitation should be clearly discussed in the Discussion section. 

We agree that this study focuses on a very specific, previously unrecognized pathway discovered in mice treated with pMHCII-NPs. Despite this apparent narrow perspective, we now have evidence that this is a naturally occurring pathway that also develops in other contexts (i.e., in mice that have not been treated with pMHCII-NPs). Furthermore, this pathway affords a unique opportunity to further understand the transcriptional and epigenetic mechanisms underpinning T cell plasticity; the findings reported can help guide/inform not only upcoming translational studies of pMHCII-NP therapy in humans, but also other research in this area. 

We have added the following text to the Discussion to address this limitation: “Although the TFH-TR1 transdifferentiation was discovered in mice treated with pMHCII-NPs, we now have evidence that this is a naturally occurring pathway that also develops in other contexts (i.e., in mice that have not been treated with pMHCII-NPs). Importantly, the discovery of this transdifferentiation process affords a unique opportunity to further understand the transcriptional and epigenetic mechanisms underpinning T cell plasticity; the findings reported here can help guide/inform not only upcoming translational studies of pMHCII-NP therapy in humans, but also other research in this area”.

We acknowledge that, in the bulk ATAC-seq studies, the differences in the number of OCRs found in tetramer+ cells or KLH-induced TFH cells vs. naïve T cells may be influenced by the intrinsic oligoclonality of the tetramer+ T cell pool arising in response to repeated pMHCII-NP challenge (Sole et al., 2023a). However, we note that our scATAC-seq studies of the tetramer+ T cell pool found similar differences between the oligoclonal tetramer+ TFH subpool and its (also oligoclonal) tetramer+ TR1 counterparts (i.e., substantially higher number of OCRs in the former vs. the latter relative to naïve T cells). 

This has been clarified in the revised version of the manuscript, by adding the following text to the last paragraph of the Results subsection entitled “Contraction of the chromatin in pMHCII-NP-induced Tet+ vs. TFH cells at the bulk level”: “We acknowledge that, in the bulk ATAC-seq studies, the differences in the number of OCRs found in tetramer+ cells or KLHinduced TFH cells vs. naïve T cells may be influenced by the intrinsic oligoclonality of the tetramer+ T cell pool arising in response to repeated pMHCII-NP challenge (6). However, we note that scATAC-seq studies of the tetramer+ T cell pool found similar differences between the oligoclonal tetramer+ TFH subpool and its (also oligoclonal) tetramer+ TR1 counterparts (i.e., substantially higher number of OCRs in the former vs. the latter relative to naïve T cells)”.

(2) This article uses peak calling to determine whether a region has histone modifications, claiming that the regions with histone modifications in TFH and TR1 are highly similar. However, they did not discuss the differences in histone modification intensities measured by ChIP-seq. For example, as shown in Figure 6C, IL10 H3K27ac modification in Tet+ cells showed significantly higher intensity than KLH-TFH, while in this article, it may be categorized as "possessing same histone modification region". This will strengthen their conclusions.

We appreciate your suggestion to discuss differences in histone modification intensities as measured by ChIP-seq. However, we respectfully disagree with the reviewer’s interpretation of these data.

Our study primarily focuses on the identification of epigenetic similarities and differences between pMHCII-NP-induced tetramer+ cells and KLH-induced TFH cells relative to naive T cells. The outcome of direct comparisons of histone deposition (ChIP-seq) between these cell types is summarized in the lower part of Figure 4B and detailed in Datasheet 5. Throughout this section, we mention the number of differentially enriched regions, their overlap with OCRs shared between tetramer+ TFH and tetramer+ TR1 cells based on scATAC-seq data, and the associated genes. Clearly, the epigenetic modifications that TR1 cells inherit from TFH cells were acquired by TFH cells upon differentiation from naïve T cell precursors. 

Regarding the specific point raised by the reviewer on differences in the intensity of the H3K27Ac peaks linked to Il10 in Figure 6C, we note that the genomic tracks shown are illustrative. Thorough statistical analyses involving signal background for each condition and p-value adjustment did not support differential enrichment for H3K27Ac deposition around the Il10 gene between pMHCII-NP-induced tetramer+ T cells and KLH-induced TFH cells. 

This has now been clarified by adding the following text to the end of the Results subsection entitled ”H3K4me3, H3K27me3 and H3K27ac marks in genes upregulated during the TFH-to-TR1 cell conversion are already in place at the TFH cell stage”: “We note that, although in the representative chromosome track views shown in Fig. 6C there appear to be differences in the intensity of the peaks, thorough statistical analyses involving signal background for each condition and p-value adjustment did not support differential enrichment for histone deposition around the Il10 gene between pMHCII-NP-induced tetramer+ T cells and KLH-induced TFH cells.” 

We have also clarified this in the corresponding section of the Methods section (“ATACseq and ChIP-seq” under “Bioinformatic and Statistical Analyses”): “Given that peak calling alone does not account for variations in the intensity of histone mark deposition, analysis of differential histone deposition includes both qualitative and quantitative assessments. Whereas qualitative assessment involves evaluating the overall pattern and distribution of the various histone marks, quantitative assessment measures the intensity and magnitude of histone mark deposition.”

(3) Last, the key findings of this study are clear and convincing, but some results and figures are unnecessary and redundant. Some results are largely a mere confirmation of the relationship between histone marks and chromatin status. I propose to reduce the number of figures and text that are largely confirmatory. Overall, I feel this paper is too long for its current contents. 

We understand your concern about the potential redundancy of some results and figures. Our aim in including these analyses was to provide a comprehensive understanding of the intricate relationships between epigenetic features and transcriptomic differences. We believe that a detailed examination of these relationships is crucial for several reasons: (i) the breadth of the data allows for a thorough exploration of the relationships between histone marks, open chromatin status and transcriptional differences. This comprehensive approach helps to ensure that our conclusions are robust and well-supported; (ii) some of the results that may appear confirmatory are, in fact, important for validating and reinforcing the consistency of our findings across different contexts. These details are intended to provide a nuanced understanding of the interactions between epigenetic features and gene expression; and (iii) By presenting a detailed analysis, we aim to offer a solid foundation for future research in this area. The extensive data presented will serve as a valuable resource for others in the field who may seek to build on our findings.

That said, we have carefully reviewed the manuscript to identify and streamline elements that might be perceived as overly redundant, while retaining the depth of analysis that we believe is essential.

Reviewer #2 (Recommendations for the authors): 

(1) In Figure 1E, the text states "94% (n=217/231) of the genes associated with chromatin regions that had closed during the TFH-TR1 conversion,", but n=231 do not match with n=1820 provided in Figure 1D as downregulated genes. This is one of the examples that do not match numbers among figures or lack sufficient explanations. Please check those numbers carefully and add some sentences if necessary. 

We note that the text referring to Figure 1D describes the total number of differentially expressed genes between Tet+ TR1 and Tet+ TFH cells using the scMultiome dataset (n = 2,086 genes downregulated in the former vs. the latter; and n = 266 genes upregulated in the former vs. the latter). The text in the paragraph that follows (referring to Figure 1E) focuses exclusively on the genes that had closed chromatin regions during the TFH-to-TR1 conversion, to ascertain whether or not chromatin closure was indeed associated with such gene downregulation. 

We have modified the first sentence in the paragraph referring to Figure 1E to clarify this point for the reader: “Further analyses focusing on the genes that had closed chromatin regions during the TFH-to-TR1 conversion, confirmed…”

eLife Assessment

This study provides important information on pre-existing epigenetic modification in T cell plasticity. The evidence supporting the conclusions is compelling, supported by comprehensive transcriptional and epigenetic analyses. The work will be of interest to immunologists and colleagues studying transcriptional regulation.

Reviewer #1 (Public review):

Summary:

Dr. Santamaria's group previously utilized antigen-specific nanomedicines to induce immune tolerance in treating autoimmune diseases. The success of this therapeutic strategy has been linked to expanded regulatory mechanisms, particularly the role of T-regulatory type-1 (TR1) cells. However, the differentiation program of TR1 cells remained largely unclear. Previous work from the authors suggested that TR1 cells originate from T follicular helper (TFH) cells. In the current study, the authors aimed to investigate the epigenetic mechanisms underlying the transdifferentiation of TFH cells into IL-10-producing TR1 cells. Specifically, they sought to determine whether this process involves extensive chromatin remodeling or is driven by pre-existing epigenetic modifications. Their goal was to understand the transcriptional and epigenetic changes facilitating this transition and to explore the potential therapeutic implications of manipulating this pathway.

The authors successfully demonstrated that the TFH-to-TR1 transdifferentiation process is driven by pre-existing epigenetic modifications rather than extensive new chromatin remodeling. The comprehensive transcriptional and epigenetic analyses provide robust evidence supporting their conclusions.

Strengths:

(1) The study employs a broad range of bulk and single-cell transcriptional and epigenetic tools, including RNA-seq, ATAC-seq, ChIP-seq, and DNA methylation analysis. This comprehensive approach provides a detailed examination of the epigenetic landscape during the TFH-to-TR1 transition.

(2) The use of high-throughput sequencing technologies and sophisticated bioinformatics analyses strengthens the foundation for the conclusions drawn.

(3) The data generated can serve as a valuable resource for the scientific community, offering insights into the epigenetic regulation of T cell plasticity.

(4) The findings have significant implications for developing new therapeutic strategies for autoimmune diseases, making the research highly relevant and impactful.

Weaknesses:

(1) While the study focuses on transcriptional and epigenetic analyses, the authors are currently undertaking efforts to validate these findings functionally. Ongoing research aims to further explore the roles of key transcription factors in the TFH-to-TR1 transition, reflecting the authors' commitment to building on the insights gained from this study.

(2) The identification of key transcription factors and epigenetic marks is a strong foundation for future work. The authors are actively investigating how these factors drive chromatin remodeling, which will enhance the mechanistic understanding of the TFH-to-TR1 process in future studies.

(3) Although the study provides a valuable snapshot of the epigenetic landscape, the authors are pursuing additional research to assess the dynamics of these changes over time. These ongoing efforts will contribute to a deeper understanding of the stability and progression of the observed epigenetic modifications.

Comments on revised version:

The authors have effectively discussed and addressed all previously raised questions. There are no further concerns.

Reviewer #2 (Public review):

Summary:

This study, based on their previous findings that TFH cells can be converted into TR1 cells, conducted a highly detailed and comprehensive epigenetic investigation to answer whether TR1 differentiation from TFH is driven by epigenetic changes. Their evidence indicated that the downregulation of TFH-related genes during the TFH to TR1 transition depends on chromatin closure, while the upregulation of TR1-related genes does not depend on epigenetic changes.

Strengths:

A significant advantage of their approach lies in its detailed and comprehensive assessment of epigenetics. Their analysis of epigenetics covers chromatin open regions, histone modifications, DNA methylation, and using both single-cell and bulk techniques to validate their findings. As for their results, observations from different epigenetic perspectives mutually supported each other, lending greater credibility to their conclusions. This study effectively demonstrates that 1. the TFH-to-TR1 differentiation process is associated with massive closure of OCRs, and 2. the TR1-poised epigenome of TFH cells is a key enabler of this transdifferentiation process. Considering the extensive changes in epigenetic patterns involved in other CD4+ T lineage commitment processes, the similarity between TFH and TR1 in their epigenetics is intriguing.

They performed correlation analysis to answer the association between "pMHC-NP-induced epigenetic change" and "gene expression change in TR1". Also, they have made their raw data publicly available, providing a comprehensive epigenomic database of pMHC-NP induced TR1 cells. This will serve as a valuable reference for future research.

Weaknesses:

A major limitation is that this study heavily relies on a premise from the previous studies performed by the same group on pMHC-NP-induced T cell responses. This significantly limits the relevance of their conclusion to a broader perspective. Specifically, differential OCRs between Tet+ and naïve T cells were limited to only 821, as compared to 10,919 differential OCRs between KLH-TFH and naïve T cells (Fig. 2A), indicating that the precursors and T cell clonotypes that responded to pMHC-NP were extremely limited. I acknowledge that this limitation has been added and discussed in the Discussion section of the revised manuscript.

eLife Assessment

The authors have developed a valuable approach that employs cell-free expression to reconstitute ion channels into giant unilamellar vesicles for biophysical characterisation. The work is convincing and will be of particular interest to those studying ion channels that primarily occur in organelles and are therefore not amenable to be studied by more traditional methods.

Reviewer #1 (Public review):

Summary:

The authors have developed a valuable method based on a fully cell-free system to express a channel protein and integrated it into a membrane vesicle in order to characterize it biophysically. The study presents a useful alternative to study channels that are not amenable to be studied by more traditional methods.

Strengths:

The evidence supporting the claims of the authors is solid and convincing. The method will be of interest to researchers working on ionic channels, allowing to study a wide range of ion channel functions such as those involved in transport, interaction with lipids or pharmacology.

Weaknesses:

The inclusion of a mechanistic interpretation how the channel protein folds into a protomer or a tetramer to become functional into the membrane, would strengthen the study.

Comments on revised version:

In the revised version, the authors did not experimentally addressed how tetrameric or protomeric proteins are actually produced. However, they performed new experiments to assess the amount of tetramers that are being actually formed. They used a size-exclusion chromatography to conclude that the protomers and tetramers species of complexes are formed and assembled.

The authors have addressed most of my minor concerns and have modified or updated the manuscript following my recommendations, so I have no further comments.

Reviewer #2 (Public review):

It is challenging to study the biophysical properties of organelle channels using conventional electrophysiology. The conventional reconstitution methods requires multiple steps and can be contaminated by endogenous ionophores from the host cell lines after purification. To overcome this challenge, in this manuscript, Larmore et al. described a fully synthetic method to assay the functional properties of the TRPP channel family. The TRPP channels are an important organelle ion channel family that natively traffic to primary cilia and ER organelles. The authors utilized cell-free protein expression and reconstitution of the synthetic channel protein into giant unilamellar vesicles (GUV), the single channel properties can be measured using voltage-clamp electrophysiology. Using this innovative method, the authors characterized their membrane integration, orientation, and conductance, comparing the results to those of endogenous channels. The manuscript is well-written and may present broad interest to the ion channel community studying organelle ion channels. Particularly because of the challenges of patching native cilia cells, the functional characterization is highly concentrated in very few labs. This method may provide an alternative approach to investigate other channels resistant to biophysical analysis and pharmacological characterization.

Comments on revised version:

The authors have addressed my concerns. This excellent method manuscript would benefit the study of organelle channels.

Author response:

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

Public reviews:

Reviewer #1 (Public Review):

Summary:

The authors have developed a valuable method based on a fully cell-free system to express a channel protein and integrate it into a membrane vesicle in order to characterize it biophysically. The study presents a useful alternative to study channels that are not amenable to being studied by more traditional methods.

Strengths:

The evidence supporting the claims of the authors is solid and convincing. The method will be of interest to researchers working on ionic channels, allowing them to study a wide range of ion channel functions such as those involved in transport, interaction with lipids, or pharmacology.

Weaknesses:

The inclusion of a mechanistic interpretation of how the channel protein folds into a protomer or a tetramer to become functional in the membrane would strengthen the study.

Work from other labs has described key factors which can improve expression and artificial lipid integration of cellfree derived transmembrane proteins (PMIDs: 35520093, 29625253, 26270393) . However, a significant number of additional experiments would be needed to elucidate the exact biophysical properties governing channel assembly of synthetically derived polycystins. We carried out additional biochemical experiments to address these concerns (see new Figure 1— figure supplement 1 D, E). We used fluorescence-detection size-exclusion chromatography (FSEC) with the goal of understanding how much of the CFE-derived protomers are biochemically folding and assembly into functional tetramers upon incorporation into SUVs. When compared to protein recombinant sources from HEK cells, the production of assembled channels is less than 4% when using the CFE+SUV approach, an estimate based on the oligomer peak fluorescence. In the absence of chaperones found in cells, the assembly of synthetically derived protomers into tetramers is likely intrinsic to the chemical properties of the proteins, and the biophysical principles governing helical membrane protein when inserted into the lipid membrane  (PMID:35133709). We have added our interpretation in lines 111-121.

Reviewer #2 (Public Review):

It is challenging to study the biophysical properties of organelle channels using conventional electrophysiology. The conventional reconstitution methods require multiple steps and can be contaminated by endogenous ionophores from the host cell lines after purification. To overcome this challenge, in this manuscript, Larmore et al. described a fully synthetic method to assay the functional properties of the TRPP channel family. The TRPP channels are an important organelle ion channel family that natively traffic to primary cilia and ER organelles. The authors utilized cell-free protein expression and reconstitution of the synthetic channel protein into giant unilamellar vesicles (GUV), the single channel properties can be measured using voltage-clamp electrophysiology. Using this innovative method, the authors characterized their membrane integration, orientation, and conductance, comparing the results to those of endogenous channels. The manuscript is well-written and may present broad interest to the ion channel community studying organelle ion channels. Particularly because of the challenges of patching native cilia cells, the functional characterization is highly concentrated in very few labs. This method may provide an alternative approach to investigate other channels resistant to biophysical analysis and pharmacological characterization.

Thank you for evaluating our manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) It would be useful to explain how the Polycystin protein is folded under the experimental conditions used. The expression data shown in Figure 1 Supplement 1B show different protein concentrations of protomer or tetramer. However, it is not described how each form is identified and distinguished. It is also important to mention in the manuscript that this method is only applicable to membrane channels that do not require chaperons for its folding and expression into the membrane. How is the tetramer mechanistically conformed? In line 184, it is stated that this method can be leveraged for studying the effects of channel subunit composition. Would this method allow the expression of two different subunit proteins in order to produce a heteromeric channel?

In Figure 1—figure supplement 1B, total fluorescence from the synthesized channel-GFP was measured. Protein concentration was calculated based on the linear regression of the GFP standards. Monomeric protein concentration was reported directly from total fluorescence. Tetrameric protein concentration was calculated by dividing the fluorescence by four, and subsequently calculating the concentration based off the GFP standards. 

This is a good point. Based on your suggestion, we carried out additional biochemical experiments (see new Figure 1— figure supplement 1 D, E). We used fluorescence-detection size-exclusion chromatography (FSEC) with the goal of understanding how much of the CFE-derived protomers are biochemically folding and assembly into functional tetramers upon incorporation into SUVs. As controls we produced recombinant PKD2-GFP and PKD2L1GFP channels as elution time standards and to compare the relative production of tetrameric channels generated when using the two expression systems. The synthetically derived polycystin channels indeed produced tetramers and protomers, which supports feasibility of using this method to assay their functional properties.  When compared to protein recombinant sources from HEK cells, the production of assembled channels is less than 4% when using the CFE+SUV approach, an estimate based on the oligomer peak fluorescence. We speculate that assembly of synthetically derived protomers into tetramers is likely intrinsic to the chemical properties of the proteins, and the biophysical principles governing helical membrane protein when inserted into the lipid membrane (PMID: 35133709). Although an interesting question, a systematic analysis of these channel-lipid interactions is beyond the scope of this eLife Report but can be addressed in future studies. The limitation of using this method to characterize channels which fold and membrane integrate without the aid of molecular chaperones is now stated in lines 201205. In principle, the CFE-GUV method can be deployed to co-express different subunits to produce heteromeric channels. We have modified the text lines 192-197 to be clearer on this point.

(2) The type of plasmid (and promoter) required for this methodology should be mentioned.

Added to the methods (lines 210-211). “PKD2 and PKD2L1 are in pET19b plasmid under T7 promoter.”

(3) Since this paper is methodological, it would be useful to have some information about the stability of the GUVs containing the synthetic channel. In Methods, it is stated that GUV vesicles are used on the same day (line 207). And in line 193 it says that the reactions (?) are placed at 4{degree sign}C for storage.

Restated in lines 226-228: GUVs are electroformed and used for electrophysiology the same day. SUVs with channel incorporated are stored at 4°C for 3 days.

(4) A comment reasoning why the PKD2 protein is more frequently incorporated into the membrane in comparison to PKD2L1 should be included. A brief description of the differences between these two proteins would also be helpful for the reader.

In terms of overall protein production and oligomeric assembly— more PKD2L1 channels are produced compared to PKD2 (see new Figure 1C, and Figure 1— figure supplement 1 D, E). In lines 149-155 we note single channel openings were frequently observed for the high expressing PKD2L1 channels, but this often resulted in patch instability. As a result, GUV patches with lower expressing PKD2-GFP channel were more stable and thus more successfully recorded from. We have revised the text to be clearer on this point.

(5) There are no methods for preparing hippocampal neurons or IMCD cells shown in Figure 4 Supplement 1. Instead, the method of mammalian cultures provided corresponds to HEK 293T cells.

This information has been added to lines 273-284.

(6) Minor:

In Figure 2C, please include the actual % of the Cell488+Surface647+Clear lumen vesicles.

Added

Line 99, 108: Figures 1B and 1C are swapped. Please correct.

Corrected in figure and figure legends.

Line 108: misspelling: effect.

Done

Line 109: check sentence: verb is missing.

Sentence now reads “Minimal changes in fluorescence were detected when a control plasmid (Ctrl) encoding a non- fluorescent protein (dihyrofolate reductase) was used in the reaction.”

Line 145: recoding. Correct.

Recoding changed to recordings

Line 169: "from" is missing (recorded from MCD cilia).

Added

Line 169: In Table 1, the PKD2 K+ conductance magnitudes recorded from IMCD cilia were significantly smaller, not larger as stated, than those assayed using CFE-GUV system. Please correct.

Corrected

Line 180: "of" is missing (adaptation of CFE derived...).

Corrected

Line 182: "to" is missing (generalized to other channels).

Corrected

Line 193: "in" 4ºC, correct at.

Corrected

Line 197: replace "mole" for "mol".

Corrected

Line 207: are used "within the" same day.

Corrected

Line 210: c-terminally. C-should be capital letter.

Corrected

Line 231: n-terminally. N- should be capital letter.

Corrected

Reviewer #2 (Recommendations For The Authors):

The authors validated their method using PKD2 and PKD2L1 channels, demonstrating the potential of this approach. However, a few points merit further clarification or validation:

(1) Stability of the protein vesicles for recording. The authors observed membrane instability during voltage transitions. It would be beneficial to discuss potential solutions to enhance stability.

In lines 197-202, we have added a discussion of potential solutions to enhance stability. CsF in the intracellular saline could be added to stabilize the GUV membranes. CsF is frequently added to stabilize whole cell membranes in HTS planer patch clamp recording. We did not explore this formulation because Cs+ would limit outward polycystin conductance. We also suggest but did not test altering the membrane formulation of GUVs with additional cholesterol to stabilize these recordings.

(2) Validation. Further discussion on how broadly this method can be applied to other channels would strengthen the manuscript.

We have included further discussion on this point in lines 190-206. 

(3) Protein production estimated by a standard GFP absorbance assay. The estimation of protein production using GFP absorption may be affected by improperly folded protein. Additional validation methods could be considered.

C-terminal GFP fluorescence has been widely used in expression systems to designate proper folding of the target protein upstream of the GFP-tag (PMID: 22848743, PMID: 21805523, PMID: 35520093). Nonetheless we have conducted additional experiments designed to estimate the amount of assembled PKD2 and PKD2L1 channels generated using the CFE method. In the new Figure 1— figure supplement 1 D, E, we carried out fluorescencedetection size-exclusion chromatography and compared channel assembly of recombinant and CFE+SUV derived PKD2-GFP and PKD2L1-GFP. Here, we clearly observed tetrameric and protomeric forms of the channels using the synthetic approach, which supports feasibility of using this method to assay their functional properties (see new Figure 1— figure supplement 1 D, E).  When compared to protein recombinant sources from HEK cells, the production of assembled channels is less than 4% when using the CFE+SUV approach, an estimate based on the oligomer peak fluorescence. 

(4) Single channels were observed more frequently from PKD2 incorporated GUVs compared to PKD2L1. Does this just randomly happen or is there a reason behind this difference?

In terms of overall protein production and oligomeric assembly— more PKD2L1 channels are produced compared to PKD2 (Figure 1C, and Figure 1— figure supplement 1 D, E). This is apparent whether the channels are produced recombinantly in cells or when using the cell-free method (Figure 1— figure supplement 1 D, E). In lines 149-155, we note single channel openings were frequently observed but that the high expression of the PKD2L1 often resulted in patch instability. As a result, GUV patches the lower expressing PKD2-GFP channel were more stable and thus more successfully recorded from. As requested, we have included a brief description of the two proteins in lines 76-78. 

(5) Additional validation or clarification for examining the channel orientation may strengthen the manuscript.

We have modified the text to make this point clearer. 

(6) Advantage and limitations. The authors compared the recordings from hippocampal primary cilia membranes, noting differences in conductance magnitudes compared to the GUV method. Further discussing the limitations and advantages of this approach for the biophysical properties of organelle channels would be beneficial.

We have revised the final paragraph to discuss the limitations of this method.

(7) Including experiments that demonstrate ligand-induced activation or inhibition to further validate the current using this method would strengthen the manuscript (optional, not required).

Despite our best attempts, exchange of the external bath to apply inhibitors (Gd3+, La3+) resulted in GUV patch instability. Our plans are to investigate ways to stabilize the high resistance seals to develop pharmacological screening using the CFE+GUV method.

Author response:

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

We would like to thank the reviewers for their interest in our studies. In response to their comments, we have conducted additional experiments and made the necessary revisions to the manuscript. The new studies included to address the reviewers’ comments are shown in Figure 1B, 1F, Figure 2—figure supplement 1, Figure 3, Figure 3—figure supplement 1, Figure 3—figure supplement 2, Figure 3—figure supplement 3, Figure 4E, Figure 4—figure supplement 1, Figure 5, Figure 5—figure supplement 1, Figure 5—figure supplement 2D, and Figure 6. We are grateful for the critiques, which have helped us substantially improve the quality of the manuscript.

Below, we have provided a point-by-point response to the reviewers’ comments.  

Public Reviews:

Reviewer #1 (Public Review):

In this paper, the authors show that disruption of calcineurin, which is encoded by tax-6 in C. elegans, results in increased susceptibility to P. aeruginosa, but extends lifespan. In exploring the mechanisms involved, the authors show that disruption of tax-6 decreases the rate of defecation leading to intestinal accumulation of bacteria and distension of the intestinal lumen. The authors further show that the lifespan extension is dependent on hlh-30, which may be involved in breaking down lipids following deficits in defecation, and nhr-8, whose levels are increased by deficits in defecation. The authors propose a model in which disruption of the defecation motor program is responsible for the effect of calcineurin on pathogen susceptibility and lifespan, but do not exclude the possibility that calcineurin affects these phenotypes independently of defecation.

We thank the reviewer for providing an excellent summary of our work. We have performed additional experiments as suggested by both the reviewers and believe we have thoroughly addressed all the reviewers' concerns.

Reviewer #2 (Public Review):

The manuscript titled "Calcineurin Inhibition Enhances Caenorhabditis elegans Lifespan by Defecation Defects-Mediated Calorie Restriction and Nuclear Hormone Signaling" by Priyanka Das, Alejandro Aballay, and Jogender Singh reveals that inhibiting calcineurin, a conserved protein phosphatase, in C. elegans affects the defecation motor program (DMP), leading to intestinal bloating and increased susceptibility to bacterial infection. This intestinal bloating mimics calorie restriction, ultimately resulting in an enhanced lifespan. The research identifies the involvement of HLH-30 and NHR-8 proteins in this lifespan enhancement, providing new insights into the role of calcineurin in C. elegans DMP and mechanisms for longevity.

The authors present novel findings on the role of calcineurin in regulating the defecation motor program in C. elegans and how its inhibition can lead to lifespan enhancement. The evidence provided is solid with multiple experiments supporting the main claims.

Strengths:

The manuscript's strength lies in the authors' use of genetic and biochemical techniques to investigate the role of calcineurin in regulating the DMP, innate immunity, and lifespan in C. elegans. Moreover, the authors' findings provide a new mechanism for calcineurin inhibitionmediated longevity extension, which could have significant implications for understanding the molecular basis of aging and developing interventions to promote healthy aging.

(1) The study uncovers a new role for calcineurin in the regulation of C. elegans DMP and a potential novel pathway for enhancing lifespan via calorie restriction involving calcineurin, HLH-30, and NHR-8 in C. elegans.

(2) Multiple signaling pathways involved in lifespan enhancement were investigated with fairly strong experimental evidence supporting their claims.

We thank the reviewer for an excellent summary of our work and for highlighting the strengths of the findings.

Weaknesses:

The manuscript's weaknesses include the lack of mechanistic details regarding how calcineurin inhibition leads to defects in the DMP and induces calorie restriction-like effects on lifespan.

The exact site of calcineurin action, i.e., whether in the intestine or enteric muscles (Lee et al., 2005), and the possible molecular mechanisms linking calcineurin inhibition, DMP defects, and lifespan were not adequately explored. Although characterization of the full mechanism is probably beyond the scope of this paper, given the relative simplicity and advantages of using C. elegans as a model organism for this study, some degree of rigor is expected with additional straightforward control experiments as listed below:

The authors state that tax-6 knockdown animals had drastically reduced expulsion events (Figure 2G), leading to irregular DMP (Lines 144-145), but did not describe the nature of DMP irregularity. For example, did the reduced expulsion events still occur with regular intervals but longer cycle lengths? Or was the rhythmicity completely abolished? The former would suggest the intestine clock is still intact, and the latter would indicate that calcineurin is required for the clock to function. Therefore, ethograms of DMP in both wild-type and tax6 mutant animals are warranted to be included in the manuscript. Along the same line, besides the cycle length, the three separable motor steps (aBoc, pBoc, EMC) are easily measurable, with each step indicating where the program goes wrong, hence the site of action, which is precisely the beauty of studying C. elegans DMP. Unfortunately, the authors did not use this opportunity to characterize the exact behavior phenotypes of the tax-6 mutant to guide future investigations. Furthermore, it is interesting that about 64% of tax-6 (p675) animals had normal DMP. The authors attributed this to p675 being a weak allele. It would be informative to further examine tax-6 RNAi as in other experiments or to make a tax-6 null mutant with CRISPR. In addition, in one of the cited papers (Lee et al., 2005), the exact calcineurin loss-of-function strain tax-6(p675) was shown to have normal defecation, including normal EMC, while the gain-of-function mutant of calcineurin tax-6(jh107) had abnormal EMC steps. It wasn't clear from Lee et al., 2005, if the reported "normal defecation" was only referring to the expulsion step or also included the cycle length. Nevertheless, this potential contradiction and calcineurin gain-of-function mutant is highly relevant to the current study and should be further explored as a follow-up to previously reported results. For some of the key experiments, such as tax-6's effects on susceptibility to PA14, DMP, intestinal bloating, and lifespan, additional controls, as the norm of C. elegans studies, including second allele and rescue experiments, would strengthen the authors' claims and conclusions.

We have now included lifespan, survival on P. aeruginosa, and DMP data using an additional knockout allele, tax-6(ok2065). Additionally, we have added ethograms of DMP for both tax-6 RNAi and the tax-6(ok2065) mutant. Our observations indicate that tax-6 inhibition leads to a complete loss of DMP rhythmicity, suggesting that calcineurin is essential for maintaining the DMP clock. While characterizing the DMP, we noticed that expulsion events appeared superficial in the tax-6(ok2065) mutant, with little to no gut content released. Consequently, we examined the movement of gut content and found that both tax-6(ok2065) mutants and tax-6 knockdown animals showed significantly reduced gut content movement. The new findings on the characterization of DMP are presented in Figure 2—figure supplement 1, Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2. The text in the results section reads (lines 160-176): “Next, we investigated whether the reduced number of expulsion events was due to regular intervals with longer cycle lengths or if rhythmicity was entirely disrupted upon tax-6 knockdown. To assess this, we obtained ethograms of the DMP for N2 animals grown on control and tax-6 RNAi. While animals on control RNAi displayed regular cycles of pBoc, aBoc, and EMC, the tax-6 RNAi animals exhibited disrupted rhythmicity (Figure 3A and Figure 3—figure supplement 1). Most tax-6 knockdown animals lacked the pBoc and aBoc steps and had sporadic expulsion events. Isolated pBoc events were occasionally observed, indicating a complete loss of rhythmicity in tax-6 knockdown animals. Ethograms for tax-6(ok2065) animals also showed disrupted rhythmicity (Figure 3B and Figure 3—figure supplement 2). Although the number of expulsion events appeared higher in tax-6(ok2065) animals compared to tax-6 RNAi animals (Figure 3—figure supplement 1 and 2), these expulsion events seemed superficial, releasing little to no gut content. This suggested slow movement of gut content in tax6(ok2065) animals, leading to constipation and intestinal bloating. We examined gut content movement by measuring the clearance of blue dye (erioglaucine disodium salt) from the gut. The clearance was significantly slower in tax-6(ok2065) animals compared to N2 animals (Figure 3C), indicating impaired gut content movement due to the loss of tax-6. Similarly, tax-6 knockdown animals also showed significantly slowed gut content movement (Figure 3D).”

Moreover, we have added a potential reason for the tax-6(p675) contradictory results from Lee et al., 2005 (lines 154-159): “At the 1-day-old adult stage, about 36% of tax-6(p675) animals showed irregular and slowed DMP, while the remainder had regular DMP (Figure 2H), suggesting that tax-6(p675) is a weak allele. The fraction of the animals with irregular DMP appeared to increase with age, indicating that this phenotype might be agedependent. This may also explain why tax-6(p675) animals were reported to have a normal defecation cycle in an earlier study (Lee et al., 2005).”

The second weakness of this manuscript is the data presentation for all survival rate curves. The authors stated that three independent experiments or biological replicates were performed for each group but only showed one "representative" curve for each plot. Without seeing all individual datasets or the averaged data with error bars, there is no way to evaluate the variability and consistency of the survival rate reported in this study.

We now provide all replicates data in the source data files.

Overall, the authors' claims and conclusions are justified by their data, but further experiments are needed to confirm their findings and establish the detailed mechanisms underlying the observed effects of calcineurin inhibition on the DMP, calorie restriction, and lifespan in C. elegans.

We have conducted additional experiments to elucidate the role of calcineurin in the DMP and to investigate the impact of the DMP on calorie restriction and lifespan in C. elegans, as described in the various responses to the reviewers’ comments. 

Recommendations for the authors:

Our specific comments to guide the authors, should they choose to revise the manuscript:

The RNAi experiments in the eat-2 mutant background are difficult to interpret. If these animals are eating fewer bacteria, it is possible that there is also less tax-6 dsRNA being ingested and therefore less tax-6 inactivation. These experiments should be conducted with a tax-6 null allele.

We have included lifespan experiments with the eat-2(ad465);tax-6(ok2065) double mutant, along with the individual single mutant controls, as shown in Figure 4E. These results demonstrate that the eat-2 mutation does not further extend the lifespan of the tax-6(ok2065) mutant. Additionally, we confirmed that the eat-2(ad465) mutants do not exhibit defects in feeding-based RNAi (Figure 4—figure supplement 1).

While aak-2, hlh-30, and nhr-8 mutants may not have an eat phenotype, the negative tax-6 RNAi results should be confirmed with a tax-6 null mutant to obviate the consideration that these background mutations reduce RNAi efficacy.

The genes hlh-30 and nhr-8 are located very close to tax-6 on chromosome IV (https://wormbase.org//#012-34-5), which made it challenging to generate double mutants. However, we tested the RNAi sensitivity of the hlh-30(tm1978) and nhr-8(ok186) mutants and confirmed that they are not defective in RNAi (Figure 5—figure supplement 1). We also found that tax-6 RNAi disrupted the DMP in both hlh-30(tm1978) and nhr-8(ok186) mutants (Figure 5—figure supplement 2). Furthermore, our results show that hlh-30(tm1978) and nhr-8(ok186) animals have increased susceptibility to P. aeruginosa upon tax-6 knockdown (Figure 6A, B), indicating that tax-6 RNAi was effective in these mutants. Since the phenotype in the aak-2 mutant was only partially observed, we did not conduct further experiments with aak-2 mutants.

Reviewer #1 (Recommendations For The Authors):

The low penetrance of defecation cycle defects in tax-6(p675) worms brings into question the role of the defecation deficits in the phenotypes caused by the disruption of tax6. At the same time, the low penetrance provides a golden opportunity to test this. Do tax6(p675) worms with a normal defecation cycle length have extended longevity? Increased susceptibility to bacterial pathogens? Smaller body size? Distended lumen? Decreased fat accumulation? Increased pha-4 and nhr-8 expression? It would be relatively straightforward to measure defecation cycle length in individual tax-6(p675) worms, bin them into normal defecation and slow defecation groups, and then compare the above-mentioned phenotypes.

We appreciate the reviewer's interesting suggestion. However, the DMP defect phenotype in tax-6(p675) worms appears to be age-dependent, with the number of DMPdefective worms increasing as they age. Additionally, we observed that exposure to P. aeruginosa accelerates the onset of DMP defects in tax-6(p675) worms. As a result, tax6(p675) worms are not suitable for the type of experiments the reviewer suggested. Nevertheless, we believe that the additional data using the tax-6(ok2065) mutant, along with the characterization of ethograms of DMP, firmly establishes the role of calcineurin in maintaining a regular DMP in C. elegans.

Another way to dissect specific effects of calcineurin disruption from phenotypes resulting from defecation motor program deficits would be to further characterize other worms with deficits in defecation (flr-1, nhx-2, pbo-1 RNAi). It is mentioned that they have decreased lifespan. Do they also show increased susceptibility to bacterial pathogens? Do they show decreased fat? Is their lifespan dependent on HLH-30 and NHR-8?

We thank the reviewer for this important suggestion. We have now included data with flr-1, nhx-2, and pbo-1 RNAi, which shows that the knockdown of these genes also enhances susceptibility to P. aeruginosa (Figure 3—figure supplement 3G). Knockdown of these genes is already known to reduce fat levels in N2 worms, and we demonstrate that they similarly reduce fat levels in hlh-30(tm1978) and nhr-8(ok186) animals (Figure 5B, C, F, G). Additionally, we found that the increased lifespan observed upon knockdown of these genes (as well as with tax-6 knockdown) is dependent on HLH-30 and NHR-8 (Figure 5A, D).

To place "enhanced susceptibility to pathogen" within the proposed model, it would be important to examine the effect of HLH-30 and NHR-8 disruption on this phenotype. The proposed model suggests that this phenotype is independent of HLH-30 and NHR-8, but this should be tested experimentally. Similarly, it would be important to test the effect of HLH-30 and NHR-8 disruption on defecation cycle length to determine if defecation deficits are upstream or downstream of deficits in the defecation motor program

We show that the knockdown of tax-6 leads to defects in the DMP in hlh30(tm1978) and nhr-8(ok186) animals (Figure 5—figure supplement 2). Moreover, we show that hlh-30(tm1978) and nhr-8(ok186) animals have increased susceptibility to P. aeruginosa upon tax-6 knockdown (Figure 6A, B). These results are described as (lines 279-285): “Given that HLH-30 and NHR-8 are essential for lifespan extension upon calcineurin inhibition, we investigated whether these pathways also influence survival in response to P. aeruginosa infection following calcineurin knockdown. Both hlh-30(tm1978) and nhr-8(ok186) animals showed significantly reduced survival upon tax-6 RNAi (Figure 6A, B). These findings suggested that the reduced survival on P. aeruginosa following calcineurin inhibition is independent of HLH-30 and NHR-8 and is more likely due to increased gut colonization by P. aeruginosa resulting from DMP defects (Figure 6C).”

Is the lifespan of tax-6(p675) increased? This would be important to measure and include in Figure 1.

Indeed, the lifespan of tax-6(p675) mutants is increased. We have included the lifespan of tax-6(p675) and tax-6(ok2065) in Figure 1F.

In Figure 2, disruption of tax-6 appears to result in a clear decrease in body size. To what extent is the decrease in fat/worm in Figure 3 simply a result of the worms being smaller? Perhaps, a measurement of Oil-Red-O intensity PER AREA would be a more appropriate measure.

The ORO intensity values we had shown per animal were already area normalized. We have now indicated this in the Figure Legends.

There are multiple long-lived mutant strains such as clk-1 and isp-1 that have an increased defecation cycle length. To what extent do these worms exhibit phenotypes similar to tax-6 disruption? isp-1 have increased resistance to bacterial pathogens suggesting that defecation motor program deficits are not sufficient to increase susceptibility to bacterial pathogens.

We have now examined the clk-1 and isp-1 mutants and found that these mutants exhibit reduced gut colonization by P. aeruginosa compared to N2 animals. This reduction in colonization may be attributed to the slowed pharyngeal pumping rates observed in these mutants. These findings suggest that the phenotypes associated with a slow DMP versus a disrupted DMP could be significantly different. The manuscript with the new data on these mutants reads (lines 177-192): “We then explored whether the disruption of DMP rhythmicity due to tax-6 knockdown affected P. aeruginosa responses similarly to longer but regular DMP cycles. To do this, we studied P. aeruginosa colonization in clk-1(qm30) and isp1(qm150) mutants, which have regular but extended DMP cycles (Feng et al., 2001; Wong et al., 1995). Interestingly, both clk-1(qm30) and isp-1(qm150) mutants showed significantly reduced intestinal colonization by P. aeruginosa compared to N2 animals (Figure 3—figure supplement 3A-D). This reduced colonization could be attributed to their significantly decreased pharyngeal pumping rates (Wong et al., 1995; Yee et al., 2014), suggesting a lower intake of bacterial food in these mutants. While the survival of clk-1(qm30) animals on P. aeruginosa was comparable to N2 animals (Figure 3—figure supplement 3E), isp1(qm150) animals exhibited significantly improved survival (Figure 3—figure supplement 3F). Conversely, knockdown of flr-1, nhx-2, and pbo-1 in N2 animals resulted in significantly reduced survival on P. aeruginosa compared to control RNAi (Figure 3—figure supplement 3G). Knockdown of these genes causes complete disruption of DMP rhythmicity, increasing gut colonization by P. aeruginosa (Singh and Aballay, 2019a). Overall, these findings demonstrated that calcineurin is crucial for maintaining the DMP ultradian clock, and its inhibition increases susceptibility to P. aeruginosa by disrupting the DMP.”

Line 192. This statement is speculative. There is no evidence that HLH-30 is mediating lipid depletion in these worms.

We have removed this statement. We observed that the knockdown of flr-1, nhx2, and pbo-1 resulted in significant fat depletion in hlh-30(tm1978) animals (Figure 5B, C). Additionally, tax-6 knockdown also caused a small but significant reduction in fat levels in hlh-30(tm1978) animals. This contrasts with our initial submission, possibly due to the increased number of animals included in the analysis. These findings suggest that the increase in lifespan due to DMP defects requires HLH-30, likely through a mechanism independent of HLH-30’s role in fat depletion. We have updated the manuscript text and model (Figure 6C) accordingly.

In Figure S2, tax-6 RNAi appears to have a more detrimental effect in pmk-1 mutants than the other mutants. The authors should comment on this.

We have added the following sentence in the manuscript (lines 123-125): “The knockdown of tax-6 appeared to have a more pronounced effect in pmk-1(km25) mutants than in other mutants, suggesting that inhibition of tax-6 might exacerbate the adverse effects observed in pmk-1(km25) mutants.”

Reviewer #2 (Recommendations For The Authors):

Line 192-193: The statement is confusing and not accurate because HLH-30 did not enhance lifespan with or without calcineurin (Figure 4A and S4A, also in Lapierre 2023). The takeaway should be along the lines of calcineurin inhibition enhancing lifespan through HLH-30 or HLH-30 being required for lifespan enhancement via calcineurin inhibition.

We have removed this statement. We now state (lines 237-239): “Knockdown of tax-6 did not extend the lifespan of hlh-30(tm1978) animals (Figure 5A), indicating that HLH-30 is required for the increased lifespan observed with calcineurin inhibition.”

Line 261: Similar to the point above. Where is the data showing NHR-8 increases lifespan with or without calcineurin?

We have removed this sentence.

Figure 1 legend line 699: animals per condition per replicate >90, but in the Method section Line 317, it says more than 80 animals per condition per replicate. Could be more accurate.

We have now specified in the Methods section that the exact number of animals per condition is provided in the source data files. Since different lifespan curves within a given figure panel had varying numbers of animals, we have indicated the lower boundary for all curves (including the replicates). The precise number of animals for each lifespan experiment is available in the source data files.

Figures 2F and G, "tax-6" should be labeled as "tax-6 RNAi" to be consistent with other figures.

We thank the reviewer for this suggestion and have updated the label to “tax-6 RNAi”.

In summary, we would like to thank the reviewers again for providing constructive critiques. We believe we have fully addressed all the concerns of the reviewers by carrying out several new experiments and modifying the text. The manuscript has undergone substantial revision and has thereby improved significantly. We do hope that the evidence in support of the conclusions is found to be complete in the revised manuscript.

eLife Assessment

This important study reveals insights into how calcineurin influences C. elegans pathogen susceptibility and lifespan through its role in controlling the defecation motor program. The authors provide convincing evidence to support a new mechanism through which calcineurin impacts longevity. This work will be of interest to investigators studying host-pathogen interactions and aging in a number of experimental systems.

Reviewer #1 (Public review):

In this paper, the authors show that disruption of calcineurin, which is encoded by tax-6 in C. elegans, results in increased susceptibility to P. aeruginosa but extends lifespan. In exploring the mechanisms involved, the authors show that disruption of tax-6 decreases the rate of defecation leading to intestinal accumulation of bacteria and distension of the intestinal lumen. The authors further show that the lifespan extension is dependent on hlh-30, which may be involved in breaking down lipids following deficits in defecation, and nhr-8, whose levels are increased by deficits in defecation. The authors propose a model in which disruption of the defecation motor program is responsible for the effect of calcineurin on pathogen susceptibility and lifespan, but do not exclude the possibility that calcineurin affects these phenotypes independently of defecation.

Reviewer #2 (Public review):

The relationships between genes and phenotypes are complex and the impact of deleting or a gene can often have multifaceted and unforeseen consequences. This paper dissected the role of calcineurin, encoded by tax-6, in various phenotypes in C. elegans, including lifespan, pathogen susceptibility, the defecation motor program, and nutrient absorption or calorie restriction, through a series of genetic and behavioral analyses. Many genes in these pathways were tested yielding robust results. Classic epistasis analyses were used to distinguish between genes operating in the same or separate pathways. Researchers in the related fields will be very interested in looking through the data presented in this paper in great detail and benefit from it.

Overall, this paper supports a model in which the increased lifespan and heightened pathogen susceptibility observed following calcineurin inhibition result from the disruptions in the defecation motor program but through distinct pathways. A defective defecation motor program leads to intestine bloating and compromised nutrient absorption. Calorie restriction resulting from poor nutrient absorption affects lifespan, whereas increased colonization in the bloated intestine heightens pathogen susceptibility. The observation that knockdown of several other DMP-related genes also results in increased lifespan and pathogen susceptibility further reinforces the proposed model.

eLife Assessment

This important study presents a high-resolution cryoEM structure of the supercomplex between photosystem I (PSI) and fucoxanthin chlorophyll a/c-binding proteins (FCPs) from the model diatom Thalassiosira pseudonana CCMP1335, revealing subunits, protein:protein interactions and pigments not previously seen in other diatoms or red/green photosynthetic lineages. Combining structural, sequence and phylogenetic analyses, the authors provide convincing evidence of conserved motifs crucial for the binding of FCPs, accompanied by interesting speculation about the mechanisms governing the assembly of PSI-FCP supercomplexes in diatoms and their implications for related PSI-LHC supercomplexes in plants. The findings set the stage for functional experiments that will further advance the fields of photosynthesis, bioenergy, ocean biogeochemistry and evolutionary relationships between photosynthetic organisms.

Reviewer #1 (Public review):

The authors present the cryo-EM structure of PSI-fucoxanthin chlorophyll a/c-binding proteins (FCPs) supercomplex from the diatom Thalassiosira pseudonana CCMP1335 at a global resolution of 2.3 Å. This exceptional resolution allows the authors to construct a near-atomic model of the entire supercomplex and elucidate the molecular details of FCPs arrangement. The high-resolution structure reveals subunits not previously identified in earlier reconstructions and models, as well as sequence analysis of PSI-FCPIs from other diatoms and red algae. Additionally, the authors use their model in conjunction with a phylogenetic analysis to compare and contrast the structural features of the T. pseudonana supercomplex with those of Chaetoceros gracilis, uncovering key structural features that contribute to the efficiency of light energy conversion in diatoms.

The study employs the advanced technique of single particle cryo-electron microscopy to visualize the complex architecture of the PSI supercomplex at near-atomic resolution and analyze the specific roles of FCPs in enhancing photosynthetic performance in diatoms.

Overall, the approach and data are both compelling and of high quality. The paper is well written and will be of wide interest for comprehending the molecular mechanisms of photosynthesis in diatoms. This work provides valuable insights for applications in bioenergy, environmental conservation, plant physiology, and membrane protein structural biology.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

The authors present the cryo-EM structure of of PSI-fucoxanthin chlorophyll a/c-binding proteins (FCPs) supercomplex from the diatom Thalassiosira pseudonana CCMP1335 at a global resolution of 2.3 Å. This exceptional resolution allows the authors to construct a near-atomic model of the entire supercomplex and elucidate the molecular details of FCPs arrangement. The high-resolution structure reveals subunits not previously identified in earlier reconstructions and models, as well as sequence analysis of PSI-FCPIs from other diatoms and red algae. Additionally, the authors use their model in conjunction with a phylogenetic analysis to compare and contrast the structural features of the T. pseudonana supercomplex with those of Chaetoceros gracilis, uncovering key structural features that contribute to the efficiency of light energy conversion in diatoms.

The study employs the advanced technique of single particle cryo-electron microscopy to visualize the complex architecture of the PSI supercomplex at near-atomic resolution and analyze the specific roles of FCPs in enhancing photosynthetic performance in diatoms.

Overall, the approach and data are both compelling and of high quality. The paper is well written and will be of wide interest for comprehending the molecular mechanisms of photosynthesis in diatoms. This work provides valuable insights for applications in bioenergy, environmental conservation, plant physiology, and membrane protein structural biology.

We thank you very much for your highly positive evaluation and comments on our manuscript.

Reviewer #2 (Public Review):

Summary:

This manuscript elucidated the cryo-electron microscopic structure of a PSI supercomplex incorporating fucoxanthin chlorophyll a/c-binding proteins (FCPs), designated as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana CCMP1335. Combining structural, sequence, and phylogenetic analyses, the authors provided solid evidence to reveal the evolutionary conservation of protein motifs crucial for the selective binding of individual FCPI subunits and provided valuable information about the molecular mechanisms governing the assembly and selective binding of FCPIs in diatoms.

Strengths:

The manuscript is well-written and presented clearly as well as consistently. The supplemental figures are also of high quality.

Weaknesses:

Only minor comments (provided in recommendations for authors) to help improve the manuscript.

We thank you very much for your highly positive evaluation and comments on our manuscript.

Reviewer #3 (Public Review):

Summary:

Understanding the structure and function of the photosynthetic machinery is crucial for grasping its mode of action. Photosystem I (PSI) plays a vital role in light-driven electron transfer, which is essential for generating cellular reducing power. A primary strategy to mitigate light and environmental stresses involves incorporating peripheral light-harvesting proteins. Among various lineages, the number of LHCIs and their protein and pigment compositions differ significantly in PSI-LHCI structures. However, it is still unclear how LHCIs recognize their specific binding sites in the PSI core. This study aims to address this question by obtaining a high-resolution structure of the PSI supercomplex, including fucoxanthin chlorophyll a/c-binding proteins (FCPs), referred to as PSI-FCPI, isolated from the diatom Thalassiosira pseudonana. Through structural and sequence analyses, distinct protein-protein interactions are identified at the interfaces between FCPI and PSI subunits, as well as among FCPI subunits themselves.

Strengths:

The primary strength of this work lies in its superb isolation and structural determination, followed by clear discussion and conclusions. However, the interactions among the protein complexes and their relevance in formulating general rules are not definitively established. While efficiency is a crucial aspect, preventing damage is equally important, and currently, we cannot infer this from the provided structures.

Weaknesses:

The interactions among the protein complexes and their relevance in formulating general rules are not definitively established. While efficiency is a crucial aspect, preventing damage is equally important, and currently, we cannot infer this from the provided structures.

We thank you very much for your highly positive evaluation and comments on our manuscript. This study is aimed to decipher the interactions among different protein subunits within the PSI-FCPI supercomplex, from which we wish to draw their relevance in formulating general rules. While we agree that damage is equally important, it is unclear to us what kind of damage you are mentioning, and we consider that this may need to be treated in another publication, as we cannot elucidate everything in one paper.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Line 69: "Diatoms are one of the most important phytoplankton in aquatic environments and contribute to the primary production in the ocean remarkably." Check the sentence, something is missing.

We modified the sentence as follow:

"Diatoms are among the most essential phytoplankton in aquatic environments, playing a crucial role in the global carbon cycle, supporting marine food webs, and contributing significantly to nutrient cycling, thus ensuring the health and sustainability of marine ecosystems"

(2) Supplementary Figure 1B: The SDS-PAGE gel shows multiple bands. Do the authors know the identity of these proteins, or have they considered analyzing the bands using mass spectrometry? The band at ~17 kDa is particularly intense. Could you comment on this? Have you tried running a Native-PAGE gel?

We did not identify protein bands by MS analysis. The protein bands in the PSI-FCPI supercomplex of this diatom have been identified by Ikeda et al. 2013. The protein bands of our sample were similar to those of Ikeda et al. 2013. To explain this, we modified the sentences and cited Ikeda et al. 2013 in the revised manuscript (lines 89-91).

"The PSI-FCPI supercomplexes were purified from the diatom T. pseudonana CCMP1335 and analyzed by biochemical and spectroscopic techniques (Fig. S1). Notably, the protein bands of PSI-FCPI closely resembled those reported in a previous study (31)."

The ~17 kDa protein band appears to be FCPIs, which was identified in Ikeda et al. 2013. We did not perform BN-PAGE of this sample; however, we performed trehalose density gradient centrifugation (Fig. S1A).

(3) Can the authors comment on the position of the FCPI subunits in the PSI supercomplex in diatoms compared to the arrangement of LHCIs in complex with PSI in cyanobacteria, green algae, and angiosperms? This information would be useful to incorporate into the text.

We previously compared the PSI-FCPI structures of the diatom C. gracilis to the PSI-LHCI structures of land plant, green alga, and red alga (Nagao et al., 2020). Also, Xu et al. 2020 compared the C. gracilis PSI-FCPI structure to the PSI-LHCI structures of land plant, green alga, and red alga. The binding sites between FCPIs and LHCIs are conserved to some extent. However, our recent study revealed that no orthologous relationship exists among LHCs bound to PSI between primitive red algae and diatoms (Kato et al., 2024). Consequently, we found that the information obtained from structural comparisons alone is extremely limited. To avoid misinterpretation, this study focused on comparing the structures and amino acid sequences of FCPIs between T. pseudonana and C. gracilis.

(4) Line 104: Despite achieving high resolution, the authors modeled only six lipid densities (the PDB model contains actually 9 lipids, you should correct it in the text). Do you believe this is due to the detergent used for purification? Can you comment on the position, identity, and potential role of the lipids within your model?

There are 6 lipids associated with the PSI core and 3 with FCP, giving rise to a total of 9 lipids. We have described it in our original text (lines 102-104 in the modified manuscript). Additionally, our structure reveals unidentified densities which likely represent lipids; they are modeled as 88 unknown lipids (UNLs). Thus, there are more lipids in the supercomplex. However, we also observed 4 β-DDM molecules (LMT) in the structure, which are used as detergents. Thus, it is possible that some lipids have dissociated and replaced by detergents. Many of the observed lipids are located between subunits, likely contributing to the stabilization of the complex.

(5) Line 111: The global resolution is very high. Why does the unknown protein have such low resolution that it was impossible to model it properly and perform de novo identification from the density map? Is it due to a lower abundance of particles with this subunit bound? Have you tried improving this with 3D classification/ focus refinement /density modification?

The Unknown subunit (UNK) is located peripherally, and its density is significantly lower compared to the neighboring subunits, which may suggest a low abundance. We applied density modification using Topaz for 3D map denoising, but the effect was minimal. As the low abundance of UNK may be the cause, 3D classification and focus refinement also had limited impact.

(6) Figure 2A: It would be useful to show the density map for the subunit together with the model, especially to demonstrate visualization of the long loop.

We added the model and map of Psa29 to Figure S4C in the revised manuscript.

(7) Given the proximity of Psa29 to PsaC, is the protein involved in electron shuttling? If so, could you comment on this? In line 131, you state that Psa29 was not found in other organisms. Can the authors speculate on the potential role of this protein in diatoms?

We have no idea about the function of Psa29 at present. However, Psa29 does not contain any cofactors, indicating no contribution of it to electron transfer reactions. To understand the function of Psa29, a deletion mutant of this gene is required for examining its functional and physiological roles in diatom photosynthesis. To explain this, we added the following sentences to the revised manuscript (lines 129-133):

"However, the functional and physiological roles of Psa29 remain unclear at present. It is evident that Psa29 does not have any pigments, quinones, or metal complexes, suggesting no contribution of Psa29 to electron transfer reactions within PSI. Further mutagenesis studies will be necessary to investigate the role of Psa29 in diatom photosynthesis."

(8) Line 163: "Among the FCPI subunits, only FCPI-1 has BCRs in addition to Fxs and Ddxs (Figure S6A). FCPI-1 is a RedCAP, which belongs to the LHC protein superfamily but is distinct from the LHC protein family (6, 7)." It would be useful if the authors could add the carotenoid model embedded in the cryoEM density map to the figure to show the features that led to modeling BCR instead of other carotenoids. Additionally, it would be helpful to include in the text why RedCAPs differ from LHCIs and their proposed role.

We added the model and map of two BCRs in FCPI-1 (RedCAP) to Figure S4F in the revised manuscript.

Phylogenetic analysis showed that RedCAPs are distinct from the LHC protein family. This has been explained in lines 163-164. Also, the functional and physiological roles of RedCAP remain unclear. To explain this, we added the sentence "; however, the functional and physiological roles of RedCAP remain unclear" to the revised manuscript (lines 164-165).

(9) Line 185: "However, it is unknown (i) whether CgRedCAP is indeed bound to the C. gracilis PSI-FCPI supercomplex and (ii) if a loop structure corresponding to the Q96-T116 loop of TpRedCAP exists in CgRedCAP." Have the authors attempted to model the protein using AlphaFold? If so, are there significant differences? Could you speculate on the absence of RedCAP in C. gracilis? Do you believe it is due to using a different detergent or related to environmental factors?

We did not model CgRedCAP using AlphaFold. Our recent study “Kato et al. 2024” proposed that CgRedCAP binds to the LHCI-1 site in the PSI-FCPI structure based on sequence comparison. There are two types of PSI-FCPI supercomplexes, one having 16 FCPIs and the other having 24 FCPs, from C. gracilis. The different antenna sizes may depend on the growth conditions of C. gracilis (Nagao et al. 2020). These explanations were already described in the manuscript (lines 243-246).

(10) Line 193: Figure 8 is mentioned before Figures 4-7.

We are sorry for the mistake of Figure number. Figure 8 is Supplementary Figure 8, so that we modified Fig. S8B in the revised manuscript.

(11) Line 223: FCPI-4 interacts only with FCPI-5, primarily through the interaction of Y196/4 with the FCPI-5 backbone. Is this interaction facilitated by other factors such as lipids, carotenoids, or other ligands? Also, FCPI-4 occupies a peculiar position compared to other LHCIs proteins (it is peripheral to FCPI-4 and FCPI-5). Do you believe this could be due to a transient interaction with the complex? Could the presence of this protein be related to the growth conditions experienced by the plant? Are there any literature reports on environmental conditions influencing FCPI arrangements? Including this information in the text would be interesting.

Y196/4 interacts with only backbones by hydrogen-bond interactions; therefore, other cofactors do not contribute to the interactions.

We do not believe that the interaction of FCPI-4 is transient; rather, this binding appears to be stable within the complex. Given that the PSI-FCPI supercomplexes were isolated by anion exchange chromatography, FCPI-4 and FCPI-5 are tightly associated within this complex. However, it is important to note that the expression of diatom FCPI proteins can indeed vary depending on growth conditions, as highlighted in our previous study (Nagao et al., 2020). While the peculiar position of FCPI-4 may not be directly related to transient interactions, environmental conditions could still influence the overall arrangement and expression levels of FCPIs. This information has already been described in the manuscript (lines 243-246).

(12) Given the high resolution of your map, the overall model quality does not seem to match the map quality. Specifically, the clash score (10) and sidechain outliers (3%) are elevated. Could you comment on this? Do you believe it is related to the high number of ligands?

Our structure contains a total of 295 ligands, including cofactors, detergents, and unknown lipids. We believe the high clash score and number of sidechain outliers are due to the large number of ligands present.

(13) Supplementary Figure 2: You should show the 3D classes that were discarded.

According to your comment, we added the 3D classes that were discarded and the sentence "Red boxes highlight selected particles from each 3D classification." to Figure S2 and its legend in the revised manuscript.

(14) Which masks were used for refinement? How were they generated, and which parameters were chosen? This information should be added to the Materials and Methods section. You should show the masks used during classification, for example.

We used a 240 Å spherical mask for refinement and classification, without applying any reference mask as input. To explain this, we added the corresponding sentence to Methods in the revised manuscript (lines 347-348) as follow:

"A 240-Å spherical mask was used during the 3D classification and refinement processes."

(15) Were any extra proteins detected in the early stages of the cryoEM analysis (i.e., 2D classification) that were discarded? Could you visualize the superior oligomeric states of the supercomplex?

In the single-particle analysis, no larger particles than the analyzed complex were detected. The results of 2D classification using a sufficiently large spherical mask with a diameter of 320 Å are shown below.

Author response image 1.

(16) Have you tried using cryoSPARC for data analysis? If so, could you comment on that?

We did not use cryoSPARC for data analysis.

Reviewer #2 (Recommendations For The Authors):

I have some minor comments below to help improve the manuscript. The line numbers below refer to those in the Word version of the manuscript.

(1) Figure 1 legend, line 559, "membrane normal"? Panel A and B, structures with the same colors, do they refer to the closely related or interacted parts? For example, the red color for FCP1-1 in A and PsaA in B. If not, the authors may want to clarify it.

The term 'membrane normal' refers to the direction perpendicular to the surface of a membrane. It is a concept frequently used in physics and biology to describe the orientation relative to the membrane's plane.

We do not refer to either the closely related or interacted parts used in Figure 1. According to your comments, the colors of subunits were revised in the revised manuscript.

(2) Line 109-117. "Psa28 is a novel subunit found in the C. gracilis PSI-FCPI structure, and its name follows the nomenclature as suggested previously (31).... After psaZ, the newly identified genes should be named psa27, psa28, etc., and the corresponding proteins are called Psa27, Psa28, etc... Psa28 was also named PsaR in the PSI-FCPI structure of C. gracilis (16)". It is confusing. Was Psa28 named twice, PsaR and Psa28? It would be helpful to add a simple explanation here.

According to your comment, we modified the sentence as follow (lines 117-118):

" However, Xu et al. named the subunit as PsaR in the PSI-FCPI structure of C. gracilis "

(3) Line 134, "One of the Car molecules in PsaJ was identified as ZXT103 in the T. pseudonana PSI-FCPI structure but it is BCR112 in the C. gracilis PSI-FCPI structure (15)". Figure S4D mentioned BCR863 but did not mention BCR112. Figure S4C, D, it may need better explanations of the colors and labels, and indicate which parts are from T. pseudonana or C. gracilis.

BCR112 was misnumbered; the correct number is BCR103. In response to your comments, we revised Figure S4C and D by labeling the characteristic pigments in the revised manuscript.

(4) Figure S7, although mentioned in the legend, it would be helpful to label interaction pairs on the figure directly with corresponding colours.

According to your comments, we modified the Figure and legends in the revised manuscript.

(5) Figure 3E, it is better to avoid red/green colours in one figure as some readers may be colour-blind. It would also be helpful to label each FCPI with the same colour as its structure on the figure directly.

According to your comments, we modified Figure 3E in the revised manuscript.

(6) Line 185, "structures similar to the Q96-T116 loop in TpRedCAP found in the present study (Figure 8B).". The authors refer to Figure S8B? I have the same comment for line 186, Figure 8C.

We are sorry for the mistake of Figure number. Figure 8 is Supplementary Figure 8, so we modified it as Fig. S8B in the revised manuscript.

(7) Line 270, "TpLhcq10 cannot bind at the FCPI-2 site". Why not use FCPI-3 for TpLhcq10?

This means that the gene product of TpLhcq10 binds at the FCPI-3 site but not at the other sites such as FCPI-2. To avoid misreading, we modified the sentence as follows:

"TpLhcq10 binds specifically at the FCPI-3 site but not at the other sites such as FCPI-2" (lines 278-279)

Reviewer #3 (Recommendations For The Authors):

I have no technical or conceptual suggestions at the current stage.

Thank you.

It feels like a William Playfair moment - the idea that numbers can be represented in graphs, charts - can now be applied to anything else. We're still imagining the forms; network/knowledge graphs are trendy (to what end though) - what else?

Information in various formats - an essay, a map, a chart, number equations, a music score, a receipt - are externalisation of some thought process, to some end.

Verene's helpful definition of the Humanities.

Author response:

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

Recommendations for the authors:

- The authors should think about revising the terminology used to describe electrophysiological data in zebrafish (Fig.5): "posterior" hair cells in a neuromast are sensitive to posterior-to-anterior flow, which is currently termed "anterior". This is confusing because when "posterior" or "anterior" is used, for instance in the labels of the figure, one may get confused about whether this applies to hair-cell position or directionality of the stimulus. It would help to always use clearer terminology for the stimulus (e.g. posterior-to-anterior (P-to-A) as in Kindig 2023, or "from the tail"). Also, the authors may want to clarify what we should see in Fig.5 demonstrating that posterior hair cells, with reversed hair-bundle polarity, actually evince transduction of similar magnitude as anterior hair cells, with normal polarity of their hair bundles. 

This nomenclature can indeed be confusing. Per the reviewers request we have changed the terminology to always refer to the direction of flow sensed by the hair cells. For example, HCs that respond to posterior-directed flow or anterior-directed flow. We now denote these HCs as (A to P) and (P to A), respectively in the Figure for clarity. We have modified Figure 5, the Figure 5 legend and Results (starting line 339) to reflect these changes.

In addition, in our results we now provide more context when comparing the response magnitude of the anterior-sensing hair cells in gpr156 mutants to the response magnitude of the two diVerent orientations of hair cells in controls.

- Also, does it make sense that there is no defect in MET for mouse otolith organs with deleted GPR156, whereas there is a diVerence in the zebrafish lateral line? It would help motivate the study on mechanoelectrical transduction (see comment of Reviewer 1 below). 

We previously discussed this point and recognized that subtle eVects remain possible in mouse (previously Discussion line 614). We have now  modified the text in the Discussion to better emphasize this point (new line 627). The Eatock lab is currently working on developing calcium imaging in the mouse utricle to revisit this question in a future study. "Subtle e)ects remain possible, however, given the variance in single-cell electrophysiological data from both control and mutant mice.  Nevertheless, current results are consistent with normal HC function in the Gpr156 mouse mutant, a prerequisite to interrogate how non-reversed HCs a)ects vestibular behavior."

To help motivate transduction studies starting in the second Result paragraph, we added a transition at Line 205 that was indeed lacking:

"Gpr156 inactivation could be a powerful model to specifically ask how HC reversal contributes to vestibular function. However, GPR156 may have other confounding roles in HCs besides regulating their orientation, similar to EMX2, which impacts mechanotransduction in zebrafish HCs (Kindig et al., 2023) and a)erent innervation  in mouse and zebrafish HCs (Ji et al., 2022; Ji et al., 2018)."

(1) One overarching objective of this study was to use the Gpr156 KO model to discover how polarity reversal informs vestibular function (Introduction, overall summary in the last paragraph) . Pairing behavioral defects with hair cell orientation is only possible if hair cell transduction is normal, which had to be tested.

(2) The notion that experiments that produced negative results are unecessary and are not properly motivated can only apply in retrospect. At early stages we performed electrophysiology because we did not know whether transduction would be normal in absence of GPR156. We also did not know whether innervation would be normal. The fact that both appear normal makes Gpr156 KO a better model to address the importance of orientation reversal (conclusion of the Discussion line 705).

See also reply to Reviewer #1 below.

Reviewer #1 (Recommendations For The Authors): 

Fig1, panel B appears to show diVerent focal planes for Gpr156del/+ and Gpr156del/del. 

Figure 1B had control and mutant panels at slightly diVerent focal planes indeed. We swapped the right (mutant) panel image and adjusted intensities in the control image to match adjustments of the new mutant image.  

Given that this work is largely about polarity and connectivity to neurons, I do not understand the need to assess mechanosensitivity in Gpr156 mutants. Please explain in the text, as follows: "After establishing normal numbers and types of mouse vestibular HCs, we assessed whether HCs respond normally to hair bundle deflections in the absence of GPR156." We did this because... 

Please see reply above in 'Recommendations for the authors' for comment about the need to assess mechanosensitivity. We agree that this transition was lacking, and we added an explanation as recommended:

"Gpr156 inactivation could be a powerful model to specifically ask how HC reversal contributes to vestibular function. However, GPR156 may have other confounding roles in HCs besides regulating their orientation, similar to EMX2, which impacts mechanotransduction in zebrafish HCs (Kindig et al., 2023) and a)erent innervation  in mouse and zebrafish HCs (Ji et al., 2022; Ji et al., 2018)."

Anyway, the data in Figures 2, 3 and 4 seems somewhat superfluous to the main message of the paper. 

Please see reply above in 'Recommendations for the authors'. This data may appear superfluous in retrospect but we could not claim that behavioral changes in Gpr156 mutants reflect the role of the line of polarity reversal if, for example, hair cell transduction was abnormal. We had to perform experiments to figure this out. We were further motivated as data began to emerge from the zebrafish lateral line that showed eVects on HC transduction. Although we did not get positive results on this question in the mouse, we think the diVerence between models should be included as a significant part of the narrative.