DOI: 10.1016/j.isci.2024.109818

Resource: (Abcam Cat# ab8226, RRID:AB_306371)

Curator: @scibot

SciCrunch record: RRID:AB_306371

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (Cell Signaling Technology Cat# 12640, RRID:AB_2629499)

Curator: @scibot

SciCrunch record: RRID:AB_2629499

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (Cell Signaling Technology Cat# 9145, RRID:AB_2491009)

Curator: @scibot

SciCrunch record: RRID:AB_2491009

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (Thermo Fisher Scientific Cat# 701283, RRID:AB_2532457)

Curator: @scibot

SciCrunch record: RRID:AB_2532457

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 103204, RRID:AB_312989)

Curator: @scibot

SciCrunch record: RRID:AB_312989

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 100704, RRID:AB_312743)

Curator: @scibot

SciCrunch record: RRID:AB_312743

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 102004, RRID:AB_312853)

Curator: @scibot

SciCrunch record: RRID:AB_312853

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 103522, RRID:AB_2566366)

Curator: @scibot

SciCrunch record: RRID:AB_2566366

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 101204, RRID:AB_312787)

Curator: @scibot

SciCrunch record: RRID:AB_312787

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 117304, RRID:AB_313773)

Curator: @scibot

SciCrunch record: RRID:AB_313773

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 100516, RRID:AB_312719)

Curator: @scibot

SciCrunch record: RRID:AB_312719

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 104512, RRID:AB_493564)

Curator: @scibot

SciCrunch record: RRID:AB_493564

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 124608, RRID:AB_1134096)

Curator: @scibot

SciCrunch record: RRID:AB_1134096

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 107626, RRID:AB_2191071)

Curator: @scibot

SciCrunch record: RRID:AB_2191071

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 105014, RRID:AB_439783)

Curator: @scibot

SciCrunch record: RRID:AB_439783

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 104706, RRID:AB_313127)

Curator: @scibot

SciCrunch record: RRID:AB_313127

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 126406, RRID:AB_1089113)

Curator: @scibot

SciCrunch record: RRID:AB_1089113

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 505810, RRID:AB_315404)

Curator: @scibot

SciCrunch record: RRID:AB_315404

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 506940, RRID:AB_2565781)

Curator: @scibot

SciCrunch record: RRID:AB_2565781

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 504303, RRID:AB_315327)

Curator: @scibot

SciCrunch record: RRID:AB_315327

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 123116, RRID:AB_893481)

Curator: @scibot

SciCrunch record: RRID:AB_893481

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 504118, RRID:AB_10898116)

Curator: @scibot

SciCrunch record: RRID:AB_10898116

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 127624, RRID:AB_10640819)

Curator: @scibot

SciCrunch record: RRID:AB_10640819

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 155508, RRID:AB_2750237)

Curator: @scibot

SciCrunch record: RRID:AB_2750237

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 101216, RRID:AB_312799)

Curator: @scibot

SciCrunch record: RRID:AB_312799

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 117308, RRID:AB_313777)

Curator: @scibot

SciCrunch record: RRID:AB_313777

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (BioLegend Cat# 103132, RRID:AB_893340)

Curator: @scibot

SciCrunch record: RRID:AB_893340

---

What is this?

DOI: 10.1016/j.isci.2024.109818

Resource: (Thermo Fisher Scientific Cat# 25-5773-82, RRID:AB_891552)

Curator: @scibot

SciCrunch record: RRID:AB_891552

---

What is this?

DOI: 10.1126/scitranslmed.adl4317

Resource: (ATCC Cat# CRL-7924, RRID:CVCL_0058)

Curator: @scibot

SciCrunch record: RRID:CVCL_0058

---

What is this?

DOI: 10.1126/scitranslmed.adl4317

Resource: (BCRC Cat# 60005, RRID:CVCL_0030)

Curator: @scibot

SciCrunch record: RRID:CVCL_0030

---

What is this?

DOI: 10.1021/jacs.3c12048

Resource: University of Pittsburgh Center for Research Computing Core Facility (RRID:SCR_022735)

Curator: @scibot

SciCrunch record: RRID:SCR_022735

---

What is this?

DOI: 10.1021/jacs.3c11511

Resource: University of California at Santa Cruz Biomolecular Cryo Electron Microscopy Core Facility (RRID:SCR_021755)

Curator: @scibot

SciCrunch record: RRID:SCR_021755

---

What is this?

DOI: 10.1126/sciadv.adj6380

Resource: Massachusetts Institute of Technology Swanson Biotechnology Center Nanotechnology Materials Core Facility (RRID:SCR_018674)

Curator: @scibot

SciCrunch record: RRID:SCR_018674

---

What is this?

DOI: 10.1088/1741-2552/ab603f

Resource: RRID:Addgene_100837

Curator: @scibot

SciCrunch record: RRID:Addgene_100837

---

What is this?

DOI: 10.1101/2024.02.15.580426

Resource: (Rockland Cat# 600-401-379, RRID:AB_2209751)

Curator: @anisehay

SciCrunch record: RRID:AB_2209751

---

What is this?

DOI: 10.21203/rs.3.rs-4166090/v1

Resource: ImageJ (RRID:SCR_003070)

Curator: @evieth

SciCrunch record: RRID:SCR_003070

---

What is this?

DOI: 10.1101/2024.01.31.578330

Resource: Fiji (RRID:SCR_002285)

Curator: @anisehay

SciCrunch record: RRID:SCR_002285

---

What is this?

DOI: 10.1101/2024.01.31.578330

Resource: (WB Cat# WBStrain00041971,RRID:WB-STRAIN:WBStrain00041971)

Curator: @anisehay

SciCrunch record: RRID:WB-STRAIN:WBStrain00041971

---

What is this?

DOI: 10.1101/2024.01.31.578239

Resource: (IMSR Cat# JAX_000664,RRID:IMSR_JAX:000664)

Curator: @anisehay

SciCrunch record: RRID:IMSR_JAX:000664

---

What is this?

DOI: 10.1016/j.isci.2024.109789

Resource: (WB Cat# WBStrain00000001,RRID:WB-STRAIN:WBStrain00000001)

Curator: @AniH

SciCrunch record: RRID:WB-STRAIN:WBStrain00000001

---

What is this?

DOI: 10.1016/j.chembiol.2024.04.003

Resource: MATLAB (RRID:SCR_001622)

Curator: @abever99

SciCrunch record: RRID:SCR_001622

---

What is this?

DOI: 10.1016/j.jns.2024.123041

Resource: RRID:Addgene_126463

Curator: @abever99

SciCrunch record: RRID:Addgene_126463

---

What is this?

DOI: 10.1016/j.isci.2024.109798

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @AniH

SciCrunch record: RRID:SCR_002798

---

What is this?

El tipo de mensaje es keyword porque son aquellos mensajes que consisten en uno o más nombres de argumentos precedidos por dos puntos (:). Su composición parte de:

Un receptor (objeto), el mensaje (método a utilizar) y el argumento que son los valores que se pasan al método para que pueda realizar la tarea. En los mensajes keyword, los argumentos están precedidos por dos puntos (:) y separados por comas (,).

Para este caso: * El objeto es: sessions. * El mensaje es: to * y el argumento pasado por el método to es el 11, Esto indica que la secuencia de números enteros debe ir desde 1 hasta 11 inclusive.

Para este ejercicio el tipo de mensaje es binario porque se compone de tres partes: receptor (objeto), selector (mensaje) y argumento.

1. Objeto: es el receptor del mensaje. En este caso, el objeto es el número 27.
2. Mensaje: es la acción que se le está enviando al objeto. Para este ejemplo, el mensaje es la operación de multiplicación representada por el selector ( * ), que indica que se debe multiplicar el objeto con el argumento.
3. Argumento: es el valor que se pasa al mensaje o con el que se realizar la operación. Para este caso, el argumento es el número 23.

En concusión, devuelve como resultado.

Es un mensaje tipo keyword

• El objeto es: memories
• El mensaje es: do
• El argumento del mensaje es: do es el bloque proporcionado entre corchetes [:doc | .... ] aquí el iterador es el do, el cual se utiliza para recorrer cada elemento de la colección o objeto memories y ejecutar el bloque proporcionado para cada uno de ellos.

Resultado devuelto al ejecutar todo el bloque:

Este mensaje es tipo Keyword y dentro de este el

• Objeto (receptor): es memories el cual invoca el método.
• Mensaje (selector): es doWithIndex: Este es un mensaje de Pharo que se utiliza para iterar sobre una colección y proporciona tanto el elemento actual como su índice en cada iteración.
• Argumento: es el mensaje doWithIndex el cual es un bloque de código que define la acción que se realizará en cada elemento de la colección. En este caso, el bloque es [:item :index | ........ ], es decir, todo lo que encierran los corchetes y recibe dos parámetros: item, que representa cada elemento de la colección, y index, que representa el índice del elemento en la colección.

Dentro del bloque: * El objeto: item es el elemento actual de la colección memories. * El mensaje enviado: es metadata, que es un mensaje enviado al objeto item. Este mensaje obtiene los metadatos asociados con el elemento actual. * El argumento del mensaje at:put: es una clave, en este caso 'title', y el nuevo valor 'Unidades semanticas, sesión ', que se asigna a la clave 'title' en los metadatos del elemento actual.

Al ejecutar todo el código se devuelve el siguiente resultado:

Este ejemplo en Pharo se refiere a la manipulación de colecciones y al uso de bloques en combinación con mensajes Keyword.

Ahora bien, a continuación se explica las dos partes del ejercicio: En mensaje en keyword es: 1. Objeto: sessions 2. Mensaje: collect<br /> 3. Argumento: [:session |

Dentro del bloque el código funciona así: 1. Objeto: para este se crea un nuevo objeto de tipo HedgeDoc utilizando el mensaje new. Este objeto se asigna a la variable doc. 2. Ahora el mensajes new envían varios mensajes al objeto doc: tales como: * url: Configura la URL del objeto doc. Toma como argumento la concatenación de commonAddress (una dirección común) y la representación de cadena de session. * Ahora utiliza retrieveContents: el cual es un mensaje que permite recuperar el contenido de la URL configurada en el objeto doc.

En general, el código utiliza el iterador collect: el cual controla la iteración sobre la colección sessions y aplica el bloque definido [:session | a cada elemento de la colección.

Al ejecutar todo este código se obtiene el siguiente resultado:

A picture of a census document from 1850, showing that the ratio of men to women in the United States 2 years following the artifact. By stating that these are the sentiments shared by HALF of the population of the country, Stanton makes a bold statement about the numbers behind her fight, as well as the injustice of the way things had been going for women for decades prior.

Wesleyan College, Middletown, Connecticut The first college to open it's doors to female students in 1836

Consider the implications being made beyond the actual statements being made within the artifact * Stanton crosses a line of distinction here to go so far as stating that men haven't only limited women politically and economically, but they have dehumanized them as well. By limiting women in the world of employment and refusing to allow them the freedom to explore, men effectively hindered women's' ability to grow, develop, think critically, etc. Stanton asserts that halting the progress of women's success and achievements within society is an agenda that has been longstanding and will continue to go on, if it is not dismantled by the efforts of the very women that it affects.

Consider the author/speaker of the artifact and their role/position on the subject matter. Does the person satisfy any aspect of ethos? * Elizabeth Cady Stanton was chosen to be the speaker for the Declaration of Sentiments primarily because of the leadership role she took on throughout the movement. She was one of the principal organizers of the convention along with Lucretia Mott and Susan B. Anthony. Stanton was solidified as one of the prominent figures of the Women's Right's Movement early on. * Stanton employs ethos by being the speaker for the artifact, as at this point, she is well known for her activism and support for Women's rights. She later does justice upon her ethos by presenting well though out, concise, and harrowing statements about the U.S. government's lack of consideration towards the female population.

Consider the pathos employed by the rhetor throughout the artifact and the contributions it makes to the overall impact * Stanton has just consecutively listed the grievances of the American women against the American government, evoking strong emotions with her choices of oppressive keywords including "denied" and "withheld." In doing so, she appeals to her female audience and unites their struggles together in one powerful voice demanding change.

Consider the economic climate of the time period your artifact takes place in * Until 1900, women in all states of America did not have protection over their wages, which meant their father or husband had ownership over their earned wages. Therefore, property is sold, bought and exchanged, and women are entirely excluded from any economic aspect of society. With that, women have been excluded economically by denying them the right to their money, politically by denying them the right to their voting opinions, and socially by denying them the respect of fellow human beings and degrading them on the sole basis of their gender.

Consider the social climate of the time period that the artifact takes place in. * In 18th and 19th century America, marriages were simple and private, usually involving the parties being married, their immediate family, and a Priest. Before marriage, a young woman was considered to be the property of her father. Following marriage, she became the property of her husband. * While the concept of a father transferring ownership of his daughter to her husband sounds traditional and "old cultured," think about wedding traditions that are still widely practiced today. The father, walking his daughter down the aisle, the removal of the garter - and consider what these practices reassert about how our society views and treats women.

A political cartoon that portrays the trivial nature of voting in early America - Captioned: "The Early Days of Voting in America: A Lot of White Men Drinking in Taverns."

With Feminist Critique Theory, identify the gendered language and the contributions made to the artifact overall * Stanton states "sufferance of women" as she appeals to a crowd of primarily white women, along with men. Jefferson wrote "sufferance of these colonies" as he appealed only to white, land owning men.

Identify the rhetorical choices made by the author and identify how they contribute to the artifact. * By echoing Thomas Jefferson's voice in "The Declaration of Independence," Stanton strategically aligns the struggles of the Women's Rights Movement with the foundational democratic principles of the United States Government. Not only does this help emphasize contradictions between the ideals of the nation and the lack of Women's rights, it underscores the legitimacy of the movement as an extension of American values. * With Feminist Critique Theory, identify the choices of gendered language. Stanton chooses to state "all men and women" rather than "all men" as Thomas Jefferson did. In doing so, Stanton deconstructs the idea of male superiority and asserts equality for men and women.

Consider the structure of the artifact and how it contributes to it's rhetorical success * Stanton follows in suit of "The Declaration of Independence," which aids her in aligning women's fights for rights with the United States' fight for independence from Great Britain. * With Feminist Critique Theory, examine the choice of words that follow: compelled, withheld, deprived etc., through which Stanton demands amendments to the grievances of women.

Consider the audience the artifact was presented to, and how it affects the purpose/goal of the artifact, how it may have been received. * The artifact was presented at the Seneca Falls Convention which had a primarily female audience and was made up of individuals who were either fighting for or had an interest in fighting for Women's rights. Frederick Douglass, a frontrunner of the abolitionist movement was in attendance as well as activists such as Lucretia Mott and Susan B. Anthony. We can assume that the sentiments and feelings evoked in the artifact were relatable to the audience and received with passion and power towards coming together to fight for the civil liberties of women. * With Feminist Critique Theory, analyzing the intersectionality's of the audience such as race and gender emphasizes the interconnected nature of gender with other social identities.

Es un mensaje tipo keyword

• El objeto es: memories
• El mensaje es: do
• El argumento del mensaje do es el bloque proporcionado entre corchetes [:doc | .... ] aquí el iterador es el do, el cual se utiliza para recorrer cada elemento de la colección o objeto memories y ejecutar el bloque proporcionado para cada uno de ellos.

Resultado devuelto al ejecutar todo el bloque:

Este es un tipo de mensaje binario en donde

Objeto:

• El objeto principal en este ejemplo es FileLocator, que es una clase en Pharo utilizada para ubicar archivos en el sistema de archivos.

Mensaje:

• El mensaje principal enviado es /, que es un mensaje binario. Este mensaje se utiliza para combinar dos rutas de archivos o directorios.

Argumento:

• El argumento del mensaje / es la cadena 'USemanticas\leidy-palma\Wiki\es\sesiones', que representa la ruta relativa de un directorio en el sistema de archivos.

Este ejercicio devuelve como resultado:

Este mensaje es tipo Keyword y dentro de este el

• Objeto (receptor): es memories el cual invoca el método.
• Mensaje (selector) es doWithIndex: Este es un mensaje de Pharo que se utiliza para iterar sobre una colección y proporciona tanto el elemento actual como su índice en cada iteración.
• Argumento: Es el mensaje doWithIndex el cual es un bloque de código que define la acción que se realizará en cada elemento de la colección. En este caso, el bloque es [:item :index | ........ ], es decir, todo lo que encierran los corchetes y recibe dos parámetros: item, que representa cada elemento de la colección, y index, que representa el índice del elemento en la colección.

Dentro del bloque: * El objeto item es el elemento actual de la colección memories. * El mensaje enviado es metadata, que es un mensaje enviado al objeto item. Este mensaje obtiene los metadatos asociados con el elemento actual. * El argumento del mensaje at:put: es una clave, en este caso 'title', y el nuevo valor 'Unidades semanticas, sesión ', que se asigna a la clave 'title' en los metadatos del elemento actual.

Al ejecutar todo el código se devuelve el siguiente resultado:

Este ejemplo en Pharo se refiere a la manipulación de colecciones y al uso de bloques en combinación con mensajes Keyword.

Ahora bien, a continuación se explica las dos partes del ejercicio: En mensaje en keyword es: 1. Objeto es: sessions 2. Mensaje es: collect<br /> 3. Argumento: [:session |

Dentro del bloque el código funciona así: 1. Objeto: para este se crea un nuevo objeto de tipo HedgeDoc utilizando el mensaje new. Este objeto se asigna a la variable doc. 2. Ahora el mensajes new envían varios mensajes al objeto doc: tales como: * url: Configura la URL del objeto doc. Toma como argumento la concatenación de commonAddress (una dirección común) y la representación de cadena de session. * Ahora utiliza retrieveContents: el cual es un mensaje que permite recuperar el contenido de la URL configurada en el objeto doc.

En general, el código utiliza el iterador collect: el cual controla la iteración sobre la colección sessions y aplica el bloque definido [:session | a cada elemento de la colección.

Al ejecutar todo este código se obtiene el siguiente resultado:

El tipo de mensaje es keyword porque son aquellos mensajes que consisten en uno o más nombres de argumentos precedidos por dos puntos (:). Su composición parte de:

Un receptor (objeto), el mensaje (método a utilizar) y el argumento que son los valores que se pasan al método para que pueda realizar la tarea. En los mensajes keyword, los argumentos están precedidos por dos puntos (:) y separados por comas (,).

Para este caso: * El objeto es sessions. * El mensaje es to * y el argumento pasado por el método to es el 11, Esto indica que la secuencia de números enteros debe ir desde 1 hasta 11 inclusive.

Para este caso es un tipo de mensaje unario en donde

• El objeto es 'https://docutopia.sustrato.red/semanticas:24A', siendo esta un literal de cadena (string).

• Seguidamente, el mensaje es la variable commonAddress que al ejecutar esta acción devuelve como resultado

看来我得去看看内核啊

入門の範囲を超えているかもしれませんが、このキャッシュをGitHubActionsなどのCIで保存できるかを知りたいです。

スクロール表示自体の長さは固定でしょうか？

MyST Notebookを単体で実行する方法があれば知りたいです。

JupytextにはMyST形式からNotebookにする方法だけではなくPythonスクリプトからNotebookにする方法もあり個人的にはそちらがおすすめです。

「コンテンツ」と「コンテンツファイル」で表記ゆれがあります。

入門であればreStructuredTextの説明は混乱するので省いても良いかと思います。

ディレクティブとロールについてここで詳しく説明してほしいです。

コンテンツの種類の説明が目次の前にあるとわかりやすいと思います。

設定しなくてもビルドはできるのか知りたいです。ビルド速度に影響があるか否かの情報もあると嬉しいです。

「に設定しない」という表現のほうがわかりやすいです。

ディレクティブの説明が必要だと思います。Sphinxのディレクティブについても言及があると背景がわかりやすくなると思います。

Listing 21から変更した部分を強調表示していただきたいです。

どのファイルの話なのかが読んでいて分からなくなりました。説明をお願いいたします。

The historical examples of crowdsourcing showcase humanity's knack for collaborative problem-solving long before the internet era. Take the Tang Dynasty's joint-stock company, a primitive form of crowdfunding born out of necessity during cold periods that disrupted agriculture. Similarly, in 1567, King Philip II of Spain dangled a cash prize to spur innovation in calculating longitude at sea, demonstrating early recognition of the power of incentives to mobilize collective effort.

The British government's longitude rewards in 1714 further underscored this concept, offering a bounty for a solution to a navigational conundrum. Even King Louis XVI of France got in on the action in 1783, offering a prize for a cost-effective method of producing alkali from sea salt. These instances illustrate how authorities leveraged financial incentives to tap into the wisdom of the masses.

Beyond monetary rewards, initiatives like Matthew Fontaine Maury's distribution of Wind and Current Charts in the 19th century and Joseph Henry's Meteorological Project in 1849 exemplify crowdsourcing's altruistic side. Maury's provision of charts in exchange for sailors' voyage logs and Henry's network of weather observers, enabled by telegraph communication, demonstrate early forms of collaborative data gathering for societal benefit.

These historical vignettes reveal that crowdsourcing isn't just a modern phenomenon; it's a timeless strategy rooted in human ingenuity and collaboration.

I read the article "Nearly All of Wikipedia is Written By Just 1 Percent of Its Editors" from Vice. This article from 2017 speaks on the work that Wikipedia editors do. It is stated that "only about 1 percent of Wikipedia's editors have generated 77 percent of the site's content." It also talks about how the number of Wikipedia editors has been on the decline. It makes me wonder how this number will change over the next 10 years. Will people want to take on the challenge? How will Wikipedia change over the next 10 years? Will AI ever be involved?

one detailed information about this website states that "Biographical information on various politicians was edited by their own staff to remove undesirable information (including pejorative statements quoted, or broken campaign promises)". Since a lot of people can edit the information at will, is there still credibility on Wikipedia? Similar Websites such as Baidu Baike also arise this problem, and ended up with little credibility.

Crowdsourcing is like a giant potluck where everyone brings a dish to share. It's this cool concept where instead of one person doing all the work, a bunch of folks chip in with their own small contributions. Think of Wikipedia – anyone with internet access can edit it. People from all walks of life come together, some are asked to join, others just jump in because they're interested.

It's fascinating because it taps into the power of the crowd. You've got this huge pool of potential talent, and by opening up the task to everyone, you're more likely to get diverse perspectives and skills. Plus, it's a win-win – the crowd gets to be part of something bigger, and the task gets done quicker and often better because of all the different inputs.

It's kind of like a modern-day barn raising, where the whole community comes together to build something awesome. Except instead of a barn, it might be designing a logo, solving a problem, or creating content. It's pretty cool how technology has made it possible for us to collaborate on such a massive scale, even if we're miles apart.

Crowdsourcing isn't limited to simple tasks. There are multiple types of crowdsourcing such as Crowd Voting, which is Gathering public opinion through ratings or votes. Or Crowd Creation, Collective production of creative content such as Youtube, Tiktok.

Lo que me llamó más la atención con respecto a la lectura es que recomiendan a los docentes recuperar conocimientos ancestrales de los pueblos indígenas y oprimidos lo que permitirá tener una educación más libre y justa dejando de lado la educación eurocentrica que solo limita a los estudiantes a un determinado conocimiento y dejando de lado valores, y cinstumnres que vienen durante años en su cultura.

コードブロックとファイルの内容の表記がどちらもListingなので両者を区別できるようにしてほしいです。

「結果ファイル」のほうがわかりやすい表現だと思います。ファイル名もあるとよりわかりやすいです。

ここまで作成したサンプルプロジェクトを使用しない理由がわからず読んでいてもやもやしました。前節までのサンプルを使用するか、理由を説明してほしいです。

文章内に組み込むのではなくシェルのコードブロックで記述したほうが読んでいてわかりやすいです。

「さまざまな」を削除しても情報量は変わらないので削除したほうがわかりやすいです。

On remarque ici la phrase de transition marquant la fin de l'introduction, et qui délimite donc le sujet. D'une part la population concernée correspond aux enfants autistes, pas aux adultes ce qui peut poser problème car nombre de patient autistes ne sont diagnostiqués que très tardivement. D'autre part la definition de lautisme ainsi que des problèmes qui lui sont associes n'est ici faite que sous le prisme de la sociabilité, or les personnes autistes peuvent rencontrer d'autres types de difficultées ( comme les Troubles du Sommeil, les troubles psychiatriques ( anxiété dépression) ).

Définition de l' autisme qui permet de mieux cerner la problématique rencontrée par les personnes autistes au quotidien : les nouvelles technologies pourraient elles donc faire partie des solutions possibles pour aider ces personnes ?

I chose this section because racial identity never was on my radar beforehand, but brought to my attention I have noticed and agree there is a lack of minority inclusion in healthcare. This directly relates to designing for equity and inclusion into the healthcare workspace due to the fact that different racial backgrounds may offer better advising and knowledge to a specific racial group than others. This relates to me because as a female, I would prefer to be advised by another female who has the same autonomy as my own.

The 710 freeway displaced a large number of people between 1965 and 1974 in West Pasadena.

A visceral example without direct connection to visualization. According to your earlier linkage of maps as the primary visualization of the slave trade, it seems that the poor passengers on the Zong would have benefited from MORE visualization.

It would be more elegant, and more respectful, if you figured out a different way to highlight aspects of the original than redrawing it. Redrawing seems to imply that the original is not enough for modern viewers. That SVG is somehow better. (It's not.)

I get a broken image.

Clear link to data, perhaps. But not clearly to data visualization. It feels that your argument would be better made against the data table, not the data visualization.

This seems more linked to relational and pictorial diagrams, not data visualization save for perhaps some later thematic maps.

also domestically at the outset of the industrial revolution.

This is a strange, and perhaps unfair, comparison. Contemporary Euro religious/royal genealogies are more similar, no?

This is a helpful idea. Deserves elevation.

I suspect few readers will know this position/title

Settler maybe, but USA isn't the exemplary colonial state.

Weak tea after the previous paragraph if your big example for visualization and slave trade settler colonialism is maps. Is there more evidence you can introduce?

Need to better qualify. Absolutes (unlinked) are easy to refute.

Science is but one source. Again it feels like strawman arguing.

This is a broader history of science, which is only one ingredient into the history of modern visualization. Feels both incomplete and too much. e.g. If you want to discuss astronomical observations and data then it is not correct to start here.

This information comes too late, and is buried mid-paragraph. If you really think that this image is a worthy counter, then you need to treat it with the same, or greater, respect, as you do the sacred cows. Who made it? Who are the Beothuk? What does it tell us? As-is, it feels like you are tokenizing this (very interesting!) graphic: introducing it just so that you can rant against the status quo.

"draftsman's" might work better as I'm not sure if any of the Russia lithography was done by hand. (Not my area of expertise!)

This is uncharitable to the great diversity of work present in Tufte's books (and other histories, particularly Neurath's autobio). Feels like you are trying to build a patriarchy golem so that you can tear him down. But I just see a pile of mud.

not Marey's! He simply relayed them in his book. (Marey is known for other contributions.)

could be misread to mean that Minard operated in the late 19th century

I've found that it is useless to reiterate Tufte's misguided advice, even if it is to reject it. The bad advice lives on. Better to let it fade away.

crop the second text blurb inline with the page. In general, they photos of his books are not good quality.

If you mention Tufte, then I would mention the likely source of the acclaim: Marey 1878.

This does not make sense. The map above is of a totally different campaign. See Friendly's paper on the anti-war message behind this compositoin.

s/b width

To my eye, not orange. Minard calls it rouge. So if anything, it is faded red.

the below animation transitions far too fast

two back-to-back ten dollar words. Use these sparingly.

Shuffle is a cute idea. But your current shuffle plan implementation is lousy. Overlaps hide more than they reveal and there's too much unproductive negative space.

This current intro seems like it is written for the author, not the audience. Start with the action, not navel-gazing. For example, open with a bold statement like: "The timeline isn't the best way of visualizing the history of data visualization."

Counter to what? This feels obnoxious for the sake of being obnoxious.

This statistic makes me wonder how representative the people who choose to do this work are. Is it mainly the same demographic of people taking on these positions? How does this change the way we intake our information? Is the information biased? Questions like this come to mind for me when hearing this information.

Would be using a Incognito window provide us with the same amount of privacy protection as DuckDuckGo or StartPage? As I believe that incognito doesn't save our information, or collect personal data.

standing out. not playing on their field

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Reply to the reviewers

Reply to the Reviewers

We sincerely thank the Referees for providing important and constructive comments. We have addressed their concerns point-by-point as described below.

Associated to Reviewer#1's comments

*- Diploid embryos are used as controls. Gynogenetic diploids seem to be better controls to ensure that the observed phenotypes are not related to loss of heterozygosity. To limit the amount of work, the use of gynogenetic diploids could be restricted to spindle polarity and centrosome number experiments. *

Response 1-1

__[Experimental plan] __Following the reviewer's suggestion, we will conduct immunostaining of a-tubulin and centrin (for visualizing the spindles and centrioles, respectively) in gynogenetic diploids that will be generated by applying heat shock to gynogenetic haploid embryos during the 1st - 2nd cleavage stage. We will observe the head area of gynogenetic diploid larvae at 3-dpf when the haploid counterparts suffer particularly drastic centrosome loss and spindle monopolarization.

• *

• *

*- As the authors discuss, it would be necessary to rescue centrosome loss to establish a causal relationship between centrosome loss and haploid viability. I certainly acknowledge that this is difficult (if not impossible), but it currently limits the significance of the results. *

Response 1-2

We agree that rescuing centrosome loss would provide an important advancement in understanding the cause of haploid syndrome in the context of our study. However, as the reviewer also pointed out in the above comment, this poses a significant technical challenge. As described in Discussion in the original manuscript, we have attempted to restore normal centrosome number through cell cycle modulations. However, we have not found a condition that rescues centrosome loss without damaging larval viability. As an alternative approach, we have also tried to induce centriole amplification by injecting mRNA encoding plk4, an essential centriole duplication inducer. However, this caused earlier embryonic death, precluding us from observing its effects on larval morphology after 1 dpf. The main challenge is that any treatment to increase centrosome number can cause centrosome overduplication, which is as deleterious to development as centrosome loss. Efforts to identify a key factor enabling the rescue of centrosome loss in haploid larvae are underway in our laboratory, which requires new explorations over several years and is beyond the scope of the present study. Reflecting on the reviewer's comment, we added a new sentence explaining the situation on this issue (line 395, page 19). To further discuss possible contributions of centrosome loss and mitotic defects to haploidy-linked embryonic defects, we also added a citation of a previous study reporting that depletion of centrosomal proteins caused mitotic defects leading to embryonic defects similar to those observed in haploid embryos in zebrafish (Novorol et al., 2013 Open Biology; line 380, page 19).

__[Experimental plan] __Meanwhile, as a new trial to induce centriole amplification in a scalable and temporally controllable manner, we plan the following experiment, which can be conducted within the time range of the revision schedule: We will investigate the effects of low dose treatment of a plk4 inhibitor centrinone B on tissue growth and viability of haploid larvae. A recent study reported that centrinone B had complicated effects on the centriole duplication process, which is highly dose-sensitive (Tkach et al., 2022 Elife, PMID: 35758262). While it blocks centriole duplication at sufficiently high concentrations for blocking plk4 activities, it paradoxically causes centriole amplification at suboptimal conditions, presumably though over-stabilizing plk4 by blocking its autophosphorylation-dependent degradation (while its centriole duplicating function remains active). Since a previous study showed that centrinone B is also effective in zebrafish embryos (Rathbun et al., 2020 Current Biology, PMID: 32916112), we try to find optimal centrinone B treatment condition that potentially restores tissue growth or viability of haploid embryos. If we find such a rescuing condition, we will address the principle of the rescuing effects by investigating the possession of centrioles in mitotic cells in these haploid larvae.

*- Some experiments are not, or arguably, quantified/statistically analyzed. *

o Figure 2, Active caspase level. Larvae are sorted into three categories, and no statistical test is performed on the obtained contingency table. A Fisher'*s exact test here, or much better, the active caspase-3 levels should be quantified, instead of sorting larvae into categories. *

Response 1-3

We apologize that we showed only "zoomed-out" images of the immunostained embryos in the original figures (Fig. 2A), which precluded a clear presentation of the haploidy-associated aggravation of apoptosis and mitotic arrest. We could clearly distinguish cleaved caspase-3- and pH3-positive cells from non-specific background staining with an enlarged view of the same immunostaining data. Therefore, to quantitatively evaluate the extent of the haploidy-linked apoptosis and mitotic arrest, we compared the density of these cells within the right midbrain. This new quantification demonstrated a statistically significant increase in cleaved caspase-3- or pH3-positive cells in haploids compared to diploids.

In the revised manuscript, we added the enlarged views of cleaved-caspase and pH3 immunostaining (Fig. 2B) and new quantifications with statistical analyses (Fig. 2C). Accompanying these revisions, we omitted the categorization of the severeness of the apoptosis, which was pointed out to be subjective in the reviewer#2's comment (see Response 2-3). We rewrote the corresponding section of the manuscript to explain the new quantitative analyses (line 143, page 7).

o Same comment for 3E-F. Larvae are scored as Scarce, Mild or Severe. Looking at Fig S3A, I see one mild p53MO embryo, but the two others are not that different from 'severe' cases, which would completely change the contingency table. Again, a proper quantification would be better.

Response 1-4

We also quantified the frequency of cleaved caspase-3-positive cells in control and p53MO larvae (original Fig. 3E and F) as described in Response 1-3. While conducting the cell counting with enlarged images, we realized that staining quality within the inner larval layers of morphants was relatively poor in these experiments. This problem precluded us from counting cleaved caspase-3-positive cells within the inner larval layers. Therefore, we tentatively quantified only the surface larval layers of these morphants and found that cleaved caspase-3-positive cells were significantly reduced in haploids upon depletion of p53. We currently show this quantification in Fig. 3G of the revised manuscript. While this quantification confirmed the trend of p53MO-dependent decrease in apoptosis, we think it more appropriate to newly conduct the same experiment with better quality of the staining to apply the same standard of quantification for Fig. 3 as Fig. 2.

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__[Experimental plan] __For the reason described above, we propose to re-conduct immunostaining of cleaved caspase-3 in control and p53MO-injected haploid larvae to improve the visibility of the inner layer of the larvae for better quality of the quantitation.

Meanwhile, we revised Fig. 3 by adding an enlarged view of immunostaining in Fig. 3F and omitting the subjective categorization shown in the original Fig. 3F and S3A. We plan to replace these data with new images and quantification to be obtained during the next revision. We also rewrote the main text to update these changes (line 166, page 8).

*o Figure 4D-E, no stats. *

Response 1-5

We conducted the ANOVA followed by the post-hoc Tukey test for new Fig. 4D and the Fisher exact test with Benjamini-Hochberg multiple testing correction for new Fig. 4E. Please note that statistical analyses were conducted after adding the data from original Fig. 6B-C following the reviewer's suggestion (see also Response 1-6).

*o Figure 6, Reversine treated haploid should be compared to haploid embryos (on the graphs and statistically). If no specific controls have been quantified for this experiment, data could be reused from previous figures, provided this is stated. *

Response 1-6

The live imaging data shown in original Fig. 4C-E and Fig. 6A-C were obtained within the same experimental series conducted in parallel at the same period under the same experimental condition. In the original manuscript, we separated them into two different figures according to the logical flow. However, following the reviewers' comments (see also Response 2-1), we realized it more appropriate to show them as a single figure panel as in the original experimental design. Therefore, we moved the reversine-treated haploid data from the original Fig. 6A-C to Fig. 4C-E to facilitate direct comparison among conditions with statistical analyses (see also Response 1-5).

*o Rescue by p53MO and Reversine, it would be nice to also include diploid measurements on the graphs, so that the reader can appreciate the extent of the rescue. *

Response 1-7

Following the reviewer's comment, we added control MO-injected or DMSO-treated diploid larval data in the corresponding graphs in Fig. 3I and 6G, respectively. Please refer to Response 2-6 for further discussion on the extent of the rescue.

Minor comments:

*- Lines 221-223, authors claim that centriole loss and spindle monopolarization commence earlier in the eyes and brain than in skin. I am note sure I see this in Fig. S5. It could as well be that the defect is less pronounced in skin. *

Response 1-8

We rewrote the manuscript to include the possible interpretation suggested by the reviewer on the result (line 225, page 11).

• *

- Lines 227-229, authors claim that 'The developmental stage when haploid larvae suffered the gradual aggravation of centrosome loss corresponded to the stage when larval cell size gradually decreased through successive cell divisions'. I did not get that. Doesn'*t cell size decrease since the first division? Fig 5D shows that cell size decreases all along development. *

Response 1-9

We agree that the original sentence implies, against our intention, that cell size does not decrease before the developmental stage mentioned here. To correct this problem, we rewrote the corresponding part of Discussion as below (line 230, page 11):

"Since the first division, embryonic cell size continuously reduces through successive cell divisions during early development (Menon et al., 2020). Cell size reduction continued at the developmental stage when we observed the gradual aggravation of the centrosome loss in haploid larvae."

*- Some correlations are used to draw conclusions: *

o Line 301-303. "The correlation between centrosome loss and spindle monopolarization indicates that haploid larval cells fail to form bipolar spindle because of the haploidy-linked centrosome loss."*. As stated by the authors, this is a correlation only. I agree it points in this direction. *

Response 1-10

We added a note to the corresponding sentence to draw readers' attention to the discussion on the limitation of the study with respect to the lack of centrosome rescue experiment (line 332, page 16).

O Line 305-308. "*Interestingly, centrosome loss occurred almost exclusively in haploid cells whose size became smaller than a certain border (Fig. 5), indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." This one is more problematic. There is no causal link established between cell size and centrosome number homeostasis. It could very well be that some unidentified problem induces both a reduction in cell size and the loss of centrioles. *

Response 1-11

To avoid an over-speculative description, we deleted the subsentence "indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." (line 336, page 17). We also added a new sentence, "Alternatively, it is also possible that other primary causes, such as the lack of second active allele producing sufficient protein pools induced cell size reduction and centrosome loss in parallel without causality between them." to discuss the possibility raised by the reviewer (line 348, page 17), in association with another comment from the reviewer #3 (see also Response 3-3).

• *

*I have concerns regarding the significance of the reported findings. Haploid zebrafish embryos show numerous developmental defects (some as early as gastrulation, as previously shown by the authors, Menon 2020), and they die by 4 dpf. That they experience massive apoptosis at day 3 does not seem very surprising, and that inhibiting p53 transiently improves the phenotype is not a big surprise. *

Response 1-12

Many reports have revealed tissue-level developmental abnormalities in haploid embryos since the discovery of haploid lethality in vertebrates more than 100 years ago. This has stimulated speculation of underlying causes of haploid intolerance for decades. However, there have been surprisingly few descriptions of cellular abnormalities underlying these tissue defects, precluding an evidence-based understanding of the principle that limits developmental ability in haploid embryos. Our findings of the haploidy-linked p53 upregulation and mitotic defects illustrate what happens in the dying haploid embryos at a cellular level. These findings would provide an evidence-based frame of reference for understanding why vertebrates cannot develop in the haploid state and also provide clues to controlling haploidy-linked embryonic defects in future studies. We added a new section in Discussion to discuss the importance of addressing the haploidy-linked defects at a cellular level (line 276, page 14).

*This reminds me of the non-specific effects of morpholino injection, which can be partially rescued by knocking down p53. *

Response 1-13

We believe the reviewer refers to the previous findings that different morpholinos generally have off-target effects activating p53-mediated apoptosis (e.g., Robu et al., 2007 PLoS Genet, PMID:17530925). However, p53 upregulation and apoptosis aggravation were also observed in uninjected haploid embryos free from morpholinos' artificial effects (Fig. 2, Fig. 3A, and B). To further address this issue, we plan to compare the frequency of cleavage caspase-3-positive cells between uninjected and control MO-injected haploids after revising the immunostaining of morphants in the original Fig. 3E-F (see Response 1-4 for details).

*The observation of mitotic arrest and mitotic defects and the observation that haploid cells often lack a centrosome is interesting. However, I felt that the manuscript suggested that these observations were novel and could explain the haploid syndrome specifically in non-mammalian embryos, when the authors reported the same observations in human haploid cells as well as in mouse haploid embryos (Yaguchi 2018). To me, this manuscript mainly confirms that their previous observation is not mammalian specific, but at least conserved in vertebrates. *

Response 1-14

As we originally wrote (line 341, page 17 in the original manuscript), we think these haploidy-linked cellular defects are conserved among mammalian and non-mammalian vertebrates. To improve the clarity of our interpretation, we rewrote a corresponding part of the manuscript (line 50, page 2).

*While I am no expert at centrosome duplication, I find the observation that haploidy leads to centrosome loss very intriguing, but have the impression that this manuscript falls short of improving our understanding of this phenomenon. *

Response 1-15

We express our gratitude to the reviewer for being interested in our findings. We hope the revisions made in the manuscript and the new results provided by the planned experiments will strengthen the contribution of this study to our understanding of haploidy-linked cellular defects.

• *

• *

Associated to Reviewer#2's comments

- Lack of proper controls in many experiments. For example, in the experiments where the authors treated haploids with reversine to suppress the SAC, there was no no-treatment control (Fig. 6A-C).

Response 2-1

We addressed the same point in__ Response 1-6__. In the original manuscript, we separately presented control and experimental conditions in the same experiment series in Fig. 4 and Fig. 6. We rejoined them in Fig. 4 as in the original experimental design. Please refer to __Response 1-6 __for further details.

• In Fig. 6D, when a DMSO control was included, the control fish were from 3 dpf while the reversine-treated fish were from 0.5-3 dpf. This is a big flaw in experimental design, especially considering the authors were looking at mitotic index, which is hugely impacted by developmental time. *

Response 2-2

In this experiment, we treated haploid larvae with either DMSO or reversine from 0.5 to 3 dpf, isolated cells from the larvae at 3 dpf, and subjected them to flow cytometry. Both DMSO- and reversine-treated larval cells were from 3-dpf larvae. Therefore, this experiment does not have the problem noted by the reviewer. To improve the clarity of the description of the experimental design, we rewrote the corresponding part of the figure legend (line 646, page 34).

- Subjective and inadequate data quantification. In the immunostaining experiments to detect caspase-3 and pH3, the authors either did not quantify at all and only showed single micrographs that might or might not be representative (for pH3), or only did very subjective and unconvincing quantification (for caspase-3). Objective measurements of fluorescence intensity could have been done, but the authors instead chose to categorize the staining into arbitrary categories with unclear standards. In example images they showed in the supplementary data, it is not obvious at all why some of the samples were classified as "mild" and others as "*severe" when their staining did not appear to be very different. *

Response 2-3

We apologize that we showed only "zoomed-out" images of the immunostained embryos in the original figures (Fig. 2A, 3E, and 6F), in which the distribution of individual cleaved caspase-3- or pH3-positive cells could not be clearly recognized. We added the enlarged view of identical immunostaining where these cells were clearly visualized in a countable manner (Fig. 2B, 3F, and 6D). Following the reviewer's suggestion, we newly conducted quantification by comparing the density of these cells within the right midbrain in haploids and diploids.

This new quantification demonstrated the haploidy-linked increase in cleaved caspase-3- or pH3-positive cells and a reversine-dependent decrease in pH3-positive cells. We added these new quantifications with statistical analyses to the revised manuscript (Fig. 2C and 6E). Accompanying these revisions, we omitted the categorization of the severeness of apoptosis, which was pointed out to be subjective. We rewrote the corresponding section of the manuscript to explain the new quantitative analyses (line 143, page 7; line 260, page 12).

While we also quantified cleaved caspase-3-positive cells in control and p53MO larvae in the original Fig. 3E, we realized that the staining quality of the inner larval layers of these morphants was relatively poor and could not apply the same standard of quantification as Fig. 2. Though we confirmed a statistically significant reduction in cleaved caspase-3-positive cells upon p53 depletion by quantified limited number of confocal sections (shown in Fig. 3G, please see also Response 1-4 for details), we decided to re-conduct this experiment for improving the staining quality to apply the same criteria of quantification for Fig 3 as Fig. 2 (Experimental plan is provided in Response 1-4).

Please note that we also tried to evaluate the extent of apoptosis and mitotic arrest based on the fluorescence intensity of organ areas. However, background staining outside the dead cell area precluded the precise quantification.

Additionally, the authors claimed that "*clusters of apoptotic cells" were only present in haploids but not diploids or p53 MO haploids, but they did not show any quantification. From the few example images (Fig.S3A), apoptotic clusters can be seen in p53 MO treated fish. Also, in some cases, the clusters were visible only because those fish were mounted in an incorrect orientation. For example, in Fig. S3A, control #2, that fish was visualized from its side, thus exposing areas around its eye that contained such clusters. These areas are not visible in other images where the fish were visualized from the top. *

__Response 2-4 __

We agree that the definition of "apoptotic clusters" was ambiguous in the original manuscript. We also agree that the visuals of the clusters could be affected by sample conditions, making them less reliable criteria for judging the severity of apoptotic upregulation in larvae. Following the reviewer's suggestion, we newly conducted apoptotic cell counting (Response 2-3), which recapitulated more reliably ploidy- or condition-dependent changes in the extent of apoptosis. Therefore, we decided to omit the description of the clusters in the new version of the manuscript.

*- Subpar data quality. Aside from issues with qualification, the IF data was not convincing as staining appeared to be inconsistent and uneven, with potential artefacts. *

Response 2-5

We apologize that the zoomed-out images in the original figures did not appropriately demonstrate the specific visualization of individual apoptotic or mitotic cells. As described in Response 2-3, we added enlarged views of the immunostaining to the revised manuscript, in which these individual cells are clearly distinguished from non-specific background staining (Fig. 2B, 3F, and 6D). Because of the poorer staining of inner layers of control and p53 morphants, we plan to re-conduct immunostaining for Fig. 3 and Fig. S3 (please refer to Response 1-4 for further detail). The current version of immunostaining and quantification in these figures will be replaced in the next revision.

- Unsupported and overstated claims. There were many overstatements. For one, in line 268, the authors claimed that "*the haploidy-linked mitotic stress with SAC activation is a primary constraint for organ growth in haploid larvae", while what they were actually showed was that reversine treatment, which suppresses the SAC, was partially rescued 2 out of the 3 growth defects they assessed, to such a small extent that the difference between haploid and haploid rescue was only Response 2-6

Following the reviewer's comment, we added control MO-injected or DMSO-treated diploid larval data in the corresponding graphs in Fig. 3I and 6G, respectively. We newly estimated the relative extent of the recovery in Results (line 174, page 8; line 268, page 13).

Reflecting the estimation, we rewrote the manuscript to discuss that haploidy-linked cell death or mitotic defects are a partial cause of organ growth retardation but that there could be other unaddressed cellular defects that also contribute to the growth retardation (line 305, page 15). We also discussed the possibility that incomplete resolution of cell death by p53MO or mitotic defects by reversine treatment may have limited their rescue effects on organ growth retardation (line 303, page 15). We also toned down several descriptions in our manuscript (lines 48 and 50, page 2; line 111, page 5; line 271, page 13; line 298, page 15; line 403, page 20) to achieve a more balanced interpretation on the potential contributions of cell death and mitotic defects to the formation of haploid syndrome.

In association with this issue, we also discussed the difficulty of assuming a priori "fully-rescued" haploid larval size in this context. This is because even normally developing haploid larvae in haplodiplontic species tend to be much smaller than their diploid counterparts. We newly cited a few cases of haplodiplontic species where haploids are smaller than or the same in size as diploids (line 307, page 15).

*With so many fundamental flaws, the data seem unreliable and the paper does not meet publishable standards. *

Response 2-7

We express our gratitude to the reviewer for providing important suggestions to improve the quality of analyses, data presentations, and interpretations in this study. We sincerely hope that one-by-one verifications of the points raised by the reviewer have improved the credibility of the paper and made it suitable for publication.

*The low quality of the analysis makes the significance low. *

*Reviewers have expertise in vertebrate embryogenesis and ploidy manipulation. *

Response 2-8

We hope that by addressing and solving the concerns pointed out by the reviewer, we could have clarified the significance of the study.

Associated to Reviewer#3's comments

*There seem to be a discrepancy between the microscopic images from Figure 2A and the quantification of pH3 positive cells using flow cytometry in Figure 4. According to the flow cytometric results the proportion of pH3 positive cells is about 3 times higher in haploid larvae compared to the control. The increase in mitotic cells in the imaging results however seems much more drastic. It would be helpful if the authors explain here. *

Response 3-1

Following comments provided by other reviewers (see also Response 1-2, 1-4, and__ 2-3__), we newly compared the frequency of pH3 positive cells between the immunostained haploid and diploid larvae. In this new analysis, pH3-positive cells were 6.4 times more frequent in haploids than in diploids, which is a more substantial difference than the one estimated based on the flow cytometric analysis.

The apparent discrepancy between the immunostaining and flow cytometric quantification would arise because pH3-positive mitotic cells tended to be more localized on the surface than in the inner region of larvae. This inevitably results in higher pH3-positive cell density in immunostaining, in which only larval surface is analyzed. To discuss this point, we newly conducted pH3 immunostaining in haploid larvae made transparent using RapiClear reagent and showed a vertical section of 3-d reconstituted larval image of pH3 immunostaining in Fig. S4E. We rewrote the manuscript to add our interpretation of this issue (line 652, page 34).

*Mitotic slippage that the authors observe to be increased in the haploid larvae to up to 5% of cells should result in an increase in the number of aneuploid cells. I am wondering why this is not recapitulated in the analyses of the DNA content in Figure S1. *

Response 3-2

A possible interpretation would be that the limited viability of newly formed aneuploid progenies precluded the detection of these populations in flow cytometric analyses. We discussed the possible generation of aneuploid progenies with our interpretation of their absence in the flow cytometric analyses in Discussion (line 293, page 14).

*Discussion: *

*I find the explanation of centrosomal loss due to depletion of centrosomal protein pools in the cytoplasm during drastic cell reduction interesting. I wonder if the reduction in size is not necessarily caused by the reduction in cells, but rather the result of the absence of a second active allele that produces centrosomal proteins? *

Response 3-3

We added the possible interpretation provided by the reviewer to the corresponding part of Discussion, in association with another comment from reviewer #1 (line 348, page 17; see also Response 1-11).

Reviewer #3 (Significance (Required)):

• *

*Overall, I find the study interesting even to a broader audience since diploid development is a fundamental feature of most animals. The authors also manage to discuss their findings on the consequences of haploidy in this bigger context of the restricted diploid development in animals. The study is very well-written even to non-experts. *

Response 3-4

We express our gratitude to the reviewer for providing positive comments on the significance of our findings. We sincerely hope that one-by-one verifications of the points raised by the reviewer further improve the quality of the paper.

I am not an expert of the literature describing previous characterizations of the consequences associated with haploid cell development in animals, which is why I cannot comment on the novelty of their study. Based on my expertise on centromeres and genome organisation I can however assess the results regarding the mitotic defects observed in haploid larvae (see comments).

Response 3-5

We sincerely thank the reviewer for providing constructive suggestions and critiques based on the expertise.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

In this study the authors aim to shed light onto the molecular reasons why most animals are restricted to diploid cell generations. In mammals, haploid intolerance has been previously attributed to defects linked to genomic imprinting, but the molecular defects associated with haploidy in non-mammalian species are unknown. To fill these gaps, the authors in this study investigate defects associated with haploidy in zebrafish larvae. They found that haploid larvae show elevated numbers of apoptotic cells that could be partially rescued by inhibition of p53. The also detected many cells with prolonged mitosis reflected by an increase of cells positive for the mitotic histone modification phospho- histone H3 (pH3) as well as cell division defects specific to the haploid larvae. These defects are likely caused by the loss of centrosomes in haploid larval cells resulting in an increase of monopolar spindle formation. Loss of centrosomes was particularly pronounced in smaller cells and occurred concomitant with a reduction in cell size through continous cell divisions. The authors could rescue the increase of cells with prolonged mitosis by inhibiting the SAC. Both restoration of mitotic length and decreased apoptosis (by p53 inhibition) also improved some organ growth defects observed in haploid larvae.

I only have some minor comments particularly regarding the mitotic defects.

There seem to be a discrepancy between the microscopic images from Figure 2A and the quantification of pH3 positive cells using flow cytometry in Figure 4. According to the flow cytometric results the proportion of pH3 positive cells is about 3 times higher in haploid larvae compared to the control. The increase in mitotic cells in the imaging results however seems much more drastic. It would be helpful if the authors explain here. Mitotic slippage that the authors observe to be increased in the haploid larvae to up to 5% of cells should result in an increase in the number of aneuploid cells. I am wondering why this is not recapitulated in the analyses of the DNA content in Figure S1.

Discussion:

I find the explanation of centrosomal loss due to depletion of centrosomal protein pools in the cytoplasm during drastic cell reduction interesting. I wonder if the reduction in size is not necessarily caused by the reduction in cells, but rather the result of the absence of a second active allele that produces centrosomal proteins?

Significance

Overall, I find the study interesting even to a broader audience since diploid development is a fundamental feature of most animals. The authors also manage to discuss their findings on the consequences of haploidy in this bigger context of the restricted diploid development in animals. The study is very well-written even to non-experts.

I am not an expert of the literature describing previous characterizations of the consequences associated with haploid cell development in animals, which is why I cannot comment on the novelty of their study. Based on my expertise on centromeres and genome organisation I can however assess the results regarding the mitotic defects observed in haploid larvae (see comments).

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Referee #2

Evidence, reproducibility and clarity

This study examined cell proliferation and death in haploid and diploid zebrafish and attempted to provide insights into cellular mechanisms underlying haploidy-linked defects in non-mammalian vertebrates. While some of the ideas were potentially interesting, the experiments were not rigorous and inadequate data analyses were performed. Major issues include: - Lack of proper controls in many experiments. For example, in the experiments where the authors treated haploids with reversine to suppress the SAC, there was no no-treatment control (Fig. 6A-C). In Fig. 6D, when a DMSO control was included, the control fish were from 3 dpf while the reversine-treated fish were from 0.5-3 dpf. This is a big flaw in experimental design, especially considering the authors were looking at mitotic index, which is hugely impacted by developmental time. - Subjective and inadequate data quantification. In the immunostaining experiments to detect caspase-3 and pH3, the authors either did not quantify at all and only showed single micrographs that might or might not be representative (for pH3), or only did very subjective and unconvincing quantification (for caspase-3). Objective measurements of fluorescence intensity could have been done, but the authors instead chose to categorize the staining into arbitrary categories with unclear standards. In example images they showed in the supplementary data, it is not obvious at all why some of the samples were classified as "mild" and others as "severe" when their staining did not appear to be very different. Additionally, the authors claimed that "clusters of apoptotic cells" were only present in haploids but not diploids or p53 MO haploids, but they did not show any quantification. From the few example images (Fig.S3A), apoptotic clusters can be seen in p53 MO treated fish. Also, in some cases, the clusters were visible only because those fish were mounted in an incorrect orientation. For example, in Fig. S3A, control #2, that fish was visualized from its side, thus exposing areas around its eye that contained such clusters. These areas are not visible in other images where the fish were visualized from the top. - Subpar data quality. Aside from issues with qualification, the IF data was not convincing as staining appeared to be inconsistent and uneven, with potential artefacts. - Unsupported and overstated claims. There were many overstatements. For one, in line 268, the authors claimed that "the haploidy-linked mitotic stress with SAC activation is a primary constraint for organ growth in haploid larvae", while what they were actually showed was that reversine treatment, which suppresses the SAC, was partially rescued 2 out of the 3 growth defects they assessed, to such a small extent that the difference between haploid and haploid rescue was only <20% of that between haploid and diploid. Again, they did not include proper controls so haploid, haploid rescue, and diploid were never in one experiment together - they were in different figures, plotted in drastically different scales - and 20% is only an estimate. With so many fundamental flaws, the data seem unreliable and the paper does not meet publishable standards.

Significance

The low quality of the analysis makes the significance low.

Reviewers have expertise in vertebrate embryogenesis and ploidy manipulation.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Yaguchi et al. investigate the causes of the "haploid syndrome" in the zebrafish embryo, the old observation that haploid embryos suffer from severe developmental defects and growth retardation of organs such as the brain and eyes (these defects are not simply a consequence of loss of heterozygosity, as they are rescued by forced diploidization of haploid larvae). Looking at apoptosis and proliferation, the authors show an increase in the number of apoptotic and mitotic cells in haploid larvae. Regarding apoptosis, they show an increase in p53 levels and demonstrate that knockdown of p53 limits apoptosis and leads to some phenotypic improvement. Regarding mitosis, they show an increase in mitotic delays and failures in haploid larvae. Inhibition of the spindle assembly checkpoint can reduce these defects and leads to some improvement in body axis length and eye size. Looking at the cause of the mitotic defects, the authors show that haploid cells often have monopolar spindles and loss of one centrosome, defects that appear to correlate with cell size.

Major comments:

While some experiments could be better quantified and/or statistically analyzed (see below), overall the results are convincing and clearly presented.

• Diploid embryos are used as controls. Gynogenetic diploids seem to be better controls to ensure that the observed phenotypes are not related to loss of heterozygosity. To limit the amount of work, the use of gynogenetic diploids could be restricted to spindle polarity and centrosome number experiments.
• As the authors discuss, it would be necessary to rescue centrosome loss to establish a causal relationship between centrosome loss and haploid viability. I certainly acknowledge that this is difficult (if not impossible), but it currently limits the significance of the results.
• Some experiments are not, or arguably, quantified/statistically analyzed.
  • Figure 2, Active caspase level. Larvae are sorted into three categories, and no statistical test is performed on the obtained contingency table. A Fisher's exact test here, or much better, the active caspase-3 levels should be quantified, instead of sorting larvae into categories.
  • Same comment for 3E-F. Larvae are scored as Scarce, Mild or Severe. Looking at Fig S3A, I see one mild p53MO embryo, but the two others are not that different from 'severe' cases, which would completely change the contingency table. Again, a proper quantification would be better.
  • Figure 4D-E, no stats.
  • Figure 6, Reversine treated haploid should be compared to haploid embryos (on the graphs and statistically). If no specific controls have been quantified for this experiment, data could be reused from previous figures, provided this is stated.
  • Rescue by p53MO and Reversine, it would be nice to also include diploid measurements on the graphs, so that the reader can appreciate the extent of the rescue.

Minor comments:

• Lines 221-223, authors claim that centriole loss and spindle monopolarization commence earlier in the eyes and brain than in skin. I am note sure I see this in Fig. S5. It could as well be that the defect is less pronounced in skin.
• Lines 227-229, authors claim that 'The developmental stage when haploid larvae suffered the gradual aggravation of centrosome loss corresponded to the stage when larval cell size gradually decreased through successive cell divisions'. I did not get that. Doesn't cell size decrease since the first division? Fig 5D shows that cell size decreases all along development.

• Some correlations are used to draw conclusions:

• Line 301-303. "The correlation between centrosome loss and spindle monopolarization indicates that haploid larval cells fail to form bipolar spindle because of the haploidy-linked centrosome loss.". As stated by the authors, this is a correlation only. I agree it points in this direction.

• Line 305-308. "Interestingly, centrosome loss occurred almost exclusively in haploid cells whose size became smaller than a certain border (Fig. 5), indicating that cell size is a key determinant of centrosome number homeostasis in the haploid state." This one is more problematic. There is no causal link established between cell size and centrosome number homeostasis. It could very well be that some unidentified problem induces both a reduction in cell size and the loss of centrioles.

Significance

I have concerns regarding the significance of the reported findings. Haploid zebrafish embryos show numerous developmental defects (some as early as gastrulation, as previously shown by the authors, Menon 2020), and they die by 4 dpf. That they experience massive apoptosis at day 3 does not seem very surprising, and that inhibiting p53 transiently improves the phenotype is not a big surprise. This reminds me of the non-specific effects of morpholino injection, which can be partially rescued by knocking down p53. The observation of mitotic arrest and mitotic defects and the observation that haploid cells often lack a centrosome is interesting. However, I felt that the manuscript suggested that these observations were novel and could explain the haploid syndrome specifically in non-mammalian embryos, when the authors reported the same observations in human haploid cells as well as in mouse haploid embryos (Yaguchi 2018). To me, this manuscript mainly confirms that their previous observation is not mammalian specific, but at least conserved in vertebrates.

While I am no expert at centrosome duplication, I find the observation that haploidy leads to centrosome loss very intriguing, but have the impression that this manuscript falls short of improving our understanding of this phenomenon.

Proyecto "Anotación PFR",

https://github.com/lmichan/PFR,

Tema/mesh/D012016/ReferenceValues,

TipoDePrueba/mesh/D002000/ForcedSpirometry,

TipoDePrueba/mesh/D010993/PlethysmographyWholeBody,

TipoDePrueba/mesh/D011653/PulmonaryDiffusingCapacity,

EtapaPrueba/Interpretacion,

PatronFuncional/Obstruccion,

PatronFuncional/PosibleRestriccion,

PatronFuncional/PosibleMixto,

PatronFuncional/Normal,

PatronFuncional/Broncodilatacion,

PatronFuncional/NoBroncodilatacion,

PatronFuncional/RestriccionSimple,

PatronFuncional/RestriccionCompleja,

PatronFuncional/TrastornoMixto,

PatronFuncional/VolumenesNormales,

PatronFuncional/Hiperinflacion,

PatronFuncional/PulmonesGrandes,

PatronFuncional/AumentoFlujoSanguineo,

PatronFuncional/AnormalidadVascularPulmonar,

PatronFuncional/PerdidaVolumenLocalizada,

PatronFuncional/PerdidaAlveoloCapilar,

PatronFuncional/DifusionNormal,

Enfermedad/mesh/D001249/Asthma,

Enfermedad/mesh/D029424/ChronicObstructivePulmonaryDisease,

test comment?

for - annotate - youtube - The Revolution will be annotated

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Reply to the reviewers

RESPONSE TO REVIEWS_RC-2024-02383

We thank all the reviewers for their comments and suggestions. Our point-by-point response is shown below, in bold.

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

Summary: the work presented by the authors detail how pharmacological inhibition of the rate limiting one carbon metabolic enzyme DHFR by the drug methotrexate increases the lifespan of yeast and worms. Furthermore, placing aged mice on dietary folate and choline restriction potentially enhanced metabolic plasticity but did not significantly increase lifespan with sex specific differences observed.

The findings in this manuscript are very interesting and important to our understanding of the conserved mechanisms that regulate longevity through one carbon metabolism. This is especially significant in light of the current folate intake and supplementation in the adult human population. The manuscript, however, requires major revisions. Please see comments below for details.

Major comments:

1. The overall tone in this manuscript is colloquial and conversational in nature. A third person academic style and tone, while avoiding the use of subjective descriptive terms would improve the quality of this text. Using terms such as "appeared less diverse", "results are remarkable ...strikingly more pronounced", "possibly positive outcomes" , "appear younger...for unknown reasons", "little Uracil", "tended to be higher", "roughly proportional", "slightly higher", "as a rough readout", and many other examples from the text should not be used in a scientific manuscript. The language should be academic, scientific, precise, and non-ambiguous. A thorough revision of the manuscript with substantial changes to the language and tone is necessary prior to publication. RESPONSE: Thank you for your feedback on the manuscript's tone. We revised most of the expressions mentioned by the reviewer. We note, however, that these phrases were used along with numbers and statistics. Hence, there was no lack of specifics, and readers could quickly evaluate the conclusions. We strive for a balance between scientific rigor and readability to maintain accessibility for a diverse audience.

In the results section, we find multiple instances where the results are interpreted and extensively discussed. This should be reserved for the discussion section. The results section should be used to simply report the findings in a detailed manner.

RESPONSE: We appreciate the suggestion on the integration of interpretation within the Results section. Upon review, we have clarified the presentation of our findings, ensuring a more distinct separation from interpretive commentary. Brief explanations remain to aid the reader's comprehension in light of the complex data, aiming to keep the flow and coherence of the manuscript and prevent overextension of the Discussion section (already ~1,300 words long). We welcome specific suggestions for further refinement.

The materials and methods section is severely lacking in details in some areas. For example, no details were provided regarding how the worm lifespans were conducted and previous work of collaborators were referenced instead. Important details such as worm numbers, biological and technical replicates, solid agar vs liquid culture, temperature, use of FUdR, antibiotics, transfer frequency, methods of scoring, etc... are lacking. Other details such as the preparation of the plates (Was MTX incorporated into the agar, seeded with the bacterial lawn, or liquid culture was used), storage conditions, age of the plates when lifespan started, how was the UV killing of the lawn verified etc...

many other methods subsections lack crucial details. Please carefully review the methodology and include sufficient pertinent details.

RESPONSE: The number of worms assayed in each case were shown in each figure, as described in the legend. We now also added all the information requested by the reviewer in the methods section. The text now reads:

“Briefly, the assays were done on solid agar nematode growth media (NGM) plates prepared fresh before each experiment. The bacterial lawn was exposed twice to a UV dose of 120mJ/cm2 using a UVC-515 Ultraviolet Multilinker (Ultra-Lum, Inc.). Streaking these UV-exposed bacteria to fresh LB agar plates (1% w/v tryptone, 0.5% w/v yeast extract, 1% w/v sodium chloride) produced no visible colonies. Methotrexate, or the ATIC inhibitor, was first dissolved in dimethyl sulfoxide (DMSO) and then added to the media used to prepare the plates after autoclaving (the media were kept in a 50°C water bath until the plates were poured). Mock-treated control plates contained only DMSO. At the start of each experiment, a sufficient number of eggs were collected from plates without any drugs and then placed on plates containing the indicated doses of each compound tested. After hatching and progression to the adult stage, animals were transferred to new plates (marked as the start of the lifespan assay) containing the drug tested and fluorodeoxyuridine (FUDR; dissolved in water), added at 50μM to block hatching of new animals. The plates were scored at least every other day until all the worms died. If an animal responded to gentle touch, it was scored as alive, otherwise a death was recorded, and the animal was removed from the plate. Worms were transferred to fresh plates as needed (e.g., if there was evidence of microbial contamination, dryness/cracks on the agar surface, consumption of the bacterial lawn, or hatching of new animals that escaped the FUDR block). The reported lifespans were compiled from several independent experiments done over several months (9-10 months for the methotrexate experiments and 4-5 months for the ATIC inhibitor), each scored by multiple individuals (4-5 persons per experiment). No experiments were excluded from the analysis.”

In the worms, interventions that impact germline proliferation can extend lifespan. Methotrexate is known to impact germline proliferation and can lead to toxic developmental effects and germline arrest. Was fecundity impacted by methotrexate using the dosages found to extend lifespan?

RESPONSE: We did not score fecundity in our experiments.

The authors stated that UV killed bacteria was used in the worm experiments but did not provide the reasoning for it. Virk had concluded that reduced bacterial pathogenicity is responsible for the lifespan extension and not the worm's OCM. How does your work agree with or refute these previous findings?

RESPONSE: The dose of methotrexate used by Virk et al was very high, so it is difficult to directly compare it to our experiment. Nonetheless, we do not think there is any contradiction. We added the following in the text to clarify this point:

“At higher doses (10-100μΜ), methotrexate did not extend lifespan (not shown), in agreement with (Virk et al., 2016), who treated adult animals with a very high dose of methotrexate (220μM). We also note that the bacteria used to feed the worms in our experiments were killed by ultraviolet radiation to exclude any impacts from bacterial folate metabolism, which is known to affect worm lifespan (Virk et al., 2016, 2012).”

The authors state that AICAR (100 uM administration to the worms (no experimental details were given) increases their lifespan and concluded that this is proof that manipulation of 1C metabolism promotes longevity. There are 2 concerns here; first, AMPK activation leads to inhibition of TOR and that has been shown to promote longevity in multiple models. While we agree that a significant crosstalk between TOR and OCM exists, this experiment does not necessarily contribute to the argument that the authors are making. Second, it has been established by multiple groups that inhibition (RNAi and pharmacological) of DHFR1, TYMS1, SAMS1 and possibly other OCM enzymes leads to lifespan extension in worms. These findings provide stronger evidence that OCM regulates organismal longevity.

RESPONSE: We acknowledged prior research on lifespan extension and do not claim our use of the ATIC inhibitor as the first evidence of 1C metabolism's impact on longevity. Rather, our findings complement existing studies from us and several other groups (including the examples mentioned by the reviewer, which we had cited) by introducing novel evidence of lifespan increase through this specific inhibitor in C. elegans. Please also note that we added a detailed description of the experiment in the Methods, as suggested in a previous comment.

In the mouse study, the authors do not provide a rationale on why a folate and choline deficient diet was adopted as opposed to only a folate deficient diet. Additionally, we assume that the diets did not contain antibiotics (succinyl sulfathiazole) to reduce microbiome folate production since it was not mentioned. Were wire bottom cages used to eliminate coprophagy? Were there any significant differences between male and female serum folate levels that could have contributed to the endpoints. Was only a subset of samples assayed for total folate? (fig 2b shows a possible n of 6 per group?). If no antibiotics and no wire bottom cages were used, mice can maintain adequate folate levels from coprophagy without developing signs of anemia. Please discuss these details as it helps clarify the conditions used.

RESPONSE: Excellent points, and we have now added this information (see Material and Methods):

“We note that when designing experiments to assess the consequences of folate limitation, it is common to control both folate and choline intake to ensure that the observed effects are due to the restriction of folate (Beaudin et al., 2011) because the presence of choline can mask the effects of folate deficiency. Choline can be oxidized to betaine, which provides methyl groups for converting homocysteine to methionine, independent of the folate cycle. Choline can also be incorporated into phosphatidylcholine, a major methyl ‘sink’ in the cell, through the Kennedy pathway. Lastly, we did not use any antibiotics to interfere with the microbiome nor wire bottom cages to eliminate coprophagy. Wire bottom cages were used only in the metabolic chamber experiments.”

Were there any significant differences between male and female serum folate levels that could have contributed to the endpoints. Was only a subset of samples assayed for total folate? (fig 2b shows a possible n of 6 per group?).

RESPONSE: ____Regarding folate levels, no significant sex differences were observed. We assayed all the animals we had at 120 weeks of age, the euthanasia endpoint, as shown in Figure 2B. There were fewer females than males in both diets.

There are instances in the results section where statements were made implying that there are differences observed "slightly higher", "negative association" when it is not statistically significant. There can be either statistically significant differences/correlation or not. please be precise in your wording.

RESPONSE: We have revised the Results section to ensure that qualitative descriptions such as "slightly higher" are only used when supported by appropriate statistical evidence. We have listed____ all the relevant numbers in each case after performing thorough and robust statistical analyses. We note, however, that mentioning qualitative descriptors is not always unwarranted, as long as they are factual.

Graying was observed less significantly in the F/C- group according to the authors. However, no quantitative assessment was made, and it is merely observational.

RESPONSE: It is not clear how to quantify graying non-invasively. Hence, we simply took photographs.

Inference to inhibition of mTOR was made, but mTOR protein and phosphorylation levels were not performed. The authors did perform western blotting on ribosomal S6 protein, however no assessment of the downstream mTOR targets P70S6k1 and 4EBP are shown.

RESPONSE: This is a good suggestion.____ We added a new experiment, looking at 4EBP1 phosphorylation (see new Figure S2). The results mirror those looking at S6 phosphorylation.

Can the change in RER in F/C- mice compared to controls be explained by the increased adiposity in these animals?

RESPONSE: We do not know. The relationship between adiposity and respiratory exchange rate can be quite complex. The increased adiposity of male mice limited for folate may lead to higher RER, reflecting perhaps a greater reliance on carbohydrate metabolism. But this is very speculative, especially since these mice are not obese. It is unclear how the improved metabolic plasticity could be associated with adiposity for the females.

How was the microbiome normalized between groups prior to the beginning of the experiment? (fecal slurry gavage, bedding exchange, cohabitation, none of the above?). There is no mention of this crucial step in the materials and methods section. Furthermore, additional details regarding the microbiome analysis are required (analysis pipeline, read depth, denoising, software, data processing, PCA analysis, etc...). it is not sufficient to state that Zymo performed the analysis.

RESPONSE: We now revised the text and added a detailed description of the methods, as follows:

“There was no microbiome normalization between groups prior to the beginning of the experiment. Mouse fecal pellets were gathered by positioning the mice on a paper towel beneath an overturned glass beaker. A minimum of three fecal pellets from each animal were transferred into cryovials using sterile forceps. The samples were preserved at -80°C and shipped to Zymo Research, where they were processed and analyzed with the ZymoBIOMICS® Shotgun Metagenomic Sequencing Service (Zymo Research, Irvine, CA).For DNA extraction, the ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used according to the manufacturer’s instructions. Genomic DNA samples were profiled with shotgun metagenomic sequencing. Sequencing libraries were prepared with Illumina® DNA Library Prep Kit (Illumina, San Diego, CA) with up to 500 ng DNA input following the manufacturer’s protocol using unique dual-index 10 bp barcodes with Nextera® adapters (Illumina, San Diego, CA). All libraries were pooled in equal abundance. The final pool was quantified using qPCR and TapeStation® (Agilent Technologies, Santa Clara, CA). The final library was sequenced on the NovaSeq® (Illumina, San Diego, CA) platform. The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each library preparation. Negative controls (i.e. blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process.

Raw sequence reads were trimmed to remove low quality fractions and adapters with Trimmomatic-0.33 (Bolger et al., 2014): quality trimming by sliding window with 6 bp window size and a quality cutoff of 20, and reads with size lower than 70 bp were removed. Antimicrobial resistance and virulence factor gene identification was performed with the DIAMOND sequence aligner (Buchfink et al., 2015). Microbial composition was profiled with Centrifuge (Kim et al., 2016) using bacterial, viral, fungal, mouse, and human genome datasets. Strain-level abundance information was extracted from the Centrifuge outputs and further analyzed to perform alpha- and beta-diversity analyses and biomarker discovery with LEfSe (Segata et al., 2011) with default settings (p > 0.05 and LDA effect size > 2).”

What is an "easily distinguishable gut microbiome" and "appeared less diverse"?

RESPONSE: To clarify these points, __w__e now edited as follows:

“The different sex and diet groups had an easily distinguishable gut microbiome, occupying different areas of principal component analysis graphs (Figure 5A), based on Bray-Curtis β-diversity dissimilarity indices (Knight et al., 2018). The intestinal microbiome of male mice on the F/C- diet was not statistically less diverse (p=0.222, based on the Wilcoxon rank sum test; Figure 5 - Supplement 1).”

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a two-dimensional plot using two principal components would be more suitable for image 5A and allow for better visualization of the clustering of the groups.

RESPONSE: We tried displaying the data on a multipanel (3 panels per group, 12 total) two-dimensional figure, but the result is more confusing. Since the sample number is small (n=6 animals per group), the 3D graphs are visually adequate and more pleasing. They are also the standard way of representing this kind of data.

Since the authors suggest that the microbiome could be a source of 1C metabolites (including natural folate), it is important to clarify if coprophagy is involved.

RESPONSE: We agree and have added the information as requested.

How are inflammatory cytokines and marker levels linked to reduced anabolism and immune function in non-challenged animals?

RESPONSE: ____We do not make any claims for such links if that is what the reviewer implied. If the intent was more towards speculation, we suspect one could imagine various situations. For instance, nutrients may be more heavily used during inflammation to support immune cell responses instead of central anabolic processes in other tissues, limiting the building blocks available for tissue growth and repair. Since we do not see major changes in inflammatory cytokines, we prefer not to speculate about possible links.

When discussing the epigenetic analysis, the authors state "no changes in the DNA methylation from liver samples.." and "groups appear younger than expected". Please clarify these statements. Additional details are needed regarding the analysis performed and the choice of methylated loci and methods. Please reference the epigenetic clock or model that was used and if was developed for the same strain and sub-strain of mice. Is it using a modified "Hovarth" mouse DNA age epigenetic clock? If so, provide the necessary details and a possible explanation for the discrepancy other than "unknown reasons"

__RESPONSE: ____The assay is based on the "Hovarth" mouse DNA age epigenetic clock, for the strain we used (C57BL/6). We have now added a detailed description, which we received from the company, as follows (see Materials and Methods): __

"Liver samples (~15mg) collected at euthanasia were placed in 0.75mL of 1X DNA/RNA Shield™ solution (Zymo Research, Irvine, CA), shipped to Zymo Research, and processed with DNAge® Service according to their established protocols. Briefly, after DNA extraction, the EZ DNA Methylation-Lightning Kit (Zymo Research, Irvine, CA) following the standard protocol was used for bisulfite conversion. Samples were enriched specifically for the sequencing of >1000 age-associated gene loci using Simplified Whole-panel Amplification Reaction Method (SWARM®), where specific CpGs are sequenced at minimum 1000X coverage. Sequencing was run on an Illumina NovaSeq instrument. Sequences were identified by Illumina base calling software then aligned to the reference genome using Bismark. Methylation levels for each cytosine were calculated by dividing the number of reads reporting a "c" by the number of reads reporting a "C" or "T". The percentage of methylation for these specific sequences were used to assess DNA age according to Zymo Research's proprietary DNAge® predictor which had been established using elastic net regression to determine the DNAge®."

As for a possible explanation for the discrepancy, since all our "groups appear younger than expected," unfortunately, other than "unknown reasons," we have none to offer. Nonetheless, the critical point for this study is that we saw no diet effects, regardless of where the company's assay draws the baseline.

Regarding Uracil misincorporation, the liver contains significant stores of folate as it is the main hub for several critical OCM reactions (Phospholipid methylation is a major one). Earlier studies used antibiotics with or without coprophagy prevention measures to induce a state of folate depletion to induce uracil incorporation in various tissues of rodent models. There is some controversy whether dietary folic acid restriction/methyl donor restriction alone will lead to uracil misincorporation when there is no apparent depletion or anemia. Please discuss your specific experimental procedures and how it agrees or disagrees with the published literature.

__RESPONSE: We have now added the experimental details, as suggested in a previous comment. Since we do not see uracil misincorporation, we prefer not to comment on the published literature for possible links between misincorporation and anemia. __

The section discussing RPS6 needs to be rewritten and it is difficult to understand.

RESPONSE: We revised the text, which now reads:

“____Immunoblot analysis of liver tissue samples gathered at the time of euthanasia revealed variability in the detected values across individual mice. When examining the male mice, we observed that, on average, those fed the F/C- diet had approximately half the amount of phosphorylated RPS6 (P-RPS6) compared to those on the F/C+ diet. However, due to high variability in the measured values, the overall differences in P-RPS6 levels between the two dietary groups did not reach statistical significance (Figure 7 - Supplement 1; p>0.05, based on the Wilcoxon rank sum test).”

Furthermore, as stated previously, considering phosphorylation of mTOR and its downstream targets 4EBP and S6K1 will give a clear indication of proliferative signaling.

RESPONSE:____ As we mentioned above, we have now added the suggested 4EBP experiment (see new Figure S2).

Additionally, these pathways are impacted by feeding status, diurnal cycles, and sex. Were these factors controlled prior to sacrifice? Were the animals sacrificed at the same time? In a fed or unfed state?

RESPONSE: The animals were sacrificed at the same time, with no feeding limitations.

The western blots provided in supplementary files show uneven protein loading across lanes (ponceau stain). No loading control is shown such as B-actin. A separate blot is used for total and phosphorylated proteins as opposed to gently stripping the membrane of the phosphorylated bolt and re-incubating with the antibody for total. While normalizing phosphorylated to total protein levels will eliminate some of the variability in the author's method. The uneven loading may introduce errors in the calculated ratios.

RESPONSE: The uneven loading across mouse samples is inconsequential. We report the ratio of phospho-RPS6 to the total amount of RPS6 ____within____ each mouse sample. These ratios were then compared among the different animals and diet groups. We also note that stripping could introduce other artifacts if it is not uniform across all the blot areas.

While the authors referenced older studies utilizing low dose methotrexate on rodents and provided a composite lifespan based on these findings, why was dietary folate and choline restriction used instead of a low dose methotrexate in mice in the current study? Please provide a rationale for this approach.

__RESPONSE: First, in the context of current folate fortification policies, we reasoned that testing dietary folate limitation late in life would be more informative. Second, three of us (M.P., B.K.K., and M.K.) proposed to the Interventions Testing Program at the National Institutes of Health to test whether low-dose methotrexate extends lifespan in mice. The proposal was accepted, and the study is ongoing (the ITP decided to test methotrexate at 0.2ppm, starting at 14 months of age; _https://www.nia.nih.gov/research/dab/interventions-testing-program-itp/supported-interventions_). __

Minor comments:

1. While the authors make compelling arguments that lower folate intake later in life may promote healthy aging, an important consideration in the human population that a considerable percentage of older individuals may be consuming an excessive amount of folate due the combination of fortification and voluntary supplementation. An alternate hypothesis that could apply to humans and lab models is that the existing levels of exposure to folate/folic acid may be accelerating the aging process and promoting disease in later life. __RESPONSE: Perhaps, but as we describe in the text (2nd paragraph in the introduction): __

“...analyses ‘did not identify specific risks from existing mandatory folic acid fortification’ in the general population (Field and Stover, 2018). This conclusion neither refutes nor contradicts the idea that a moderate decrease in folic acid intake among older adults may improve healthspan. Merely because high folic acid intake does not harm the health of older adults does not negate the possibility that a lower folic acid intake might enhance health.”

The common C57BL/6j is being referred to as the "long lived strain". Is this relative to mice in wild conditions? There are many transgenic C57bl/6 strains that live considerably longer. Please clarify if this is meant to describe the aged mice used in the experimental process.

RESPONSE: ____This was from a comprehensive comparison of many different inbred strains. We apologize for omitting the citation, which we have now added____ (Yuan et al, 2009).

While the authors state early in the manuscript that longevity was not a measured outcome in the mouse study, the manuscript contains statements discussing animal survival in the results and survival curves (figure 2). This gives the impression that the study was planned as a survival analysis initially and since no difference was observed between the experimental groups during the earlier stages, the secondary endpoints of health span analysis were adopted. Either approach does not detract from the significance of the study's findings. Further clarity on the approach would be beneficial to the readers.

RESPONSE: The study was designed, and the Animal Use Protocol was institutionally approved for healthspan, not lifespan. The number of animals we used did not have sufficient power to detect lifespan differences. Note that, at least for males, very few animals had died by 120 weeks, our approved euthanasia endpoint. However, it was important to report that folate limitation did not adversely affect overall survival during the analysis time frame.

For yeast culture conditions, what are the folate sources and content? Is there added folic acid similar to cell culture conditions where supraphysiological concentrations are used in standard mediums (RPMI and DMEM).

RESPONSE: The yeast media we used ____were undefined (YPD, see Materials and Methods). The source of folate in this media is “yeast extract,” which is generally considered to contain very high amounts of folate (it was used decades ago to treat anemia and folate deficiency in pregnant women). Note also that, unlike animals, yeast can synthesize folate.

In the metabolism section, the authors make statements such as "the differences were minimal" , "probably were due..", "minimal effects", "apparent increase", "tended to be", "little uracil" etc.. please refrain from using subjective language and use precise scientific terms.

RESPONSE: Please see our earlier response to this comment.

Figure 2-c, there is a typo, Weeks not months

RESPONSE: Corrected. Thank you!

** Referees cross-commenting**

while we generally agree with the other reviewer's concerns, we find that reviewer 3 rejection of the authors conclusion without considering the evidence presented in the context of what is currently known in the field potentially limiting. Multiple groups have shown that manipulation of OCM enzymes (DHFR, TYMS, SAMS) can extend lifespan in worms. the recent report Antebi's group (Annibal et al. Nature Com, 2021) provides strong evidence that OCM is central to longevity regulation in worms and mice and that folate intake can interact with and modulate organismal longevity. while this manuscript findings are not conclusive, I think it is premature to dismiss it completely. perhaps the alternative is to discuss the limitations of this approach and interpret the results (or the lack of significant differences) in order to help guide future research into this important subject. generalizing rodent results to human is always going to be a limiting factor in this type of work. Mice have significantly higher circulating folate. additionally, DHFR activity (the rate limiting enzyme in folate OCM) in rodents can be up to 100 times higher than its human equivalent. another consideration is that mice, similar to other rodents, engage in coprophagy, thereby recycling and supplementing bacterially produced folate in the absence of antibiotics in the diet. Therefore, mice placed of dietary folate restriction in the absence of antibiotics do not develop signs of anemia or deficiency. Therefore, it could be argued that there is no loss of nutrients in mice in this scenario and that supplementation at the arbitrarily recommended level of synthetic folic acid (2mg/kg day) or higher could impact health and aging. Similarly , in humans excess folate intake has been controversially associated with a number of deleterious health effects. It is important not to dismiss these reports and encourage further research into this subject that impacts a significant percentage of the human population due to the widespread use of supplements.

RESPONSE: We thank the reviewers for their evaluation of the work we presented. We have also added the following in the discussion, expanding the limitations of the study:

“Since mice engage in coprophagy, microbiome contributions to folate metabolism are bound to be substantial in this species. There are also significant differences in folate status between mice and people. For example, people have lower levels (~10-15 ng/mL) of serum folate than mice (Bailey et al., 2015), and the activity of DHFR, an enzyme essential for maintaining tetrahydrofolate pools -the folate form used in 1C reactions, maybe only 2% of that in rodents (Bailey and Ayling, 2009). Hence, mice are likely more refractory to a low folate dietary intake.”

Reviewer #1 (Significance (Required)):

Significance:

A major strength of this study is that the authors show that manipulation of OCM either through pharmacological inhibition or dietary restriction can impact organismal longevity in a conserved manner across species from yeast to worms and mammals. These findings provide compelling evidence that folate intake and metabolism in humans should be rigorously researched as potential regulator of aging. These findings complement and agree with a recent report by Antebi's group (Annibal et al. Nature Com, 2021) highlighting that long-lived worm and mice strains exhibit similar metabolic regulation of one carbon metabolism. In the same report low levels of folate supplementation partially or completely abrogated the lifespan extension in some models. This study provides additional evidence that restricting OCM through drugs or dietary restriction can significantly impact healthspan and lifespan. Additionally, it raises the question whether excessive folate intake in aged adults may have potentially deleterious effects on health and longevity. The limitations of this study can be seen in the overall lack of significant impact of the dietary intervention on the health metrics that were measured in mice. The study does not provide strong evidence that restricting folate and choline intake will produce favorable effects on health. Similarly, no significant impact on mice lifespan was observed based on the partial lifespan analysis. Further clarity is needed regarding the experimental procedures and methods used. The study, nonetheless, is an important step towards investigating the role of folate and OCM in regulating mammalian healthspan and lifespan. Future studies can expand on these findings and investigate whether OCM interventions that are started in early life can produce significant and measurable effects on longevity and health in mammals. The findings here provide a conceptual and incremental advance in our understanding of these complex interactions.

These findings are important to the research communities especially in the areas of longevity, metabolism, and nutrition.

RESPONSE: We appreciate the recognition of our work's significance in furthering understanding of longevity, metabolism, and nutrition. We would also like to stress that this study is not an incremental advance. We believe our study's focus on dietary folate limitation ____in aged mice____ represents a novel and more radical contribution, considering the lack of prior research in this specific context, underscoring the distinctiveness and importance of our findings.

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

Summary: In this manuscript they investigate whether disruption of the folate cycle can slow ageing/improve health in yeast, worms and mice. There are a few experiments in yeast and C. elegans but the rest is a meta analysis of some old data on folate-deprived mice and their own study of mice on a diet with and without folic acid and choline. The find that various interventions of the folate cycle extend lifespan in yeast and worms, that the old study suggest mice live longer without folic acid supplementation and that there is no change to healthspan with mice without folic acid and choline in the diet late in life and that these mice show some positive benefits. Analysis of the microbiome and the transcriptomics suggest small changes to the microbiota and changes in gene expression. Overall the authors conclude that biosynthetic processes have been inhibited without negative effects on healthspan.

Major comments

1. The two worm lifespan experiments in Fig 1 show very different controls despite the methods stating that the conditions were the same. Controls can vary from one experiment to another but the difference is striking. It would be good to have supplementary data about the number of repeats and other data about these experiments. RESPONSE: We also noted the difference. However, we believe our conclusions are valid and robust because we used only experiment-matched controls for each comparison. We now describe in detail how the experiments were done (see revised Materials and Methods). Lastly, the two compounds were tested years apart from different individuals, and the different lifespans of the controls could arise from differences in the media batches, temperature control, etc.

The diet lack folic acid and choline yet the conclusions are only about folate. The choline aspect of the diet needs to be acknowledged as a potential factor.

RESPONSE: As we mentioned above, we have now added this information (see Material and Methods):

“We note that when designing experiments to assess the consequences of folate limitation, it is common to control both folate and choline intake to ensure that the observed effects are due to the restriction of folate (Beaudin et al., 2011) because the presence of choline can mask the effects of folate deficiency. Choline can be oxidized to betaine, which provides methyl groups for converting homocysteine to methionine, independent of the folate cycle. Choline can also be incorporated into phosphatidylcholine, a major methyl ‘sink’ in the cell, through the Kennedy pathway. Lastly, we did not use any antibiotics to interfere with the microbiome nor wire bottom cages to eliminate coprophagy. Wire bottom cages were used only in the metabolic chamber experiments.”

The authors argue that the effects on the mice are not mediated effects on the diet by the microbiome because there is not a statistical effect on diversity. However they do show a clear difference at the metagenomic level that fits with a metabolic difference. It also ignores work in C. elegans showing that inhibition of bacterial folate synthesis increases lifespan, not by decreasing folate supply but because lowered bacterial folate prevents an age-accelerating activity in the bacteria (Virk et al 2016). It has also been shown that a breakdown product of folic acid can be taken up by bacteria and influence ageing (Maynard et al 2018). I do not think the evidence is strong enough to discounted that the changes seen in the mice are not mediated by microbes.

RESPONSE: We do not state that “changes seen in the mice are not mediated by microbes”. On the contrary, we agree with the reviewer that the microbiome likely contributes significantly, and we hope this is conveyed in the text. We also agree with the references the reviewer pointed out, which we cite (see also our response to point#5 of reviewer 1).

Minor comments

1. It had been shown a long time ago that sams-1 mutants in C. elegans extend lifespan. MTX is likely to influence SAMS levels. This point needs to mentioned. RESPONSE: Thank you. We added the reference.

Page - 6 "folate accelerates worm aging". This statement is not correct and is not what Virk et al 2016 suggests.

RESPONSE: We revised it to the following: “____It has been reported that treating worms with high levels of methotrexate (220μΜ) at the adult stage did not extend their lifespan ____(Virk et al., 2016)____”.

Page 7. "at 100μM, a dose similar to the one used in mice with metabolic syndrome (Asby et al., 2015)." It's not valid to compare the concentration of a drug in the media in a C. elegans experiment to a dose given to mice.

RESPONSE: We appreciate the reviewer's point on comparing drug dosages across species. The intention was to provide a reference point for the concentration used rather than suggesting a direct equivalence with outcomes. We recognize the complexities of cross-species dosage comparisons and have amended the text to clarify that the mention of dosage is for contextual purposes only.

** Referees cross-commenting**

I would like to add that it is important to consider whether there are in fact negative effects of folic acid given in later life and this is one of the only studies that addresses this question in a mammalian model, and thus needs to be reported, once the issues raised have been addressed.

__RESPONSE: As we mentioned in a comment from reviewer 1 and describe in the text (2nd paragraph in the introduction): __

“...analyses ‘did not identify specific risks from existing mandatory folic acid fortification’ in the general population (Field and Stover, 2018). This conclusion neither refutes nor contradicts the idea that a moderate decrease in folic acid intake among older adults may improve healthspan. Merely because high folic acid intake does not harm the health of older adults does not negate the possibility that a lower folic acid intake might enhance health.”

Reviewer #2 (Significance (Required)):

The main strength of this manuscript is that it examines the effect of mice given a folate and choline deficient diet late in life and finds mostly positive effects. This finding challenges the dogma that folate

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

Blank/Polymenis and colleagues explore how reduced folate metabolism impacts aging. While folate supplementation is known to benefit the development and health of young people, little is known about the impact of this substrate at advanced ages. The paper consists of two parts: 1) blocking folate metabolism in yeast and C. elegans while measuring lifespan (reproductive or age of death); 2) measuring a vast array of traits in mice where folate (and choline) is removed from the diet starting at age 1 year. The second approach is most central to the paper's theme, and the authors conclude their 'data raise the exciting possibility that ... reduced folate intake later in life might be beneficial." However, I do accept this conclusion. Instead, the overwhelming fact is that there were no changes in any phenotype due to the absence of F/C in the older animals. Loss of this nutrient is neutral, although perhaps bad for the kidney. In my view, the authors misinterpret their very basic results: loss of dietary folate has no impact on aged mice (one strain, at that). And there is no way to generalize this simple conclusion to humans.

RESPONSE: ____We respectfully disagree with the reviewer's assessment of our study's conclusions and its significance. With the primary focus on evaluating the effects of reduced folate intake in aged mice, we explored a comprehensive range of healthspan markers and molecular analyses. Contrary to the reviewer's assertion, our data demonstrate significant outcomes such as altered body weight and metabolic parameters in mice subjected to folate restriction, along with insights into molecular changes indicative of lower anabolism.

The reviewer's interpretation that folate limitation has no observable impact on aged mice overlooks the nuanced findings presented in our study. While acknowledging the neutral effects observed in some phenotypes, we contend that our results collectively contribute to a deeper understanding of the implications of late-life folate restriction. It is unwarranted to dismiss these findings.

Generalizing findings from model systems to humans is indeed complex, as noted by the reviewer. However, our study, alongside existing literature, provides valuable insights that warrant consideration and further exploration. We stand by the rigor of our methodology, the diversity of data presented, and the significance of our results in enhancing knowledge on the impact of folate metabolism in aging models.

There are other issues throughout the work that need to be addressed but given weakness on its key argument, I will not elaborate these points.

__RESPONSE: Since the reviewer offered no specifics on “other issues,” we cannot respond. We hope, however, that we have addressed them in our response to the other reviewers’ comments. __

Reviewer #3 (Significance (Required)):

Blank/Polymenis and colleagues explore how reduced folate metabolism impacts aging. While folate supplementation is known to benefit the development and health of young people, little is known about the impact of this substrate at advanced ages.

RESPONSE: ____We concur with the reviewer's observation regarding the knowledge gap surrounding the impact of reduced folate metabolism on aging, particularly in advanced stages of life, which ____is why our study significantly contributes to the field. As we mentioned above, not only do we report that some healthspan metrics were improved in folate-limited animals (e.g., body weight, improved metabolic plasticity), but our study also offers for the first time a comprehensive biomarker analysis of folate limitation late in life (e.g., metabolite and mRNAs changes associated with lower anabolism, lower IGF1 levels in females). ____This original contribution enhances our understanding of the complex interplay between folate metabolism and aging.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Referee #3

Evidence, reproducibility and clarity

Blank/Polymenis and colleagues explore how reduced folate metabolism impacts aging. While folate supplementation is known to benefit the development and health of young people, little is known about the impact of this substrate at advanced ages. The paper consists of two parts: 1) blocking folate metabolism in yeast and C. elegans while measuring lifespan (reproductive or age of death); 2) measuring a vast array of traits in mice where folate (and choline) is removed from the diet starting at age 1 year. The second approach is most central to the paper's theme, and the authors conclude their 'data raise the exciting possibility that ... reduced folate intake later in life might be beneficial." However, I do accept this conclusion. Instead, the overwhelming fact is that there were no changes in any phenotype due to the absence of F/C in the older animals. Loss of this nutrient is neutral, although perhaps bad for the kidney. In my view, the authors misinterpret their very basic results: loss of dietary folate has no impact on aged mice (one strain, at that). And there is no way to generalize this simple conclusion to humans. There are other issues throughout the work that need to be addressed but given weakness on its key argument, I will not elaborate these points.

Significance

Blank/Polymenis and colleagues explore how reduced folate metabolism impacts aging. While folate supplementation is known to benefit the development and health of young people, little is known about the impact of this substrate at advanced ages.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

Summary: In this manuscript they investigate whether disruption of the folate cycle can slow ageing/improve health in yeast, worms and mice. There are a few experiments in yeast and C. elegans but the rest is a meta analysis of some old data on folate-deprived mice and their own study of mice on a diet with and without folic acid and choline. The find that various interventions of the folate cycle extend lifespan in yeast and worms, that the old study suggest mice live longer without folic acid supplementation and that there is no change to healthspan with mice without folic acid and choline in the diet late in life and that these mice show some positive benefits. Analysis of the microbiome and the transcriptomics suggest small changes to the microbiota and changes in gene expression. Overall the authors conclude that biosynthetic processes have been inhibited without negative effects on healthspan.

Major comments

1. The two worm lifespan experiments in Fig 1 show very different controls despite the methods stating that the conditions were the same. Controls can vary from one experiment to another but the difference is striking. It would be good to have supplementary data about the number of repeats and other data about these experiments.
2. The diet lack folic acid and choline yet the conclusions are only about folate. The choline aspect of the diet needs to be acknowledged as a potential factor.
3. The authors argue that the effects on the mice are not mediated effects on the diet by the microbiome because there is not a statistical effect on diversity. However they do show a clear difference at the metagenomic level that fits with a metabolic difference. It also ignores work in C. elegans showing that inhibition of bacterial folate synthesis increases lifespan, not by decreasing folate supply but because lowered bacterial folate prevents an age-accelerating activity in the bacteria (Virk et al 2016). It has also been shown that a breakdown product of folic acid can be taken up by bacteria and influence ageing (Maynard et al 2018). I do not think the evidence is strong enough to discounted that the changes seen in the mice are not mediated by microbes.

Minor comments

1. It had been shown a long time ago that sams-1 mutants in C. elegans extend lifespan. MTX is likely to influence SAMS levels. This point needs to mentioned.
2. Page - 6 "folate accelerates worm aging". This statement is not correct and is not what Virk et al 2016 suggests.
3. Page 7. "at 100μM, a dose similar to the one used in mice with metabolic syndrome (Asby et al., 2015)." It's not valid to compare the concentration of a drug in the media in a C. elegans experiment to a dose given to mice.

** Referees cross-commenting**

I would like to add that it is important to consider whether there are in fact negative effects of folic acid given in later life and this is one of the only studies that addresses this question in a mammalian model, and thus needs to be reported, once the issues raised have been addressed.

Significance

The main strength of this manuscript is that it examines the effect of mice given a folate and choline deficient diet late in life and finds mostly positive effects. This finding challenges the dogma that folate

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

Summary: the work presented by the authors detail how pharmacological inhibition of the rate limiting one carbon metabolic enzyme DHFR by the drug methotrexate increases the lifespan of yeast and worms. Furthermore, placing aged mice on dietary folate and choline restriction potentially enhanced metabolic plasticity but did not significantly increase lifespan with sex specific differences observed. The findings in this manuscript are very interesting and important to our understanding of the conserved mechanisms that regulate longevity through one carbon metabolism. This is especially significant in light of the current folate intake and supplementation in the adult human population. The manuscript, however, requires major revisions. Please see comments below for details.

Major comments:

1. The overall tone in this manuscript is colloquial and conversational in nature. A third person academic style and tone, while avoiding the use of subjective descriptive terms would improve the quality of this text. Using terms such as "appeared less diverse", "results are remarkable ...strikingly more pronounced", "possibly positive outcomes" , "appear younger...for unknown reasons", "little Uracil", "tended to be higher", "roughly proportional", "slightly higher", "as a rough readout", and many other examples from the text should not be used in a scientific manuscript. The language should be academic, scientific, precise, and non-ambiguous. A thorough revision of the manuscript with substantial changes to the language and tone is necessary prior to publication.
2. In the results section, we find multiple instances where the results are interpreted and extensively discussed. This should be reserved for the discussion section. The results section should be used to simply report the findings in a detailed manner.
3. The materials and methods section is severely lacking in details in some areas. For example, no details were provided regarding how the worm lifespans were conducted and previous work of collaborators were referenced instead. Important details such as worm numbers, biological and technical replicates, solid agar vs liquid culture, temperature, use of FUdR, antibiotics, transfer frequency, methods of scoring, etc... are lacking. Other details such as the preparation of the plates (Was MTX incorporated into the agar, seeded with the bacterial lawn, or liquid culture was used), storage conditions, age of the plates when lifespan started, how was the UV killing of the lawn verified etc... many other methods subsections lack crucial details. Please carefully review the methodology and include sufficient pertinent details.
4. In the worms, interventions that impact germline proliferation can extend lifespan. Methotrexate is known to impact germline proliferation and can lead to toxic developmental effects and germline arrest. Was fecundity impacted by methotrexate using the dosages found to extend lifespan?
5. The authors stated that UV killed bacteria was used in the worm experiments but did not provide the reasoning for it. Virk had concluded that reduced bacterial pathogenicity is responsible for the lifespan extension and not the worm's OCM. How does your work agree with or refute these previous findings?
6. The authors state that AICAR (100 uM administration to the worms (no experimental details were given) increases their lifespan and concluded that this is proof that manipulation of 1C metabolism promotes longevity. There are 2 concerns here; first, AMPK activation leads to inhibition of TOR and that has been shown to promote longevity in multiple models. While we agree that a significant crosstalk between TOR and OCM exists, this experiment does not necessarily contribute to the argument that the authors are making. Second, it has been established by multiple groups that inhibition (RNAi and pharmacological) of DHFR1, TYMS1, SAMS1 and possibly other OCM enzymes leads to lifespan extension in worms. These findings provide stronger evidence that OCM regulates organismal longevity.
7. In the mouse study, the authors do not provide a rationale on why a folate and choline deficient diet was adopted as opposed to only a folate deficient diet. Additionally, we assume that the diets did not contain antibiotics (succinyl sulfathiazole) to reduce microbiome folate production since it was not mentioned. Where wire bottom cages used to eliminate coprophagy? Were there any significant differences between male and female serum folate levels that could have contributed to the endpoints. Was only a subset of samples assayed for total folate? (fig 2b shows a possible n of 6 per group?). If no antibiotics and no wire bottom cages were used, mice can maintain adequate folate levels from coprophagy without developing signs of anemia. Please discuss these details as it helps clarify the conditions used.
8. There are instances in the results section where statements were made implying that there are differences observed "slightly higher", "negative association" when it is not statistically significant. There can be either statistically significant differences/correlation or not. please be precise in your wording.
9. Graying was observed less significantly in the F/C- group according to the authors. However, no quantitative assessment was made, and it is merely observational. Inference to inhibition of mTOR was made, but mTOR protein and phosphorylation levels were not performed. The authors did perform western blotting on ribosomal S6 protein, however no assessment of the downstream mTOR targets P70S6k1 and 4EBP are shown.
10. Can the change in RER in F/C- mice compared to controls be explained by the increased adiposity in these animals?
11. How was the microbiome normalized between groups prior to the beginning of the experiment? (fecal slurry gavage, bedding exchange, cohabitation, none of the above?). There is no mention of this crucial step in the materials and methods section. Furthermore, additional details regarding the microbiome analysis are required (analysis pipeline, read depth, denoising, software, data processing, PCA analysis, etc...). it is not sufficient to state that Zymo performed the analysis. What is an "easily distinguishable gut microbiome" and "appeared less diverse"? a two-dimensional plot using two principal components would be more suitable for image 5A and allow for better visualization of the clustering of the groups. Since the authors suggest that the microbiome could be a source of 1C metabolites (including natural folate), it is important to clarify if coprophagy is involved.
12. How are inflammatory cytokines and marker levels linked to reduced anabolism and immune function in non-challenged animals?
13. When discussing the epigenetic analysis, the authors state "no changes in the DNA methylation from liver samples.." and "groups appear younger than expected". Please clarify these statements. Additional details are needed regarding the analysis performed and the choice of methylated loci and methods. Please reference the epigenetic clock or model that was used and if was developed for the same strain and sub-strain of mice. Is it using a modified "Hovarth" mouse DNA age epigenetic clock? If so, provide the necessary details and a possible explanation for the discrepancy other than "unknown reasons"
14. Regarding Uracil misincorporation, the liver contains significant stores of folate as it is the main hub for several critical OCM reactions (Phospholipid methylation is a major one). Earlier studies used antibiotics with or without coprophagy prevention measures to induce a state of folate depletion to induce uracil incorporation in various tissues of rodent models. Theres is some controversy whether dietary folic acid restriction/methyl donor restriction alone will lead to uracil misincorporation when there is no apparent depletion or anemia. Please discuss your specific experimental procedures and how it agrees or disagrees with the published literature.
15. The section discussing RPS6 needs to be rewritten and it is difficult to understand. Furthermore, as stated previously, considering phosphorylation of mTOR and its downstream targets 4EBP and S6K1 will give a clear indication of proliferative signaling. Additionally, these pathways are impacted by feeding status, diurnal cycles, and sex. Were these factors controlled prior to sacrifice? Where the animals sacrificed at the same time? In a fed or unfed state?
16. The western blots provided in supplementary files show uneven protein loading across lanes (ponceau stain). No loading control is shown such as B-actin. A separate blot is used for total and phosphorylated proteins as opposed to gently stripping the membrane of the phosphorylated bolt and re-incubating with the antibody for total. While normalizing phosphorylated to total protein levels will eliminate some of the variability in the author's method. The uneven loading may introduce errors in the calculated ratios.
17. While the authors referenced older studies utilizing low dose methotrexate on rodents and provided a composite lifespan based on these findings, why was dietary folate and choline restriction used instead of a low dose methotrexate in mice in the current study? Please provide a rationale for this approach.

Minor comments:

1. While the authors make compelling arguments that lower folate intake later in life may promote healthy aging, an important consideration in the human population that a considerable percentage of older individuals may be consuming an excessive amount of folate due the combination of fortification and voluntary supplementation. An alternate hypothesis that could apply to humans and lab models is that the existing levels of exposure to folate/folic acid may be accelerating the aging process and promoting disease in later life.
2. The common C57BL/6j is being referred to as the "long lived strain". Is this relative to mice in wild conditions? There are many transgenic C57bl/6 strains that live considerably longer. Please clarify if this is meant to describe the aged mice used in the experimental process.
3. While the authors state early in the manuscript that longevity was not a measured outcome in the mouse study, the manuscript contains statements discussing animal survival in the results and survival curves (figure 2). This gives the impression that the study was planned as a survival analysis initially and since no difference was observed between the experimental groups during the earlier stages, the secondary endpoints of health span analysis were adopted. Either approach does not detract from the significance of the study's findings. Further clarity on the approach would be beneficial to the readers.
4. For yeast culture conditions, what are the folate sources and content? Is there added folic acid similar to cell culture conditions where supraphysiological concentrations are used in standard mediums (RPMI and DMEM).
5. In the metabolism section, the authors make statements such as "the differences were minimal" , "probably were due..", "minimal effects", "apparent increase", "tended to be", "little uracil" etc.. please refrain from using subjective language and use precise scientific terms.
6. Figure 2-c, there is a typo, Weeks not months

** Referees cross-commenting**

while we generally agree with the other reviewer's concerns, we find that reviewer 3 rejection of the authors conclusion without considering the evidence presented in the context of what is currently known in the field potentially limiting. Multiple groups have shown that manipulation of OCM enzymes (DHFR, TYMS, SAMS) can extend lifespan in worms. the recent report Antebi's group (Annibal et al. Nature Com, 2021) provides strong evidence that OCM is central to longevity regulation in worms and mice and that folate intake can interact with and modulate organismal longevity. while this manuscript findings are not conclusive, I think it is premature to dismiss it completely. perhaps the alternative is to discuss the limitations of this approach and interpret the results (or the lack of significant differences) in order to help guide future research into this important subject. generalizing rodent results to human is always going to be a limiting factor in this type of work. Mice have significantly higher circulating folate. additionally, DHFR activity (the rate limiting enzyme in folate OCM) in rodents can be up to 100 times higher than its human equivalent. another consideration is that mice, similar to other rodents, engage in coprophagy, thereby recycling and supplementing bacterially produced folate in the absence of antibiotics in the diet. Therefore, mice placed of dietary folate restriction in the absence of antibiotics do not develop signs of anemia or deficiency. Therefore, it could be argued that there is no loss of nutrients in mice in this scenario and that supplementation at the arbitrarily recommended level of synthetic folic acid (2mg/kg day) or higher could impact health and aging. Similarly , in humans excess folate intake has been controversially associated with a number of deleterious health effects. It is important not to dismiss these reports and encourage further research into this subject that impacts a significant percentage of the human population due to the widespread use of supplements.

Significance

A major strength of this study is that the authors show that manipulation of OCM either through pharmacological inhibition or dietary restriction can impact organismal longevity in a conserved manner across species from yeast to worms and mammals. These findings provide compelling evidence that folate intake and metabolism in humans should be rigorously researched as potential regulator of aging. These findings complement and agree with a recent report by Antebi's group (Annibal et al. Nature Com, 2021) highlighting that long-lived worm and mice strains exhibit similar metabolic regulation of one carbon metabolism. In the same report low levels of folate supplementation partially or completely abrogated the lifespan extension in some models. This study provides additional evidence that restricting OCM through drugs or dietary restriction can significantly impact healthspan and lifespan. Additionally, it raises the question whether excessive folate intake in aged adults may have potentially deleterious effects on health and longevity. The limitations of this study can be seen in the overall lack of significant impact of the dietary intervention on the health metrics that were measured in mice. The study does not provide strong evidence that restricting folate and choline intake will produce favorable effects on health. Similarly, no significant impact on mice lifespan was observed based on the partial lifespan analysis. Further clarity is needed regarding the experimental procedures and methods used. The study, nonetheless, is an important step towards investigating the role of folate and OCM in regulating mammalian healthspan and lifespan. Future studies can expand on these findings and investigate whether OCM interventions that are started in early life can produce significant and measurable effects on longevity and health in mammals. The findings here provide a conceptual and incremental advance in our understanding of these complex interactions.

These findings are important to the research communities especially in the areas of longevity, metabolism, and nutrition.

For LGBTQIA+ designers and advocates, as well as those representing other minority groups, the focus is on creating spaces that are not just physically accessible, but also emotionally resonant and culturally sensitive. This approach prioritizes the needs, experiences, and comfort of people, ensuring that spaces are welcoming and supportive for everyone, regardless of their identity. It’s a holistic process that involves active listening, empathy, and a deep commitment to diversity and inclusion at every level of design and interaction.

for - climate crisis - voting - citizen action

from - LinkedIn post - https://hyp.is/rVHY0A46Ee-nAxPjir47lA/www.linkedin.com/feed/update/urn:li:activity:7194083120868921345/?commentUrn=urn:li:comment:(activity:7194083120868921345,7194083739939815424)&replyUrn=urn:li:comment:(activity:7194083120868921345,7194300883474481154)&dashCommentUrn=urn:li:fsd_comment:(7194083739939815424,urn:li:activity:7194083120868921345)&dashReplyUrn=urn:li:fsd_comment:(7194300883474481154,urn:li:activity:7194083120868921345)

test4

test3

Cette phrase me rappelle que nous voyons l'être humain comme un tout plutôt que comme un assemblage. Cela nous pousse peut-être à chercher une intelligence artificielle qui soit aussi un tout, en mesure d'effectuer tout ce que l'humain entier peut faire. Mais l'humain n'est pas un tout. C'est un assemblage.

Dans "Saussure meets the brain", Jan Koster décrit deux types d'interprétation. La t-interpretation est le passage d'une représentation à une autre (voir intermediation de Hayles, 2019), et la u-interpretation, c'est la création de sens par la conscience d'un être humain. La u-interprétation permet de nous libérer de la régression infinie de t-interpretations qui ne parviennent jamais au sens, pluisque ces dernières ne font que passer d'un système à un autre. Koster, qui modélise ainsi la division entre sens et syntaxe, la dépendance de plusieurs théories de la conscience sur la u-interpretation n'est pas en accord avec la neuroscience ou la grammaire générative de Chomsky.

If anything material is arbitrary with respect to u-interpretation and if u-interpretation is the core of consciousness, perception, knowledge, experience and other such qualia-invested phenomena, we seem, in practice to be doomed to a form of epistemological (but not necessarily ontological) dualism. [@koster_saussure_1996]

Et lorsqu'on aura modéliser les sentiments, on dira qu'il lui manque une qualia (ne nous demandez surtout pas ce qu'est une qualia, nous ne voulons pas que vous ayez la chance de la modéliser aussi).

L'un comme l'autre force la question : un système connectiviste est-il à même de produire une représentation de ce sur quoi il fut entraîné?

Quelle est la différence entre la chose, et une approximation de la chose? Cette question importe moins que celle de la valeur épistémique ou scientifique de l'approximation. Sur ce point reposent la majorité des succès des sciences naturelles et des sciences sociales.

Should check

Should look up this Saarland case.

Proyecto "Anotación PFR",

https://github.com/lmichan/PFR,

Tema/mesh/D006666/HistoryOfMedicine,

TipoDePrueba/mesh/D002000/ForcedSpirometry,

EtapaPrueba/Interpretacion,

PatronFuncional/Obstruccion,

PatronFuncional/PosibleRestriccion,

PatronFuncional/PosibleMixto,

PatronFuncional/Normal,

PatronFuncional/Broncodilatacion,

PatronFuncional/NoBroncodilatacion,

Enfermedad/mesh/D001249/Asthma,

Enfermedad/mesh/D029424/ChronicObstructivePulmonaryDisease,

Enfermedad/mesh/D017563/InterstitialLungDisease,

Enfermedad/mesh/D003550/CysticFibrosis,

Enfermedad/mesh/D054990/IdiopathicPulmonaryFibrosis,

Enfermedad/mesh/D000092122/BronchiolitisObliteransSyndrome,

for - voting - climate crisis - citizen action - Environmental Voter Project

to - Environmental Voter Project - voter action on climate crisis - https://hyp.is/AXsq4g47Ee-GbAc3PtbGuA/www.outrageandoptimism.org/episodes/moments-of-truth

for - annotate - Environmental Voter Project

from - Post - LinkedIn - https://hyp.is/rVHY0A46Ee-nAxPjir47lA/www.linkedin.com/feed/update/urn:li:activity:7194083120868921345/?commentUrn=urn:li:comment:(activity:7194083120868921345,7194083739939815424)&replyUrn=urn:li:comment:(activity:7194083120868921345,7194300883474481154)&dashCommentUrn=urn:li:fsd_comment:(7194083739939815424,urn:li:activity:7194083120868921345)&dashReplyUrn=urn:li:fsd_comment:(7194300883474481154,urn:li:activity:7194083120868921345)

TipoDePrueba/mesh/D002000/ForcedSpirometry

EtapaPrueba/Estandar

PatronFuncional/Obstruccion

PatronFuncional/PosibleRestriccion

PatronFuncional/PosibleMixto

PatronFuncional/Normal

PatronFuncional/NoBroncodilatacion

PatronFuncional/Broncodilatacion

for - communities and individuals - liberalism

from - .book - liberalism and the challenge of climate change - https://hyp.is/NDACig4VEe-ci1Oome4_kw/bafybeibgduwvv4dya4nwez5bcy24z5ya27oisiixpioafnxjjx56jgkv4m.ipfs.localhost:8080/

journal article details - title - Communities and the individual: Beyond the liberal-communitarian divide - date - May 11, 2021 - authors - Volker Kaul

a lightly packed form of chromatin (DNA, RNA, and protein) that is enriched in genes, and is often (but not always) under active transcription - Wikipedia

Perhaps the earlier cohorts would be scientists and actuaries

Instead of two negatives, there is opportunity to highlight a positive/progressive use of visualization, e.g. "colonialism and abolitionism."

retelling compared to what?

There may be a better verb than this. As-is, it characterizes the power as an off-the-shelf stock, rather than an evolving/emerging creative generative force. The latter is more interesting.

This strikes me as an odd construction as the 18th century does not feel that distant to me. (Also, a similar statement could be made about the 17th century.)

cada dedo é um número por que está ligado a outros

As human we need steps to follow to get a result. When we have a problem and want to solve it there are some steps to follow to resolve it. Using the Figure 1.1.1 could help a problem-solving seeing in the effective way in a business interaction.

test

test2

test

• so, because of Immer, we can 'mutate' states directly. but we can also return state as well.
• 有了Immer，可以直接在reducer里改state，也可以return state。

Musings of a San Diego SEO Expert https://www.tumblr.com/freshjen

https://www.linkedin.com/feed/update/urn%3Ali%3Aactivity%3A7182106044301557760/?commentUrn=urn%3Ali%3Acomment%3A%28activity%3A7182106044301557760%2C7182123565037617152%29&dashCommentUrn=urn%3Ali%3Afsd%5Fcomment%3A%287182123565037617152%2Curn%3Ali%3Aactivity%3A7182106044301557760%29&midToken=AQFVYkd8ftYDiA&midSig=1pWGb6HsgXurc1&trk=eml-email_notification_single_mentioned_you_in_this_01-hero_notification_cta-0-1ep~cta&trkEmail=eml-email_notification_single_mentioned_you_in_this_01-hero_notification_cta-0-1ep~cta-null-3xzxs~lun6078t~hp-null-null&eid=3xzxs-lun6078t-hp

• this is why extraReducers exists.
• 为什么extraReducers存在的原因。

Just Imagine, 1947. http://archive.org/details/JustImag1947.

This is Coffee by Vision Associates

Look through the text to see what supports the first paragraph, making this the most violent war in history. What specifically does the author say that leads this to be the case?

Why did Marshall plan to expand the army?

What was the death toll? What made it armed and violent? See if you can find evidence of this when you begin reading.

The Onion Sold by G/O Media by [[Katie Robertson]]