¿Por que en IBM no conocian los trabajos de Bush y Nelson o no les importaban o de modo concomitante/anterior?

Me parece interesante la contrapostura de Tudor Girba y los desarrolladores del Glamorous Toolkit respecto a hacer sentido del código sin leerlo, al menos no como prosa, sino, por el contrario, usando visualizaciones y consultas debido a su caracter altamente estructurado.

Lo digital en conexión y extensión con lo análogo me recuerda proyectos como Hypercard in the world de Bret Victor y su equipo donde literalmente lo digital y lo análogo están en ese diálogo y usan el ejemplo del catálogo y la biblioteca como lugares prominentes y ejemplares de tales conexiones.

Desafortunadamente, con la computación complicada y "separatista"/enclaustrado de hoy en día las tradiciones análogas y digitales se ven en contraposición más que en complemento (unas habitando la pantalla y otras por fuera, sin vínculos evidentes).

Y también la marginalia digital provista por Hypothesis, usada para la lecutra anotada durante la evaluación de esta tesis, o durante la producción de la mía, o en varios ejercicios educativos institucionalizados y en el Hackerspace, aunque desvinculados de los intereses monásticos.

Y también existe un interés por las reflexiones al respecto del acceso, la democratización y la accesibilidad de los archivos, y en general una postura a favor del software y la ciencia abierta.

Aunque a este nivel de zoom o alguno cercano hacia afuera (zoom out) no se alcanza a ver dicha postura en las palabras que el mapa muestra.

Muy interesantes los interactivos a lo largo del texto. Su dependencia de JavaScript, también para los textos acompañantes hace difícil anotarlos con Hypothesis. Me pregunto si herramientas hipérmediales como HTMX, Data Star e incluso AlpineJS permitirían que cierta parte del texto sea estática y embebida en la salida HTML, así corresponda a visualizaciones dinámicas que se presentan progresivamente.

De allí lo desafortunado del término "inteligencia artificial2 cuando otros términos anteriores, pero menos taquilleros, como Inteligencia Aumentada de Engelbart capturaban mejor la noción de una computación creado por humanos para aumentarles (como el paratexto) en lugar de para reemplazarles.

Una definición enactiva o performante: se define a partir de la práctica.

De nuevo, un sesgo de género, en el material publicado/seleccionado.

Interesante experimento de humanidades digitales en pequeño.

Me recuerda la idea de Isin y Ruppert frente a una ciudadanía que va más allá del Estado Nación o la ciudad (cfg otro comentario al respecto).

Es inevitable percibir un sesgo de género, no forzósamente del autor de la tesis, como de la literatura publicada en latinoamérica al respecto.

Esto me recuerda de la noción del conocimiento "conversacional sobre programación" que mencionaban en un Podcast (si no estoy mal, Future of Coding), de personas que, sin ser programadores profesionales, saben lo suficiente como para poder charlar con ellos, alrededor de proyectos que requieren dichos saberes.

como lo afirma Liu,

Esto me recuerda los proyectos como los que hacemos de memoria viva para la revitalización lingüística en el Amazonas o para auditar el discurso en Twitter de los candidatos a la presidencia en Colombia que entrarían en esas otras formas de ciudadanía y de mezcla entre la memoria análoga y la digital en nuestros tiempos y que fueron concebidos en diálogo con la academia, pero con tecnologías y prácticas desarrolladas por fuera.

Además de situarse por fuera de la oda al gigantismo de datos antes criticado. Lo cuantitativo y lo algorítmico, unido a lo interpretativo está ocurriendo a distintas escalas y contextos, en distintos lugares y prácticas (como los espacios hacker/maker), de modos más alineados con esta postura.

Precisamente, en ese sentido valdría la pena visibilizar métodos digitales que provienen de periferias y lugares no académicos, como los espacios hacker/maker.

Y sin embargo, este bonito propósito, es ortogonal al tamaño de las bases de datos. Otras hermenéuticas computacionales podrían ocurrir y de hecho ocurren, en lo pequeño.

De nuevo esa oda al gigantismo, tan presente en las humanidades digitales y muchos de sus exponentes.

Aunque Lisa Gitelman en "Raw Data" Is an Oxymoron" critica esta idea del dato como objetivo y dado, mostrando las relaciones entre dato y cultura.

Por supuesto, hay datos objetivos (la velocidad de la luz), pero son sujetos, inmersos en culturas, quienes se formulan las preguntas que conducen a ellos y crean los instrumentos de medida. Datos y letras son escritos por humanos, si bien hayamos algunos datos objetivos por el camino.

Algunas personas vemos con preocupación esta oda a la escala que ocurre en las humanidades digitales y sus exponentes más visibles (ejp: Manovich). Parece ser que, si no se tiene un supercomputador con teraflops de procesamiento y "chochentamil" teras de información, no se puede entrar en diálogo con las humanidades digitales.

Quizás es por eso que alternativas como la permacomputación, las infraestructuras de bolsillo, la computación convivencial no se ubican a sí mismas dentro de las DH.

Precisamente a eso me refería en comentarios previos frente a cómo la defensa aborda los problemas que se plantean, pues si bien ratifica 5 puntos a favor de las humanidades, ellos no configuran una respuesta a muchas de las críticas acá planteadas.

Sin embargo, salvo la 2 y la 5, el resto de argumentos pareciera no dialogar con las críticas hechas a la democracia y su caracter centrado en lo humano o una preocupación genuina por otras formas de felicidad.

Otro tanto se podría decir del párrafo siguiente y su relación institucionalizada con lo humano, re-enfatizando lugares en los que se ha centrado la "cultura" y lo "culto", en sus sentido más excluyentes que incluyentes. Esfuerzos por redefinir estas instituciones o por conectarlas con otras extituciones (espacios maker/hacker, Malokas, etc, precisamente intentan ver los valores positivos que se atribuyen a las humanidades, más allá de estas.

Resuena con varias de mis comentarios a lo largo del capítulo. Quizás valdría la pena colocar esta crítica antes. Incluso al comienzo del capítulo para que futuros lectores dialoguen con ella antes y también se pregunten por cómo toda la tesis realiza el diálogo con esta crítica tan contemporanea y pertinente.

También están los discursos post-humanistas que no recurren a lo humano, ni al "hombre" como centro de lo discursivo, y las llamadas "nuevas materialidades" que cuestionan lo textual o la lectoescrita como el elemento discursivo por omisión, con sus fuertes tendencias academicistas.

Me recuerda una aproximación del Ronin Institute y una forma de asociación quizás más cercana a ese origen y no mediada por una filiación institucional.

Sería conveniente poner a charlar esta idea, con la del museo como lugar exotisante y de expropiación, así como con las discusiones sobre el regreso de los objetos museísticos expropiados a las culturas y naciones de las que fueron sacados.

Este desconocimiento de unas tradiciones, en favor de la europa clásica y su "cuna" es lo que algunos critican cuando se refieren al humanismo como una forma atávica y colonial de eurocentrismo.

Precisamente la idea de lectura más allá de la textualidad se podría criticar en cómo lo computacional nos permite pensar con todo el cuerpo, en el espacio y en comunidad, en lugar de un proceso del logos y lo textual. Proyectos como Dynamiclad, el Folk computing y los desarrollos propios de Computación Convivencial critican ese énfasis en lo textual, que puede ubicarse tanto de los copistas mediavales como en los textos anotados computacionalmente.

De ahí el peligro del proyecto "Humano" en mayúsculas y definido desde un solo centro y mirada y un trivium heredado via dinámicas coloniales. En ese sentio l acrítica de Spivak puede resonar con una mirada más adecuada para las posturas propias que cuestione esa tradición. (ver comentario anterior)

Esto de por sí, coloca al humano en ese lugar privilegiado que se critica recientemente y la idea del resto (lo animal, lo vejetal) sin voluntad propia y, por tanto, sujeto a la voluntad humana.

Otras tradiciones autóctonas del continente, antes de la conquista, no se paran en ese lugar.

Además de no tener en cuenta las oralituras, las inscripciones no textuales rituales ampliamente expandidos y como formas de participación en lo colectivo que no tienen la idea de "Plaza Pública".

¿Qué pasa con la mirada sobre los 3 propósitos del humanismo, antes mencionados fuera de la mirada institucional e incluso del estado-nación? En particular me recuerda la noción de ciudadanía de Isin y Ruppert que reconoce la crítica a la noción de estado-nación y su mirada colonizante y piensa el ejercicio de deberes y derechos en clave más allá de la constitución, pensando en 3 fuerzas: performativas, legales e imaginativas.

¿Cómo la potencia de mirar por fuera de los confines instituicionales, disciplinares y del estado-nación los 3 propósitos de las humanidades podría dialogar con esta tesis?

Interesante. Pensé en lo autoreferencia y la autopoieses.

En esta pregunta me encuentro yo. Incluso me pregunto por visiones posthumanas o que cuestionan el centro de lo humano en discursos que deberían ser más holísticos, hibridados y ciborg, por ejemplo.

También está la inquietud por dónde está lo propio desde incluso el nombre del contienente "descubierto" y otras acepciones como las del Abya Yala.

E incluso, ¿en qué sentido las humanidades son las únicas guardianas de la memoria? ¿Qué pasa con tradiciones indígenas, hacktivistas u otras que se instauran en el dicho resguardo, pero no forzosamente se adscriben a un proyecto humanista o no ponen lo humano en el centro?

Interesante esta forma de escritura. Me pregunto qué tipos de tensiones explicita y cuáles supera y si lo hace a partir de dualismos.

¿Alguna razón para no considerar las experiencias endógenas de creación de tecnología en Colombia, por ejemplo las que ocurren en comunidades hacktivistas?

Esta tensión la habitamos en la comunidad de Grafoscopio. El abordaje dado acá ha sido reconfigurar ensamblajes o "stacks" tecnológicos para que den cuenta de contextos y necesidades locales y la incorporación de metaherramientas digitales y programación intersticial para extenderlas desde los límites/conexiones entre el stack, en lugar de desde adentro de los componentes (salvo en el caso de las metaherramientas, pues esa precisamente es su función).

Precisamente en clave de esas sostenibilidad, resistencia y no uniformidad es que planteo la pregunta anterior.

Interesante. Particularmente desde las periferias que pueden estar en diálogo, pero no se quieren ver subsumidas dentro de las llamadas Humanidades Digitales, como los hacktivismos, grupos de tecnologías cívicas, estudios críticos de datos y código, enmarcados en los llamados estudios críticos de ciencia, tecnología y sociedad

A la luz de estas lecturas decoloniales es que me interesa saber cómo se mantiene la paradoja bárbaro/civilizado.

Curioso por leer más en detalle. Particularme por la pregunta de si se pueden superar dichos binarismos heredados y colonizantes.

Me recuerda la perspectiva en diseño desde la perspectiva de Wolfgang Jonas, en la que el diseño no tiene un saber fundante "debajo" que le "sostenga", sino que es sostenido por los saberes en red que se encuentran al lado.

¿Cuál sería la forma de que los interactivos se puedan reproducir sin estar fuertemente ligados a la plataforma de publicación específica? Es decir, dado que se trata de trozos de JavaScript embebidos dentro del código Markdown, ¿podrían estos pasarse por conversores como Pandoc y aún así lograr páginas relativamente autónomas y transportables?

HackBo se ha sostenido durante 14 años. ¿Qué explicaciones hay detrás?

¿Cómo esta teoría se refleja en la práctica, particularmente en la elección de una licencia para el repositorio de la tesis? Si bien encontré un archivo README en la raíz del repositorio con una alusión breve:

Este repositorio es libre y abierto y se puede adaptar para crear proyectos similares.

no dice cómo ocurre en particular esa apertura y libertad entre el centenar de opciones disponibles. Tampoco encontré un archivo LICENSE en la raíz del repositorio que explicitara la manera concreta en que estas libertades toman cuerpo.

Valdría la pena problematizar estos binarios. ¿Pueden ser superados?

Algo similar pasa en el caso de Grafoscopio, en el que estamos en diálogo, pero no subsumidos, a los referentes internacionales. Mostrar las resonancias y distancias ayuda a ubicar esa tensión constructiva discursiva.

Debido a la perspectiva Latinoamericana a la que apuesta esta investigación, sería bueno contar con un desarrollo acerca de cómo se ubica esta tesis respecto a estas historias difractivas y no anglo-europeas de las Humanidades Digitales y los campos relacionados

Sería bueno que el mapa incluya algún tipo de convención de calor que indique no sólo el idioma de la búsqueda por color sino la cantidad de items buscados. De igual manera en enlace a Google Trends aparece roto y tiene fechas relativas al día de la búsqueda (today -5) en lugar de abosolutas con las fechas exactas.

interesante cómo se explicitan las materialidades digitales detrás de la publicación multiformato como parte del argumento mismo del libro.

Es raro que en ese relacionamiento activista y preocupación por las infraestructuras y comunidades, las comunidades hacktivistas no parecen ser mencionadas, ni siquiera por contraste con la red de humanidades digitales

Dado que ya existe la versión web/HTML, la impresa/PDF podría alimentarse, en ediciones posteriores, de un maquetado (tipo Tufte, por ejemplo) que haga explícitos los vínculos entre los dos formatos

¿Dónde estarían los hacktivistas y otras comunidades de práctica que también nos ofreces y enriqueces esas aproximaciones mixtas?

¿Qué pasa con las comunidades de práctica pequeñas o las redes conformadas informalmente como los espacios hacker/maker en distintas latitudes de Latinoamérica? ¿Por qué ellos no están listados en esas maneras propias de hacer y entender que enlazan a este párrafo con el siguiente? ¿Es referido a la denominación dentro de esta red o temática de humanidades digitales?

La versión digital del texto podría, al menos en notas al pie de página aprovechar la naturaleza hipertextual para enlazar las versiones en video de Nature of Code en línea (al menos el video introductorio).

Sería bueno agregar la lectura hipertextual a cada una de las páginas de la edición en línea

provienen

Sin embargo, al no estar explícito el licenciamiento de esta tesis y su código fuente, no es posible saber si, efectivamente, se encaja o no con las condiciones de este proyecto, cuando la licencia del mismo no está clara.

De hecho el formato digital web (en contraposición al digital PDF) permite anotaciones que no sólo pueden ser hechas en los márgenes, como esta, sino habilitar una conversación en línea abierta, como indicaba en otro comentario. Esto implica repensar el formato y la infraestructura de publicación para habilitar dihas ramas de lectura por omisión (como indiqué allí).

Mucho de este caracter dual se puede apreciar en el cuidado estilo escritural que es accesible a un público general, sin dejar de ser riguroso para uno especializado.

Por ello recomendaría que se habiliten, por omisión en la versión web, sistemas de lectura hipertextual, como Hypothesis y ramas de lectura (versiones que puedan ser bifurcadas y activadas para el comentario público, pero que habiten enlaces distintos, como es posible usando Fossil SCM). No sé si la infraestructura lo permita, a pesar del formato Markdown detrás, debido a llamados a JavaScript que no son muy portables, al menos en mis experimentos preliminares.

Precisamente esta posibilidad no llega hasta Manovich, sino que varios de los autores detrás del Dynabook (Kay, Goldberg, Ingalls) ya hablaban de estas características propias e incluso precursoras como Ada Lovelace ya hablaban de un medio cuya música son las ideas.

Diría que la acepción de Goldberg y Kay va más allá de la imitación (vía la simulación computacional) y coloca atributos propios (por ejemplo la programación).

Interesante. Algo similar ocurrió en mi caso con el desarrollo de Grafoscopio.

Todas esas dificultades resuenan mucho con lo que hemos experimentando en HackBo y Grafoscopio.

Una tesis muy rica en referencias. Creo que los hiperenlaces en la versión PDF e incluso la versión web no le hacen justicia. Quizás otro tema impreso y en web como Tufte CSS, para futuras ediciones de la tesis (luego del grado, por supuesto) pueda resaltar toda esa riqueza en la marginalia, mostrando algunas capturas de pantalla a modo de provocación de los enlaces a los que se hace referencia acá, como está:

Por ejemplo, frente a la pregunta:

Dimensión de la epistemología digital. ¿Si tuviera que posicionarse en este eje de la epistemología de las HD, en qué lugar se ubicaría?

Yo no me ubicaría en ninguno de los dos extremos, ni en la mitad. Precisamente siento que ese enunciado no recoge la postura de académicos activistas, como yo, y me pregunto si no habrían muchos en el mismo lugar.

¿Cómo se entiende entonces el activismo realizado por estas personas? Las Humanidades Digitales son un término abarcante, que se puede usar para quienes no se reconocen como tales y se discute ampliamente cómo da cuenta de la dualidad mencionada, pero el activismo y las prácticas comunitarias parecieran verse subsumidas dentro de ese término sin discutirse por qué, incluso ante el no reconocimiento como humanistas digitales (con la crítica o apatía al humanismo como tradición) y sí como activistas no se alcanza a recuperar hasta el momento y se pasa de largo.

Esto responde a la pregunta que me formulé en el capítulo anterior.

Una nota en la introducción podría aclarar el surgimiento de estas dimensiones y tensiones a partir de estos elementos puntuales.

¿Cuáles activistas? Es una perspectiva que no he podido detectar claramente hasta el momento.

La mención a las personas entrevistas muestra una filiación principalmente al mundo académico (salvo la persona asociada a las escuelas digitales, que para mí sería más un miembro de organización de base) y no está clara una doble condición de académicos y activistas, por ejemplo. Quizás tengan esa doble condición muchos, pero precisamente el no enunciarla hace parte de ese caracter más bien tácito del activismo que no se percibe con claridad en las genealogías para las humanidades digitales, ni en las posturas y materialidades críticas sobre las mismas (que son más académicas que activistas).

Esta investigación se fundamenta en una metodología de investigación mixta y multimétodo, es decir, es una mezcla de

• métodos cualitativos,
  • teoría fundamentada,
  • revisión bibliográfica y de proyectos,
  • y observación participante;
• métodos digitales
  • análisis de textos,
  • análisis de redes
  • y visualización de datos
• y de investigación-creación.
  • escritura como investigación
  • y piezas de programación creativa.

Voici un sommaire minuté des idées fortes concernant le microbiote, basé sur la transcription de la vidéo de France Culture :

• 0:00-1:10: Introduction au microbiote intestinal, constitué de milliards de micro-organismes (bactéries, virus, levures) logés dans nos intestins, interagissant avec notre cerveau. Le microbiote fascine les chercheurs car ses déséquilibres pourraient expliquer certaines maladies digestives, inflammatoires ou neurologiques. L'idée est de le modifier ou même de le transplanter pour traiter diverses pathologies.

• 1:10-2:20: Définition du microbiote intestinal comme l'ensemble des micro-organismes colonisant notre tube digestif dès la naissance. Ces micro-organismes reçoivent le gîte et le couvert en échange de services rendus à notre santé. La composition du microbiote varie selon l'environnement intestinal, comme entre le haut de l'intestin et le colon.

• 2:20-3:15: Chaque individu possède un microbiote spécifique, un peu comme des empreintes digitales. Bien qu'il existe des différences selon les régions du monde, notamment une diversité moindre dans les pays développés par rapport aux populations traditionnelles, il y a aussi des aspects communs. Le microbiote se constitue après la naissance lors des premières interactions avec le monde microbien.

• 3:15-4:20: La naissance par voie basse ou césarienne influence le microbiote initial du bébé. Le microbiote évolue et se mature jusqu'à l'âge de 3 à 5 ans, en parallèle avec le développement du système immunitaire. Avec l'âge, des perturbations peuvent survenir.

• 4:20-5:00: Le microbiote joue un rôle important dans l'immunité, en stimulant et en éduquant notre système de défense. Un déséquilibre précoce du microbiote peut augmenter le risque de développer des maladies liées à l'immunité plus tard.

• 5:00-5:49: La découverte du rôle du microbiote est récente, car les bactéries intestinales sont difficiles à cultiver. L'avènement de la biologie moléculaire et du séquençage de l'ADN a permis d'analyser le microbiote intestinal à partir des années 2000.

• 5:49-7:14: De nombreux facteurs impactent le microbiote intestinal, notamment l'alimentation, l'exposition aux antibiotiques et le lieu de vie. L'alimentation est le facteur environnemental le plus important.

• 7:14-8:07: Les perturbations du microbiote peuvent jouer un rôle dans les maladies inflammatoires chroniques de l'intestin. Un microbiote déséquilibré envoie des signaux altérés au système immunitaire, entraînant une activation inappropriée. Le microbiote des patients atteints de ces maladies est altéré en termes de composition et de fonctions. Le rôle du microbiote dans le syndrome de l'intestin irritable est moins clair.

• 8:07-9:02: L'intestin communique avec le cerveau de manière bidirectionnelle. Les bactéries produisent des métabolites qui peuvent atteindre le cerveau via la circulation générale, influençant ainsi son fonctionnement. Au moins 30 % des molécules présentes dans le sang sont produites par des bactéries ou issues de leur transformation.

• 9:02-10:00: Le microbiote est impliqué dans diverses maladies neurologiques, le diabète, l'obésité, les cancers et les maladies rhumatismales. Cependant, son rôle varie d'une maladie à l'autre. Une bonne alimentation, riche en fibres végétales (fruits et légumes), est essentielle pour un microbiote sain. Il faut éviter les aliments ultra-transformés, la viande rouge et la charcuterie. Les aliments fermentés peuvent être bénéfiques.

• 10:00-10:53: Les probiotiques en prévention ne sont pas forcément nécessaires, il est préférable de privilégier une bonne alimentation. L'impact de l'alimentation bio sur le microbiote est peu documenté. Le tabac peut influencer positivement le microbiote lors de l'arrêt, tandis que l'alcool a des effets plus indirects. Les tests disponibles actuellement pour analyser le microbiote n'ont pas d'intérêt clinique.

• 10:53-12:00: La transplantation fécale consiste à remplacer un microbiote altéré par celui d'un sujet sain. Cette pratique est ancienne, utilisée notamment dans la médecine chinoise. Les vétérinaires l'utilisent également. Les donneurs doivent passer de nombreux tests pour éviter la transmission de maladies.

• 12:00-13:03: La transplantation fécale se fait par les voies naturelles, après un nettoyage intestinal. Elle peut se faire par la bouche (gélules) ou par le bas (coloscopie, lavement). Il n'y a pas de rejet car on ne donne pas de traitement immunosuppresseur. L'efficacité de la transplantation dépend du donneur et du receveur.

• 13:03-14:38: La transplantation fécale est efficace à 90 % dans les infections récidivantes à Clostridium difficile. Dans d'autres situations, la recherche est en cours. Le microbiote n'est qu'un facteur parmi d'autres pour la santé. La transplantation fécale à domicile est fortement déconseillée en raison des risques de transmission de maladies et d'aggravation de l'état du patient. Le tourisme de la greffe fécale est également déconseillé.

Author response:

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

Reviewer #1 (Public Review):

Summary:

his study shows a new mechanism of GS regulation in the archaean Methanosarcina mazei and clarifies the direct activation of GS activity by 2-oxoglutarate, thus featuring another way in which 2-oxoglutarate acts as a central status reporter of C/N sensing.

Mass photometry and single particle cryoEM structure analysis convincingly show the direct regulation of GS activity by 2-OG promoted formation of the dodecameric structure of GS. The previously recognized small proteins GlnK1 and Sp26 seem to play a subordinate role in GS regulation, which is in good agreement with previous data. Although these data are quite clear now, there remains one major open question: how does 2-OG further increase GS activity once the full dodecameric state is achieved (at 5 mM)? This point needs to be reconsidered.

Weaknesses:

It is not entirely clear, how very high 2-OG concentrations activate GS beyond dodecamer formation.

The data presented in this work are in stark contrast to the previously reported structure of M. mazei GS by the Schumacher lab. This is very confusing for the scientific community and requires clarification. The discussion should consider possible reasons for the contradictory results.

Importantly, it is puzzling how Schumacher could achieve an apo-structire of dodecameric GS? If 2-OG is necessary for dodecameric formation, this should be discussed. If GlnK1 doesn't form a complex with the dodecameric GS, how could such a complex be resolved there?

In addition, the text is in principle clear but could be improved by professional editing. Most obviously there is insufficient comma placement.

We thank Reviewer #1 for the professional evaluation and raising important points. We will address those comments in the updated manuscript and especially improve the discussion in respect to the two points of concern.

(1) How can GlnA1 activity further be stimulated with further increasing 2-OG after the dodecamer is already fully assembled at 5 mM 2-OG.

We assume a two-step requirement for 2-OG, the dodecameric assembly and the priming of the active sites. The assembly step is based on cooperative effects of 2-OG and does not require the presence of 2-OG in all 2-OG-binding pockets: 2-OG-binding to one binding pocket also causes a domino effect of conformational changes in the adjacent 2-OG-unbound subunit, as also described for Methanothermococcus thermolithotrophicus GS in Müller et al. 2023. Due to the introduction of these conformational changes, the dodecameric form becomes more favourable even without all 2-OG binding sites being occupied. With higher 2-OG concentrations present (> 5mM), the activity increased further until finally all 2-OG-binding pockets were occupied, resulting in the priming of all active sites (all subunits) and thereby reaching the maximal activity.

(2) The contradictory results with previously published data on the structure of M. mazei by Schumacher et al. 2023.

We certainly agree that it is confusing that Schumacher et al. 2023 obtained a dodecameric structure without the addition of 2-OG, which we claim to be essential for the dodecameric form. 2-OG is a cellular metabolite that is naturally present in E. coli, the heterologous expression host both groups used. Since our main question focused on analysing the 2-OG effect on GS, we have performed thorough dialysis of the purified protein to remove all 2-OG before performing MP experiments. In the absence of 2-OG we never observed significant enzyme activity and always detected a fast disassembly after incubation on ice. We thus assume that a dodecamer without 2-OG in Schumacher et al. 2023 is an inactive oligomer of a once 2-OG-bound form, stabilized e.g. by the presence of 5 mM MgCl2.

The GlnA1-GlnK1-structure (crystallography) by Schumacher et al. 2023 is in stark contrast to our findings that GlnK1 and GlnA1 do not interact as shown by mass photometry with purified proteins. A possible reason for this discrepancy might be that at the high protein concentrations used in the crystallization assay, complexes are formed based on hydrophobic or ionic protein interactions, which would not form under physiological concentrations.

Reviewer #2 (Public Review):

Summary:

Herdering et al. introduced research on an archaeal glutamine synthetase (GS) from Methanosarcina mazei, which exhibits sensitivity to the environmental presence of 2-oxoglutarate (2-OG). While previous studies have indicated 2-OG's ability to enhance GS activity, the precise underlying mechanism remains unclear. Initially, the authors utilized biophysical characterization, primarily employing a nanomolar-scale detection method called mass photometry, to explore the molecular assembly of Methanosarcina mazei GS (M. mazei GS) in the absence or presence of 2-OG. Similar to other GS enzymes, the target M. mazei GS forms a stable dodecamer, with two hexameric rings stacked in tail-to-tail interactions. Despite approximately 40% of M. mazei GS existing as monomeric or dimeric entities in the detectable solution, the majority spontaneously assemble into a dodecameric state. Upon mixing 2-OG with M. mazei GS, the population of the dodecameric form increases proportionally with the concentration of 2-OG, indicating that 2-OG either promotes or stabilizes the assembly process. The cryo-electron microscopy (cryo-EM) structure reveals that 2-OG is positioned near the interface of two hexameric rings. At a resolution of 2.39 Å, the cryo-EM map vividly illustrates 2-OG forming hydrogen bonds with two individual GS subunits as well as with solvent water molecules. Moreover, local side-chain reorientation and conformational changes of loops in response to 2-OG further delineate the 2-OG-stabilized assembly of M. mazei GS.

Strengths & Weaknesses:

The investigation studies the impact of 2-oxoglutarate (2-OG) on the assembly of Methanosarcina mazei glutamine synthetase (M mazei GS). Utilizing cutting-edge mass photometry, the authors scrutinized the population dynamics of GS assembly in response to varying concentrations of 2-OG. Notably, the findings demonstrate a promising and straightforward correlation, revealing that dodecamer formation can be stimulated by 2-OG concentrations of up to 10 mM, although GS assembly never reaches 100% dodecamerization in this study. Furthermore, catalytic activities showed a remarkable enhancement, escalating from 0.0 U/mg to 7.8 U/mg with increasing concentrations of 2-OG, peaking at 12.5 mM. However, an intriguing gap arises between the incomplete dodecameric formation observed at 10 mM 2-OG, as revealed by mass photometry, and the continued increase in activity from 5 mM to 10 mM 2-OG for M mazei GS. This prompts questions regarding the inability of M mazei GS to achieve complete dodecamer formation and the underlying factors that further enhance GS activity within this concentration range of 2-OG.

Moreover, the cryo-electron microscopy (cryo-EM) analysis provides additional support for the biophysical and biochemical characterization, elucidating the precise localization of 2-OG at the interface of two GS subunits within two hexameric rings. The observed correlation between GS assembly facilitated by 2-OG and its catalytic activity is substantiated by structural reorientations at the GS-GS interface, confirming the previously reported phenomenon of "funnel activation" in GS. However, the authors did not present the cryo-EM structure of M. mazei GS in complex with ATP and glutamate in the presence of 2-OG, which could have shed light on the differences in glutamine biosynthesis between previously reported GS enzymes and the 2-OG-bound M. mazei GS.

Furthermore, besides revealing the cryo-EM structure of 2-OG-bound GS, the study also observed the filamentous form of GS, suggesting that filament formation may be a universal stacking mechanism across archaeal and bacterial species. However, efforts to enhance resolution to investigate whether the stacked polymer is induced by 2-OG or other factors such as ions or metabolites were not undertaken by the authors, leaving room for further exploration into the mechanisms underlying filament formation in GS.

We thank Reviewer #2 for the detailed assessment and valuable input. We will address those comments in the updated manuscript and clarify the message.

(1) The discrepancy of the dodecamer formation (max. at 5 mM 2-OG) and the enzyme activity (max. at 12.5 mM 2-OG). We assume that there are two effects caused by 2-OG: 1. cooperativity of binding (less 2-OG needed to facilitate dodecamer formation) and 2. priming of each active site. See also Reviewer #1 R.1). We assume this is the reason why the activity of dodecameric GlnA1 can be further enhanced by increased 2-OG concentration until all catalytic sites are primed.

(2) The lack of the structure of a 2-OG and ATP-bound GlnA1. Although we strongly agree that this would be a highly interesting structure, it seems out of the scope of a typical revision to request new cryo-EM structures. We evaluate the findings of our present study concerning the 2-OG effects as important insights into the strongly discussed field of glutamine synthetase regulation, even without the requested additional structures.

(3) The observed GlnA1-filaments are an interesting finding. We certainly agree with the referee on that point, that the stacked polymers are potentially induced by 2-OG or ions. However, it is out of the main focus of this manuscript to further explore those filaments. Nevertheless, this observation could serve as an interesting starting point for future experiments.

Reviewer #3 (Public Review):

Summary:

The current manuscript investigates the effect of 2-oxoglutarate and the Glk1 protein as modulators of the enzymatic reactivity of glutamine synthetase. To do this, the authors rely on mass photometry, specific activity measurements, and single-particle cryo-EM data.

From the results obtained, the authors convey that glutamine synthetase from Methanosarcina mazei exists in a non-active monomeric/dimeric form under low concentrations of 2-oxoglutarate, and its oligomerization into a dodecameric complex is triggered by higher concentration of 2-oxoglutarate, also resulting in the enhancement of the enzyme activity.

Strengths:

Glutamine synthetase is a crucial enzyme in all domains of life. The dodecameric fold of GS is recurrent amongst prokaryotic and archaea organisms, while the enzyme activity can be regulated in distinct ways. This is a very interesting work combining protein biochemistry with structural biology.

The role of 2-OG is here highlighted as a crucial effector for enzyme oligomerization and full reactivity.

Weaknesses:

Various opportunities to enhance the current state-of-the-art were missed. In particular, omissions of the ligand-bound state of GnK1 leave unexplained the lack of its interaction with GS (in contradiction with previous results from the authors). A finer dissection of the effect and role of 2-oxoglurate are missing and important questions remain unanswered (e.g. are dimers relevant during early stages of the interaction or why previous GS dodecameric structures do not show 2-oxoglutarate).

We thank Reviewer #3 for the expert evaluation and inspiring criticism.

(1) Encouragement to examine ligand-bound states of GlnK1. We agree and plan to perform the suggested experiments exploring the conditions under which GlnA1 and GlnK1 might interact. We will perform the MP experiments in the presence of ATP. In GlnA1 activity test assays when evaluating the presence/effects of GlnK1 on GlnA1 activity, however, ATP was always present in high concentrations and still we did not observe a significant effect of GlnK1 on the GlnA1 activity.

(2) The exact role of 2-OG could have been dissected much better. We agree on that point and will improve the clarity of the manuscript. See also Reviewer #1 R.1.

(3) The lack of studies on dimers. This is actually an interesting point, which we did not consider during writing the manuscript. Now, re-analysing all our MP data in this respect, GlnA1 is likely a dimer as smallest species. Consequently, we will add more supplementary data which supports this observation and change the text accordingly.

(4) Previous studies and structures did not show the 2-OG. We assume that for other structures, no additional 2-OG was added, and the groups did not specifically analyse for this metabolite either. All methanoarchaea perform methanogenesis and contain the oxidative part of the TCA cycle exclusively for the generation of glutamate (anabolism) but not a closed TCA cycle enabling them to use internal 2-OG concentration as internal signal for nitrogen availability. In the case of bacterial GS from organisms with a closed TCA cycle used for energy metabolism (oxidation of acetyl CoA) like e.g. E. coli, the formation of an active dodecameric GS form underlies another mechanism independent of 2-OG. In case of the recent M. mazei GS structures published by Schumacher et al. 2023, the dodecameric structure is probably a result from the heterologous expression and purification from E. coli. (See also Reviewer #1 R.2). One example of methanoarchaeal glutamine synthetases that do in fact contain the 2-OG in the structure, is Müller et al. 2023.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Specific issues:

L 141: 2-OG levels increase due to slowing GOGAT reaction (due to Gln limitation as a consequence of N-starvation).... (2-OG also increases in bacteria that lack GDH...)

As the GS-GOGAT cycle is the major route of ammonium assimilation, consumption of 2-OG by GDH is probably only relevant under high ammonium concentrations.

In Methanoarchaea, GS is strictly regulated and expression strongly repressed under nitrogen sufficiency - thus glutamate for anabolism is mainly generated by GDH under N sufficiency consuming 2-OG delivered by the oxidative part of the TCA cycle (Methanogenesis is the energy metabolism in methanoarchaea, a closed TCA cycle is not present) thus 2-OG is increasing under nitrogen limitation, when no NH3 is available for GDH.

L148: it is not clear what is meant by: "and due to the indirect GS activity assay"

We apologize for not being clear here. The GS activity assay used is the classical assay by Sahpiro & Stadtman 1970 and is a coupled optical test assay (coupling the ATP consumption of the GS activity to the oxidation of NADH by lactate dehydrogenase). Based on the coupled test assay the measurements of low activities show a high deviation. We now added this information in the revised MS respectively.

L: 177: arguing about 2-OG affinities: more precisely, the 0.75 mM 2-OG is the EC50 concentration of 2-OG for triggering dodecameric formation; it might not directly reflect the total 2-OG affinity, since the affinity may be modulated by (anti)cooperative effects, or by additional sites... as there may be different 2-OG binding sites involved... (same in line 201)

Thank you for the valuable input. We changed KD to EC50 within the entire manuscript. Concerning possible additional 2-OG binding sites: we did not see any other 2-OG in the cryo-EM structure aside from the described one and we therefore assume that the one described in the manuscript is the main and only one. Considering the high amounts of 2-OG (12.5 mM) used in the structure, it is quite unlikely that additional 2-OG sites exist since they would have unphysiologically low affinities.

In this respect, instead of the rather poor assay shown in Figure 1D, a more detailed determination of catalytic activation by different 2-OG concentrations should be done (similar to 1A)... This would allow a direct comparison between dodecamerization and enzymatic activation.

We agree and performed the respective experiments, which are now presented in revised Fig. 1D

Discussion: the role of 2-OG as a direct activator, comparison with other prokaryotic GS: in other cases, 2-OG affects GS indirectly by being sensed by PII proteins or other 2-OG sensing mechanisms (like 2OG-NtcA-mediated repression of IF factors in cyanobacteria)

We agree and have added that information in the discussion as suggested.

290. Unclear: As a second step of activation, the allosteric binding of 2-OG causes a series of conformational.... where is this site located? According to the catalytic effects (compare 1A and 1D) this site should have a lower affinity …

Thank you very much for pointing this out. Binding of 2-OG only occurs in one specific allosteric binding-site. Binding however, has two effects on the GlnA1: dodecamer assembly and priming of the active site (with two specific EC50, which are now shown in Fig. 1A and D).

See also public comment #1 (1).

Reviewer #2 (Recommendations For The Authors):

The primary concern for me is that mass photometry might lead to incorrect conclusions. The differences in the forms of GS seen in SEC and MP suggest that GS can indeed form a stable dodecamer when the concentration of GS is high enough, as shown in Figure S1B. I strongly suggest using an additional biophysical method to explore the connection between GS and 2-OG in terms of both assembly and activity, to truly understand 2-OG's role in the process of assembly and catalysis.

We apologize if we did not present this clear enough, however the MP analysis of GlnA1 in the absence of 2-OG showed always (monomers/) dimers, dodecamers were only present in the presence of 2-OG. The SEC analysis in Fig. S1B has been performed in the presence of 12.5 mM 2-OG, we realized this information is missing in the figure legend - we now added this in the revised version. The 2-OG is in addition visible in the Cryo EM structure. Thus, we do not agree to perform additional biophysical methods.

As for the other experimental findings, they appear satisfactory to me, and I have no reservations regarding the cryoEM data.

(1) Mass photometry is a fancy technique that uses only a tiny amount of protein to study how they come together. However, the concentration of the protein used in the experiment might be lower than what's needed for them to stick together properly. So, the authors saw a lot of single proteins or pairs instead of bigger groups. They showed in Figure S1B that the M. mazei GS came out earlier than a 440-kDa reference protein, indicating it's actually a dodecamer. But when they looked at the dodecamer fraction using mass photometry, they found smaller bits, suggesting the GS was breaking apart because the concentration used was too low. To fix this, they could try using a technique called analytic ultracentrifuge (AUC) with different amounts of 2-OG to see if they can spot single proteins or pairs when they use a bit more GS. They could also try another technique called SEC-MALS to do similar tests. If they do this, they could replace Figure 1A with new data showing fully formed GS dodecamers when they use the right amount of 2-OG.

Thank you for this input. In MP we looked at dodecamer formation after removing the 2-OG entirely and re-adding it in the respective concentration. We think that GlnA1 is much more unstable in its monomeric/dimeric fraction and that the complete and harsh removal of 2-OG results in some dysfunctional protein which does not recover the dodecameric conformation after dialysis and re-addition of 2-OG. Looking at the dodecamer-peak right after SEC however, we exclusively see dodecamers, which is now included as an additional supplementary figure (suppl. Fig. 1C). Consequently, we did not perform additional experiments.

(2) Building on the last point, the estimated binding strength (Kd) between 2-OG and GS might be lower than it really is, because the GS often breaks apart from its dodecameric form in this experiment, even though 2-OG helps keep the pairs together, as seen with cryoEM. What if they used 5-10 times more GS in the mass photometry experiment? Would the estimated bond strength stay the same? Could they use AUC or other techniques like ITC to find out the real, not just estimated, strength of the bond?

We agree that the term KD is not suitable. We have changed the term KD to EC50 as suggested by reviewer #1, which describes the effective concentration required for 50 % dodecamer assembly. Furthermore, we disagree that the dodecamer breaks apart when the concentrations are as low as in MP experiments. The actual reason for the breaking is rather the harsh dialysis to remove all 2-OG before MP experiments. Right after SEC, the we exclusively see dodecamer in MP (suppl. Fig. S1C). See also #2 (1).

(3) The fact that the GS hardly works without 2-OG is interesting. I tried to understand the experiment setup, but it wasn't clear as the protocol mentioned in the author's 2021 FEBS paper referred to an old paper from 1970. The "coupled optical test assay" they talked about wasn't explained well. I found other papers that used phosphometry assays to see how much ATP was used up. I suggest the authors give a better, more detailed explanation of their experiments in the methods section. Also, it's unclear why the GS activity keeps going up from 5 to 12.5 mM 2-OG, even though they said it's saturated. They suggested there might be another change happening from 5 to 12.5 mM 2-OG. If that's the case, they should try to get a cryo-EM picture of the GS with lots of 2-OG, both with and without ATP/glutamate (or the Met-Sox-P-ADP inhibitor), to see what's happening at a structural level during this change caused by 2-OG.

We agree with the reviewer that the GS assay was not explained in detail (since published and known for several years). However, we now added the more detailed description of the assay in the revised MS, which also measures the ATP used up by GS, but couples the generation of ADP to an optical test assay producing pyruvate from PEP with the generated ADP catalysed by pyruvate kinase present in the assay. This generated pyruvate is finally reduced to lactate by the present lactate dehydrogenase consuming NADH, the reduction of which is monitored at 340 nm.

The still increasing activity of GS after dodecamer formation (max. at 5 mM 2-OG) and the continuously increasing enzyme activity (max. at 12.5 mM 2-OG): See also public reviews, we assume that there are two effects caused by 2-OG: 1. cooperativity of binding (less 2-OG needed to facilitate dodecamer formation) and 2. priming of each active site.

The suggested additional experiments with and without ATP/Glutamate: Although we strongly agree that this would be a highly interesting structure, it seems out of the scope of a typical revision to request new cryo-EM structures. We evaluate the findings of our present study concerning the 2-OG effects as important insights into the strongly discussed field of glutamine synthetase regulation, even without the requested additional structures.

(4) Please remake Figure S2, the panels are too small to read the words. At least I have difficulty doing so.

We assume the reviewer is pointing to Suppl. Fig S3, we now changed this figure accordingly.

Line 153, the reference Schumacher et al. 23, should be 2023?

Yes, thank you. We corrected that.

Line 497. I believe it's UCSF ChimeraX, not Chimera.

We apologize and corrected accordingly.

Reviewer #3 (Recommendations For The Authors):

Recent studies on the Methanothermococcus thermolithotrophicus glutamine synthetase, published by Müller et al., 2024, have identified the binding site for 2-oxoglutarate as well as the conformational changes that were induced in the protein by its presence. In the present study, the authors confirm these observations and additionally establish a link between the presence of 2-oxoglutarate and the dodecameric fold and full activation of GS.

Curiously, here, the authors could not confirm their own findings that the dodecameric GS can directly interact with the PII-like GlnK1 protein and the small peptide sP26. However, the lack of mention of the GlnK-bound state in these studies is very alarming since it certainly is highly relevant here.

We agree with the reviewer that we have not observed the interaction with GlnK1 and sP26 in the recent study. Consequently, we speculate that yet unknown cellular factor(s) might be required for an interaction of GlnA1 with GlnK1 and sP26, which were not present in the in vitro experiments using purified proteins, however they were present in the previous pull-down approaches (Ehlers et al. 2005, Gutt et al. 2021). Another reason might be that post-translational modifications occur in M. mazei, which might be important for the interaction, which are also not present in purified proteins expressed in E. coli.

The manuscript interest could have been substantially increased if the authors had done finer biochemical and enzymatic analyses on the oligomerization process of GS, used GlnK1 bound to known effectors in their assays and would have done some more efforts to extrapolate their findings (even if a small niche) of related glutamine synthetases.

We thank the reviewer for their valuable encouragement to explore ligand-bound-states of GlnK1. However, in this manuscript we mainly focused on 2-OG as activator of GlnA1 and decided to dedicate future experiments to the exploration of conditions that possibly favor GlnK1-binding.

In principle, we have explored the ATP bound GlnK1 effects on GlnA1 activity in the activity assays (Fig. 2E) since ATP (3.6 mM) is present. GlnK1 however showed no effects on GlnA1 activity.

In general, the manuscript is poorly written, with grammatically incorrect sentences that at times, which stands in the way of passing on the message of the manuscript.

Particular points:

(1) It is mentioned that 2-OG induces the active oligomeric (dodecamer, 12-mer) state of GlnA1 without detectable intermediates. However, only 62 % of the starting inactive enzyme yields active 12-mers. Note that this is contradicted in line 212.

Thanks for pointing out this discrepancy. After removing all 2-OG as we did before MP-experiments, GlnA1 doesn’t reach full dodecamers anymore when 2-OG is re-added. This is not because the 2-OG amount is not enough to trigger full assembly, but because the protein is much more unstable in the absence of 2-OG, so we predict that some GlnA1 breaks during dialysis. See also answer reviewer #2 (1) and supplementary figure S1C.

Is there any protein precipitation upon the addition of 2-OG? Is all protein being detected in the assay, meaning, is monomer/dimer + dodecamer yields close to 100% of the total enzyme in the assay?

There is no protein precipitation upon the addition of 2-OG, indeed, GlnA1 is much more stable in the presence of 2-OG. In the mass photometry experiments, all particles are measured, precipitated protein would be visible as big entities in the MP.

Please add to Figure 1 the amount of monomer/dimer during titration. Some debate why there is no full conversion should be tentatively provided.

We agree with the reviewer and included the amount of monomer/dimer in the figure, as well as some discussion on why it is not fully converted again. GlnA1 is unstable without 2-OG and it was dialysed against buffer without 2-OG before MP measurements. This sample mistreatment resulted in no full re-assembly after re-adding 2-OG (although full dodecamers before dialysis (suppl. Fig. S1C).

(2) Figure 1B reflects an exemplary result. Here, the addition of 0.1 mM 2-OG seems to promote monomer to dimer transition. Why was this not studied in further detail? It seems highly relevant to know from which species the dodecamer is assembled.

We thank the reviewer for their comment. However, we would like to point out that, although not shown in the figure, GlnA1 is always mainly present as dimers as the smallest entity. As suggested earlier, we have added the amount of monomers/dimers to Figure 1A, which shows low monomer-counts at all 2-OG concentrations (Fig.1A). Although not depicted in the graph starting at 0.01 mM OG, we also see mainly dimers at 0 mM 2-OG.

How does the y-axis compare to the number and percentage of counts assigned to the peaks? In line 713, it is written that the percentage of dodecamer considers the total number of counts, and this was plotted against the 2-OG concentration.

We thank the reviewer for addressing this unclarity. Line 713 corresponds to Figure 1A, where we indeed plotted the percentage of dodecamer against the 2-OG-concentration. Thereby, the percentage of dodecamer corresponds to the percentage calculated from the Gaussian Fit of the MP-dodecamer-peak. In Figure 1 B, however, the y-axis displays the relative amount of counts per mass, multiple similar masses then add up to the percentage of the respective peak (Gaussian Fit above similar masses).

(3) Lines 714 and 721 (and elsewhere): Why only partial data is used for statistical purposes?

We in general only show one exemplary biological replicate, since the quality of the respective GlnA1 purification sometimes varied (maximum activity ranging from 5 - 10 U/mg). Therefore, we only compared activities within the same protein purification. For the EC50 calculations of all measurements, we refer to the supplement.

(4) Lines 192-193: It is claimed that GlnK1 was previously shown to both regulate the activity of GlnA1 and form a complex with GlnA1. Please mention the ratio between GlnK1 and GlnA1 in this complex.

We now included the requested information (GlnA1:GlnK1 1:1, (Ehlers et al. 2005); His6-GlnA1 (0.95 μM), His6-GlnK1 (0.65 μM); 2:1,4, Gutt et al. 2021).

It is also known that PII proteins such as GlnK1 can bind ADP, ATP, and 2-OG. Interestingly, however, for various described PII proteins, 2-OG can only bind after the binding of ATP.

So, the crucial question here is what is the binding state of GlnK1? 

Were these assays performed in the absence of ATP? This is key to fully understand and connect the results to the previous observations. For example, if the GlnK1 used was bound to ADP but not to ATP, then the added 2-OG might indeed only be able to affect GlnA1 (leading to its activation/oligomerization). If this were true and according to the data reported, ADP would prevent GlnK1 from interacting with any oligomeric form of GlnA1. However, if GlnK1 bound to ATP is the form that interacts with GlnA1 (potentially validating previous results?) then, 2-OG would first bind to GlnK1 (assuming a higher affinity of 2-OG to GlnK1), eventually causing its release from GlnA1 followed by binding and activation of GlnA1.

These experiments need to be done as they are essential to further understand the process. Given the ability of the authors to produce the protein and run such assays, it is unclear why they were not done here. As written in line 203, in this case, "under the conditions tested" is not a good enough statement, considering what is known in the field and how many more conclusions could easily be taken from such a setup.

Thanks for the encouragement to investigate the ligand-bound states of GlnK1. We agree and plan to perform the suggested mass photometry experiments exploring the conditions under which GlnA1 and GlnK1 might interact in future work. In GlnA1 activity test assays, when evaluating the presence/effects of GlnK1 on GlnA1 activity, however, ATP was always present in high concentrations and still we did not observe a significant effect of GlnK1 on the GlnA1 activity.

(5) Figure 2D legend claims that the graphic shows the percentage of dodecameric GlnA1 as a function of the concentration of 2-OG. This is not what the figure shows; Figure 2D shows the dodecamer/dimer (although legend claims monomer was used, in line 732) ratio as a function of 2-OG (stated in line 736!). If this is true, a ratio of 1 means 50 % of dodecamers and dimers co-exist. This appears to be the case when GlnK1 was added, while in the absence of GlnK1 higher ratios are shown for higher 2-OG concentration implying that about 3 times more dodecamers were formed than dimers. However, wouldn´t a 50 % ratio be physiologically significant?

We apologize for the partially incorrect and also misleading figure legend and corrected it. Indeed, the ratio of dodecamers and dimers is shown. Furthermore, we did not use monomeric GlnA1 (the smallest entity is mainly a dimer, see Fig 1A), however, the molarity was calculated based on the monomer-mass. Concerning the significance of the difference between the maximum ratio of GlnA1 and GlnK1: The ratio does appear higher, but this is mostly because adding large quantities of GlnK1 broadens all peaks at low molecular weight. This happens because the GlnK1 signal starts overlapping with the signal from GlnA1, leading to inflated GlnA1 dimer counts. We therefore do not think that this is biologically significant, especially as the activities do not differ under these conditions.

(6) Is it possible that the uncleaved GlnA1 tag is preventing interaction with GlnK1? This should be discussed.

This is of course a very important point. We however realized that Schumacher et al. also used an N-terminal His-tag, so we assume that the N-terminal tag is not hampering the interaction.

(7) Line 228: Please detail the reported discrepancies in rmsd between the current protein and the gram-negative enzymes.

The differences in rmsd between our M.mazei GlnA1 structure and the structure of gram-negative enzymes is caused by a) sequence similarity: E.g. M.mazei GlnA1 compared to B.subtilis GlnA have a sequence percent identity of 58.47; b) ligands in the structure: The B.Subtilis structure contains L-Methionine-S-sulfoximine phosphate, a transition state inhibitor, while the M. mazei  structure contains 2OG; c) Methodology: The structural determination methods also contribute to these differences. B. subtilis GlnA was determined using X-ray crystallography, while the M. mazei GlnA1 structure was resolved using Cryo-EM, where the protein behaves differently in ice compared to a crystal.

(8) Line 747: The figure title claims "dimeric interface" although the manuscript body only refers to "hexameric interface" or "inter-hexamer interface" (line 224). Moreover, the figure 4 legend uses terms such as vertical and horizontal dimers and this too should be uniformized within the manuscript.

Thank you for your valuable feedback. We have updated both the figure title and the figure legend as well in the main text to ensure consistency in the description.

(9) Line 752: The description of the color scheme used here is somehow unclear.

Thanks for pointing this out. We changed the description to make it more comprehensive.

(10) Please label H14/15 and H14´/H15´in Fig 4C zoom.

We agree that this has not been very clear. We added helix labels.

(11) In Figure 4D legend, make sure to note that the binding sites for the substrate are based on homologies with another enzyme poised with these molecules.

The same should be clear in the text: sites are not known, they are assumed to be, based on homologies (paragraph starting at line 239).

Concerning this comment we want to point out that we studied the exact same enzyme as the Schumacher group, except that we used 2-OG in our experiments, which they did not.

(12) Figure 3 appears redundant in light of Figure 4. 

(13) Line 235: When mentioning F24, please refer to Figure 5.

Thank you, we changed that accordingly.

(14) Please provide the distances for the bonds depicted in Figure 4B.

Thanks for pointing this out, we added distance labels to Figure 4B. For reasons of clarity only to three H-bonds.

(15) Line 241: D57 is likely serving to abstract a proton from ammonium, what is residue Glu307 potentially doing? The information seems missing in light of how the sentence is built.

Thanks for pointing this out. According to previous studies both residues are likely involved in proton abstraction - first from ammonium, and then from the formed gamma-ammonium group. Additionally, they contribute in shielding the active site from bulk solvent to prevent hydrolysis of the formed phospho-glutamate.

(16) Why do the authors assume that increased concentrations of 2-OG are a signal for N starvation only in M. mazei and not in all prokaryotic equivalent systems (line 288)?

In line 288, we did not claim that this is a unique signal for M. mazei. It is also the central N-starvation signal in Cyanobacteria but not directly perceived by the cyanobacterial GS through binding directly to GS.

The authors should look into the residues that bind 2-OG and check if they are conserved in other GS. The results of this sequence analysis should be discussed in line with the variable prokaryotic glutamine synthetase types of activity modulation that were exposed in the introduction and Figure 7.

Please refer to supplementary figure S5, where we already aligned the mentioned glutamine synthetase sequences. Since this was also already discussed in Müller et al. 2024, we did not want to repeat their observations and refer to our supplementary figure in too much detail.

(17) Figure 5 title: Replace TS by transition state structures of homology enzymes, or alike.

Thank you for this suggestion. We did not change the title however, since it is not a homologue but the exact same glutamine synthetase from Methanosarcina mazei.

(18) Line 249: D170 is not shown in Figure 5A or elsewhere in Figure 5.

Thank you for pointing this out. We added D170 to figure 5A.

(19) Representative density for the residues binding 2-OG should be provided, maybe in a supplemental figure.

Thank you for the suggestion. We added the densities of 2-OG-binding residues to figure 4B

(20) Line 260: Please add a reference when describing the phosphoryl transfer.

We thank the reviewer for this important point and added that accordingly.

(21) Line 296: The binding of 2-OG indeed appears to be cooperative, such that at concentrations above its binding affinity to the protein, only dodecamers are seen (under experimental conditions). However, claiming that the oligomerization is fast is not correct when the experimental setup includes 10 minutes of incubation before measurements are done. Please correct this within the entire manuscript.

A (fast) continuous kinetic assay could have confirmed this point and revealed the oligomerization steps and the intermediaries in the process (maybe monomer/dimers, then dimers/hexamers, and then hexamers/dodecamers). Such assays would have been highly valuable to this study.

We thank the reviewer for this suggestion, but disagree. It is indeed a rather fast regulation (as activity assays without pre-incubation only takes 1 min longer to reach full activity, see the newly included suppl. Fig S6). Considering other regulation mechanisms like e.g. transcription or translation regulation, an activation that takes only 60 s is actually quite quick.

(22) Line 305 (and elsewhere in the manuscript): the authors state that 2-OG primes the active site for a transition state. This appears incorrect. The transition state is the highest energy state in an enzymatic reaction progressing from substrate to product. Meaning, the transition state is a state that has a more or less modified form of the original substrate bound to the active site. This is not the case.

In line 366 an "active open state" appears much more adequate to use. 

We agree and changed accordingly throughout the manuscript.

(23) Line 330: Please delete "found". Eventually replace it with "confirmed": As the authors write, others have described this residue as a ligand to glutamine.

Thanks, we changed that accordingly, although previous descriptions were just based on homologies without the experimental validation.

(24) The discussion in at various points summarizing again the results. It should be trimmed and improved.

(25) Line 381: replace "two fast" with "fast"?

We thank the reviewer for this suggestion, but disagree on this point. We especially wanted to highlight that there are two central nitrogen-metabolites involved in the direct regulation of GlnA1, that means TWO fast direct processes mediated by 2-OG and glutamine.

Voici un sommaire minuté des idées fortes qui ressortent de l'entretien avec Éric Debarbieux:

• 0:00-1:13 Introduction Éric Debarbieux, spécialiste du climat scolaire, a publié "Zéro pointé ? Une histoire politique de la violence à l'école". L'ouvrage dresse un bilan mitigé des politiques menées pour prévenir la violence scolaire et s'inquiète des difficultés croissantes de l'école à gérer les troubles du comportement.
• 1:14-2:41 Intérêt pour la question de la violence à l'école: Debarbieux explique son intérêt pour la violence à l'école par son expérience de praticien en tant qu'éducateur spécialisé et instituteur spécialisé auprès de jeunes en difficulté. Il a voulu comprendre la violence plutôt que de se laisser submerger par elle.
• 2:42-3:41 Ce livre n'est pas sur "comment on fait" mais "comment on fait politiquement": Debarbieux précise que son livre ne se concentre pas sur des solutions pédagogiques, mais plutôt sur une analyse politique de la violence à l'école. Il continue d'intervenir sur le terrain, mais souhaite laisser la place aux jeunes chercheurs.
• 3:42-6:25 Évolution du regard sur la violence à l'école: Historiquement, la violence à l'école n'était pas une question politique et personne ne voulait la voir. Debarbieux retrace l'évolution du regard sur la violence à l'école, en commençant par le phénomène du chahut dans les années 60, qui était une forme de violence acceptée et ritualisée contre les professeurs.
• 6:26-7:42 Démocratisation du lycée et nouveaux publics: L'arrivée de nouveaux publics dans les lycées, suite à une volonté politique de démocratisation, a entraîné une contestation de l'ordre et une perte de sens pour certains élèves. Cette violence est devenue une violence antiscolaire.
• 7:43-9:17 Massification, exclusion sociale et ghettoïsation: La massification de l'école, l'exclusion sociale, les problèmes d'exclusion urbaine ont également eu un impact sur la violence à l'école. Initialement, cette violence était perçue comme venant uniquement de l'extérieur, ce qui était une erreur.
• 9:18-10:21 La violence vient aussi de l'intérieur: Une rupture claire se produit avec les manifestations de lycéens au début des années 90 contre la violence dans les établissements scolaires et pour plus de sécurité. Cela marque le début des politiques publiques et des plans antiviolence.
• 10:22-11:01 La puissance publique s'y intéresse: La puissance publique commence à s'intéresser à la question de la violence à l'école en raison de faits divers et de la pression médiatique.
• 11:02-12:55 Mesurer la violence: Un des grands combats scientifiques des années 90 est la mesure de la violence à l'école, ce qui implique de la définir. Un premier appel d'offre est lancé pour mieux comprendre le phénomène. Debarbieux et son équipe mènent une enquête auprès de 14000 élèves sans financement initial.
• 12:56-13:41 Expérience du terrain: L'expérience de Debarbieux est partie du terrain et il est resté en contact avec le terrain en permanence, ce qui lui a permis de poser de nouvelles questions et d'articuler la recherche avec la pratique.
• 13:42-16:21 Être un médiateur dans les équipes: Face à des enseignants qui ne veulent pas entendre parler de pédagogie coopérative ou d'élèves difficiles, il fallait être en mesure d'être un médiateur dans les équipes. La question de la violence n'est pas seulement celle de la violence des élèves, mais aussi des conflits d'équipe.
• 16:22-17:07 Enquête de victimation et de climat scolaire: Debarbieux et son collègue Yves Montoya ont créé une méthode d'enquête de victimation et de climat scolaire pour recueillir l'avis de tous les élèves. L'objectif était de restituer les enquêtes au terrain et de réfléchir avec les personnels sur ce qu'il pouvait faire.
• 17:08-18:39 Violence en milieu scolaire: La violence en milieu scolaire est souvent présentée comme un problème lié au comportement des élèves ou aux problèmes familiaux, mais rarement comme un problème de relations entre adultes. Or, le premier facteur de risque de violence à l'école est l'instabilité des équipes éducatives et la qualité de ces équipes, liée à la conflictualité.
• 18:40-19:52 Instabilité des équipes éducatives: Denise Godfredson a montré que le premier facteur de risque sur la violence à l'école est l'instabilité des équipes éducatives. Les équipes qui se déchirent ne peuvent pas prendre en charge les problèmes de violence, ce qui entraîne un repli dans la classe et une incivilité.
• 19:53-21:06 Violences entre adultes: Des travaux montrent l'importance des violences entre adultes. Une enquête menée par Debarbieux en Seine-Saint-Denis a quantifié le lien entre les conflits en équipe et les agressions subies par les élèves.
• 21:07-22:20 Aggravation des conflits: On observe une aggravation des conflits entre les directions et les enseignants, avec une augmentation du nombre de personnels qui se disent harcelés. Les enseignants se plaignent d'être harcelés par la hiérarchie, et les personnels de direction, par les enseignants.
• 22:21-23:05 Méfiance envers la hiérarchie: Ce que révèle aussi cette situation est la méfiance envers la haute hiérarchie et le personnel politique à la tête du ministère de l'Éducation nationale. Une grande majorité du personnel ne se sent pas soutenue, voire méprisée, par la haute hiérarchie.
• 23:06-24:28 Climat scolaire et conflits internes: Le climat scolaire, qui inclut la bonne entente d'équipe, est un facteur de protection contre la violence. Les conflits au sein de l'administration et des cabinets ministériels ont un impact direct sur les politiques publiques. Derrière cette conflictualité, il y a une conflictualité sociétale globale.
• 24:29-26:13 Harcèlement à l'école: Le harcèlement à l'école est un phénomène de groupe où des individus se liguent contre un autre, souvent motivé par le racisme, la xénophobie ou la transphobie. Les discours haineux tenus dans la société ont des répercussions dans les cours de récréation.
• 26:14-27:25 Difficulté à gérer les enfants en difficulté: L'institution a de plus en plus de mal à gérer les enfants en grande difficulté comportementale.
• 27:26-28:22 Stabilité des enquêtes de victimation: Les enquêtes de victimation montrent une stabilité, voire une petite aggravation récente, de la violence à l'école. Un des phénomènes préoccupants est la difficulté de l'école primaire face aux enfants à troubles du comportement, en lien avec l'école inclusive.
• 28:23-29:22 Augmentation des problèmes avec les enfants à troubles: On observe une augmentation du nombre d'enseignants qui disent avoir des problèmes fréquents avec des enfants à troubles du comportement, passant de 40% à plus de 70%.
• 29:23-30:47 Craintes des enseignants: Les enseignants expriment un cri de désespoir et demandent de l'aide. En 2023, une partie d'entre eux souhaitent que ces enfants soient placés dans des centres spécialisés. Debarbieux souligne que cela ne se fera pas pour des raisons économiques et que l'école inclusive ne suffit pas.
• 30:48-32:36 Désespoir des enseignants: Le désespoir d'un nombre incroyable d'enseignants du premier degré fait craindre un danger réel pour le maintien de l'offre éducative dans le primaire. Il y a une désaffection pour le métier d'enseignant, notamment en raison de la difficulté à gérer les enfants difficiles.
• 32:37-33:12 Sentiment d'impuissance: Pour Debarbieux, cette désaffection est liée au découragement et au sentiment d'impuissance des enseignants. Une majorité d'entre eux estime ne pas avoir été suffisamment formée.
• 33:13-34:09 Formation continue: Il est important de proposer une formation continue de qualité, dispensée par des personnes connaissant le terrain et capables de sortir des discours théoriques.
• 34:10-35:27 Désintérêt pour les questions scientifiques: Debarbieux constate un désintérêt pour les questions scientifiques dans les sphères politiques et un intérêt pour le court-termisme. Il nuance en précisant qu'il a souvent été appelé à la rescousse, mais que l'intérêt pour la science arrive souvent tardivement.
• 35:28-36:24 Claude Allègre: Claude Allègre a été le premier à s'intéresser véritablement au point de vue scientifique, mais sa communication maladroite a nui à ses efforts.
• 36:25-37:22 La communication l'emporte: La communication l'emporte souvent sur la science, surtout depuis l'avènement du web 2.0 et de la réponse immédiate. Luc Chatel, par exemple, était dans le court-termisme et la réponse musclée.
• 37:23-38:02 Tentative d'informer les politiques publiques par la science: Luc Chatel a ensuite tenté d'informer les politiques publiques par la science, avec les États généraux sur la sécurité à l'école et les Assises contre le harcèlement. Cette politique a été continuée par la gauche au pouvoir.
• 38:03-39:02 Arrivée de Blanquer: L'arrivée de Blanquer a mis fin à cette continuité et a imposé un autre programme, cassant notamment la délégation ministérielle dirigée par Debarbieux.
• 39:03-40:00 Instabilité ministérielle: L'instabilité ministérielle et la volonté de chaque ministre de laisser sa marque lassent le terrain et nuisent à l'action publique. Même ceux qui essaient de faire quelque chose deviennent prisonniers de ce climat de rejet et d'autoritarisme. Gabriel Attal, par exemple, a commencé par des plans sur l'empathie et a terminé par une loi contre la jeunesse.
• 40:01-41:13 Moment harcèlement en politique: Le "moment harcèlement" en politique marque une bascule où l'on comprend que la violence n'est pas forcément extérieure aux établissements et qu'il faut la prévenir. Il y a un intérêt pour les victimes que l'on ne sentait pas trop avant.
• 41:14-42:27 Vision de la violence à l'école: Jusqu'en 2010-2011, la vision de la violence à l'école est celle d'une violence provenant de l'extérieur, nécessitant de se protéger en renforçant la sécurité et le partenariat avec la police et la justice.
• 42:28-43:12 Essentiel de la violence à l'école: L'essentiel de la violence à l'école n'est pas constitué d'intrusions, mais de violences banales et ordinaires qui, lorsqu'elles s'accumulent, ont des effets délétères sur les victimes, les témoins et les agresseurs.
• 43:13-44:40 Enquête pour l'UNICEF: Une enquête menée par Debarbieux pour l'UNICEF a révélé qu'environ 10% des élèves sont victimes à répétition de harcèlement. Ces chiffres ont été fortement médiatisés et ont conduit à l'organisation des Assises nationales contre le harcèlement.
• 44:41-46:17 Basculement: Ce moment a aussi été la révélation d'un phénomène où tout le monde s'est dit "c'est bien sûr", en lien avec le harcèlement au travail et le mouvement #MeToo. On ne supporte plus les micro-violences qui étaient auparavant considérées comme banales. Il y a une évolution sociétale intéressante, mais on observe un retour de bâton dramatique.
• 46:18-47:06 Mesures inefficaces: Certaines mesures politiques prises sont inefficaces, comme la sanctuarisation de l'école ou le regroupement des enfants difficiles.
• 47:07-48:01 Discipline militaire: L'exemple de la discipline militaire, proposée par différents responsables politiques, a été essayé et évalué, et s'est avéré inefficace et coûteux. Les militaires eux-mêmes reconnaissent ne pas savoir faire.
• 48:02-50:04 Boot camps: Les "boot camps" aux États-Unis ont également montré leur inefficacité. Le regroupement des individus difficiles, par exclusion interne ou externe, augmente leur capacité à faire bande. C'est un principe de criminologie de base.
• 50:05-51:13 Internat: Debarbieux n'est pas contre l'idée d'internat, mais celui-ci doit être souhaité et ne pas devenir une punition. De même, l'enseignement professionnel ne doit pas devenir une punition pour les mauvais élèves.
• 51:14-53:07 Programmes importés: La transposition directe de programmes contre le harcèlement ou la violence provenant d'autres pays, notamment d'Europe du Nord, est également inefficace. Il n'y a pas de programme miracle et il faut tenir compte des contextes et de la manière dont les équipes s'en emparent.
• 53:08-54:27 Responsabilité sur le harcelé: Mettre toute la responsabilité du harcèlement sur le harcelé est un effet pervers. Les adultes doivent être présents et aider les élèves à s'aider eux-mêmes.
• 54:28-55:11 Prévention indirecte: Une prévention indirecte, basée sur des choses triviales mais montrant que l'on fait attention aux élèves et à leur bien-être corporel, peut être plus efficace. L'exemple des toilettes est souvent cité.
• 55:12-56:17 Question politique: Quoi qu'il en soit, la question politique doit être posée majoritairement. Debarbieux se dit en désaccord avec l'idée que la violence est due à l'ensauvagement de la jeunesse. Il ne s'agit pas d'être laxiste, mais de ne pas voir la situation uniquement à travers le prisme de la répression policière.
• 56:18-57:12 Syndicalisme policier: Debarbieux s'inquiète de l'évolution du syndicalisme policier, où l'on a tendance à réduire le rôle du policier à "petites tête, gros bâton".
• 57:13-58:30 Ce qui fonctionne: Il faut agir sur le terrain en favorisant une approche climat scolaire globale, où l'on veille au bien-être de l'équipe, à la communication, à la qualité du leadership, et où l'on met en place un système disciplinaire cohérent et appliqué par tous.
• 58:31-59:06 Climat scolaire: Le climat scolaire ne doit pas être enfermé dans l'établissement, mais doit prendre en compte l'environnement extérieur, les parents et le quartier. Il faut se demander si l'on veut une école "du" quartier ou une école "dans" le quartier.
• 59:07-1:00:05 Actions possibles au ministère: Au niveau du ministère, il est possible d'agir à condition d'éviter la circularité et les discours théoriques, et en apportant une aide maximale à la formation de long terme.
• 1:00:06-1:00:13 Être combatif: Malgré les difficultés, il faut rester combatif et continuer à se battre.

Éric Debarbieux, chercheur en sciences de l'éducation, aborde la question de la violence scolaire et de son évolution. Voici les points clés de son analyse :

• Évolution de la perception de la violence scolaire Historiquement, la violence à l'école n'était pas considérée comme une question politique ou sociale importante. Elle était parfois acceptée ou ritualisée, comme le chahut. L'évolution a été marquée par l'arrivée de nouveaux publics dans les lycées, la massification scolaire, le chômage de masse et l'exclusion sociale. La violence était initialement perçue comme venant de l'extérieur de l'école, mais une rupture s'est produite dans les années 1990 avec les manifestations lycéennes contre la violence, menant à des politiques publiques et des plans de prévention.

• La violence entre adultes Debarbieux souligne que la violence en milieu scolaire n'est pas seulement liée au comportement des élèves, mais aussi aux relations entre adultes. L'instabilité des équipes éducatives et la conflictualité entre adultes sont des facteurs de risque importants. Des études montrent un lien entre les conflits entre adultes et les agressions subies par les élèves. Il y a une aggravation des conflits entre directions et enseignants, avec des sentiments de harcèlement. La méfiance envers la hiérarchie et le personnel politique au ministère de l'Éducation nationale complexifie la situation.

• Difficultés face aux troubles du comportement Les écoles primaires rencontrent des difficultés croissantes avec les enfants ayant des troubles du comportement, ce qui crée un sentiment de désespoir chez les enseignants. L'école inclusive est un facteur, mais il ne faut pas assimiler handicap et violence. Le manque de formation et de soutien adéquat pour les enseignants contribue à ce problème.

• Inefficacité de certaines mesures politiques Debarbieux critique certaines mesures politiques comme la discipline militaire, les "boot camps", et le regroupement d'élèves difficiles. Il remet en question la transposition directe de programmes contre le harcèlement venant d'autres pays, soulignant l'importance de les adapter aux contextes locaux. Il insiste sur la nécessité de ne pas déresponsabiliser les adultes et de prendre en compte la dimension politique et sociétale de la violence.

• Pistes pour des solutions Debarbieux met en avant l'importance de l'approche du climat scolaire, qui prend en compte le bien-être de l'équipe éducative, la communication, la qualité du leadership et un système disciplinaire cohérent. Il souligne également la nécessité d'impliquer les parents et la communauté.

Reviewer #1 (Public review):

Wang et al., recorded concurrent EEG-fMRI in 107 participants during nocturnal NREM sleep to investigate brain activity and connectivity related to slow oscillations (SO), sleep spindles, and in particular their co-occurrence. The authors found SO-spindle coupling to be correlated with increased thalamic and hippocampal activity, and with increased functional connectivity from the hippocampus to the thalamus and from the thalamus to the neocortex, especially the medial prefrontal cortex (mPFC). They concluded the brain-wide activation pattern to resemble episodic memory processing, but to be dissociated from task-related processing and suggest that the thalamus plays a crucial role in coordinating the hippocampal-cortical dialogue during sleep.

The paper offers an impressively large and highly valuable dataset that provides the opportunity for gaining important new insights into the network substrate involved in SOs, spindles, and their coupling. However, the paper does unfortunately not exploit the full potential of this dataset with the analyses currently provided, and the interpretation of the results is often not backed up by the results presented.

I have the following specific comments.

(1) The introduction is lacking sufficient review of the already existing literature on EEG-fMRI during sleep and the BOLD-correlates of slow oscillations and spindles in particular (Laufs et al., 2007; Schabus et al., 2007; Horovitz et al., 2008; Laufs, 2008; Czisch et al., 2009; Picchioni et al., 2010; Spoormaker et al., 2010; Caporro et al., 2011; Bergmann et al., 2012; Hale et al., 2016; Fogel et al., 2017; Moehlman et al., 2018; Ilhan-Bayrakci et al., 2022). The few studies mentioned are not discussed in terms of the methods used or insights gained.

(2) The paper falls short in discussing the specific insights gained into the neurobiological substrate of the investigated slow oscillations, spindles, and their interactions. The validity of the inverse inference approach ("Open ended cognitive state decoding"), assuming certain cognitive functions to be related to these oscillations because of the brain regions/networks activated in temporal association with these events, is debatable at best. It is also unclear why eventually only episodic memory processing-like brain-wide activation is discussed further, despite the activity of 16 of 50 feature terms from the NeuroSynth v3 dataset were significant (episodic memory, declarative memory, working memory, task representation, language, learning, faces, visuospatial processing, category recognition, cognitive control, reading, cued attention, inhibition, and action).

(3) Hippocampal activation during SO-spindles is stated as a main hypothesis of the paper - for good reasons - however, other regions (e.g., several cortical as well as thalamic) would be equally expected given the known origin of both oscillations and the existing sleep-EEG-fMRI literature. However, this focus on the hippocampus contrasts with the focus on investigating the key role of the thalamus instead in the Results section.

(4) The study included an impressive number of 107 subjects. It is surprising though that only 31 subjects had to be excluded under these difficult recording conditions, especially since no adaptation night was performed. Since only subjects were excluded who slept less than 10 min (or had excessive head movements) there are likely several datasets included with comparably short durations and only a small number of SOs and spindles and even less combined SO-spindle events. A comprehensive table should be provided (supplement) including for each subject (included and excluded) the duration of included NREM sleep, number of SOs, spindles, and SO+spindle events. Also, some descriptive statistics (mean/SD/range) would be helpful.

(5) Was the 20-channel head coil dedicated for EEG-fMRI measurements? How were the electrode cables guided through/out of the head coil? Usually, the 64-channel head coil is used for EEG-fMRI measurements in a Siemens PRISMA 3T scanner, which has a cable duct at the back that allows to guide the cables straight out of the head coil (to minimize MR-related artifacts). The choice for the 20-channel head coil should be motivated. Photos of the recording setup would also be helpful.

(6) Was the EEG sampling synchronized to the MR scanner (gradient system) clock (the 10 MHz signal; not referring to the volume TTL triggers here)? This is a requirement for stable gradient artifact shape over time and thus accurate gradient noise removal.

(7) The TR is quite long and the voxel size is quite large in comparison to state-of-the-art EPI sequences. What was the rationale behind choosing a sequence with relatively low temporal and spatial resolution?

(8) The anatomically defined ROIs are quite large. It should be elaborated on how this might reduce sensitivity to sleep rhythm-specific activity within sub-regions, especially for the thalamus, which has distinct nuclei involved in sleep functions.

(9) The study reports SO & spindle amplitudes & densities, as well as SO+spindle coupling, to be larger during N2/3 sleep compared to N1 and REM sleep, which is trivial but can be seen as a sanity check of the data. However, the amount of SOs and spindles reported for N1 and REM sleep is concerning, as per definition there should be hardly any (if SOs or spindles occur in N1 it becomes by definition N2, and the interval between spindles has to be considerably large in REM to still be scored as such). Thus, on the one hand, the report of these comparisons takes too much space in the main manuscript as it is trivial, but on the other hand, it raises concerns about the validity of the scoring.

(10) Why was electrode F3 used to quantify the occurrence of SOs and spindles? Why not a midline frontal electrode like Fz (or a number of frontal electrodes for SOs) and Cz (or a number of centroparietal electrodes) for spindles to be closer to their maximum topography?

(11) Functional connectivity (hippocampus -> thalamus -> cortex (mPFC)) is reported to be increased during SO-spindle coupling and interpreted as evidence for coordination of hippocampo-neocortical communication likely by thalamic spindles. However, functional connectivity was only analysed during coupled SO+spindle events, not during isolated SOs or isolated spindles. Without the direct comparison of the connectivity patterns between these three events, it remains unclear whether this is specific for coupled SO+spindle events or rather associated with one or both of the other isolated events. The PPIs need to be conducted for those isolated events as well and compared statistically to the coupled events.

(12) The limited temporal resolution of fMRI does indeed not allow for easily distinguishing between fMRI activation patterns related to SO-up- vs. SO-down-states. For this, one could try to extract the amplitudes of SO-up- and SO-down-states separately for each SO event and model them as two separate parametric modulators (with the risk of collinearity as they are likely correlated).

(13) L327: "It is likely that our findings of diminished DMN activity reflect brain activity during the SO DOWN-state, as this state consistently shows higher amplitude compared to the UP-state within subjects, which is why we modelled the SO trough as its onset in the fMRI analysis." This conclusion is not justified as the fact that SO down-states are larger in amplitude does not mean their impact on the BOLD response is larger.

(14) Line 77: "In the current study, while directly capturing hippocampal ripples with scalp EEG or fMRI is difficult, we expect to observe hippocampal activation in fMRI whenever SOs-spindles coupling is detected by EEG, if SOs- spindles-ripples triple coupling occurs during human NREM sleep". Not all SO-spindle events are associated with ripples (Staresina et al., 2015), but hippocampal activation may also be expected based on the occurrence of spindles alone (Bergmann et al., 2012).

References:

Bergmann TO, Molle M, Diedrichs J, Born J, Siebner HR (2012) Sleep spindle-related reactivation of category-specific cortical regions after learning face-scene associations. Neuroimage 59:2733-2742.<br /> Caporro M, Haneef Z, Yeh HJ, Lenartowicz A, Buttinelli C, Parvizi J, Stern JM (2011) Functional MRI of sleep spindles and K-complexes. Clin Neurophysiol.<br /> Czisch M, Wehrle R, Stiegler A, Peters H, Andrade K, Holsboer F, Samann PG (2009) Acoustic oddball during NREM sleep: a combined EEG/fMRI study. PLoS One 4:e6749.<br /> Fogel S, Albouy G, King BR, Lungu O, Vien C, Bore A, Pinsard B, Benali H, Carrier J, Doyon J (2017) Reactivation or transformation? Motor memory consolidation associated with cerebral activation time-locked to sleep spindles. PLoS One 12:e0174755.<br /> Hale JR, White TP, Mayhew SD, Wilson RS, Rollings DT, Khalsa S, Arvanitis TN, Bagshaw AP (2016) Altered thalamocortical and intra-thalamic functional connectivity during light sleep compared with wake. Neuroimage 125:657-667.<br /> Horovitz SG, Fukunaga M, de Zwart JA, van Gelderen P, Fulton SC, Balkin TJ, Duyn JH (2008) Low frequency BOLD fluctuations during resting wakefulness and light sleep: a simultaneous EEG-fMRI study. Hum Brain Mapp 29:671-682.<br /> Ilhan-Bayrakci M, Cabral-Calderin Y, Bergmann TO, Tuscher O, Stroh A (2022) Individual slow wave events give rise to macroscopic fMRI signatures and drive the strength of the BOLD signal in human resting-state EEG-fMRI recordings. Cereb Cortex 32:4782-4796.<br /> Laufs H (2008) Endogenous brain oscillations and related networks detected by surface EEG-combined fMRI. Hum Brain Mapp 29:762-769.<br /> Laufs H, Walker MC, Lund TE (2007) 'Brain activation and hypothalamic functional connectivity during human non-rapid eye movement sleep: an EEG/fMRI study'--its limitations and an alternative approach. Brain 130:e75; author reply e76.<br /> Moehlman TM, de Zwart JA, Chappel-Farley MG, Liu X, McClain IB, Chang C, Mandelkow H, Ozbay PS, Johnson NL, Bieber RE, Fernandez KA, King KA, Zalewski CK, Brewer CC, van Gelderen P, Duyn JH, Picchioni D (2018) All-Night Functional Magnetic Resonance Imaging Sleep Studies. J Neurosci Methods.<br /> Picchioni D, Horovitz SG, Fukunaga M, Carr WS, Meltzer JA, Balkin TJ, Duyn JH, Braun AR (2010) Infraslow EEG oscillations organize large-scale cortical-subcortical interactions during sleep: A combined EEG/fMRI study. Brain Res.<br /> Schabus M, Dang-Vu TT, Albouy G, Balteau E, Boly M, Carrier J, Darsaud A, Degueldre C, Desseilles M, Gais S, Phillips C, Rauchs G, Schnakers C, Sterpenich V, Vandewalle G, Luxen A, Maquet P (2007) Hemodynamic cerebral correlates of sleep spindles during human non-rapid eye movement sleep. Proc Natl Acad Sci U S A 104:13164-13169.<br /> Spoormaker VI, Schroter MS, Gleiser PM, Andrade KC, Dresler M, Wehrle R, Samann PG, Czisch M (2010) Development of a large-scale functional brain network during human non-rapid eye movement sleep. J Neurosci 30:11379-11387.<br /> Staresina BP, Bergmann TO, Bonnefond M, van der Meij R, Jensen O, Deuker L, Elger CE, Axmacher N, Fell J (2015) Hierarchical nesting of slow oscillations, spindles and ripples in the human hippocampus during sleep. Nat Neurosci 18:1679-1686.

briefing détaillé basé sur les extraits de la vidéo ARTE "Notre microbiote nous domine-t-il ?":

Briefing Document : "Notre microbiote nous domine-t-il ?"

Source : Extraits de la vidéo ARTE "Notre microbiote nous domine-t-il ? | 42 - La réponse à presque tout" (14 février 2025).

Thèmes Principaux :

L'importance du Microbiote : Le documentaire met en évidence le rôle crucial du microbiote (ensemble des micro-organismes) pour la santé humaine. Il souligne que nous sommes totalement dépendants de ces bactéries : "On est totalement dépendant de ces bactéries sans son microbiote l'être humain ne survivrait pas". Il y a plus de micro-organismes vivant dans et sur un seul adulte que d'humains sur la planète. Diversité et Santé : Une grande diversité microbienne est corrélée à une meilleure santé. Le documentaire souligne que " plus il est diversifié, meilleure est notre santé." Cette diversité est essentielle pour la création de réseaux complexes au sein du microbiote, comparée à un chantier où différents ouvriers spécialisés sont nécessaires pour construire un bâtiment solide. La diversité du microbiote est bénéfique pour l'organisme et le système immunitaire : "une grande diversité est bénéfique à notre organisme et notre système immunitaire". Une baisse de la diversité est observée dans beaucoup de maladies chroniques. Menaces sur la Biodiversité Microbienne : L'urbanisation, les règles d'hygiène, l'évolution de l'alimentation et des modes de vie conduisent à une perte de biodiversité microbienne : "L’urbanisation, les règles d’hygiène ainsi que l’évolution de l’alimentation et des modes de vie conduisent cependant à une perte de biodiversité microbienne". L'environnement dans lequel on vit a une influence directe sur notre microbiote. Le recours aux pesticides est identifié comme une menace majeure. Urbanisation et Microbiote : La vidéo aborde l'impact de l'urbanisation sur le microbiote, suggérant que le microbiote d'un citadin est très différent de celui d'un campagnard. Avec l'augmentation de la population urbaine, il est crucial de comprendre ces changements. Il est supposé que l'appauvrissement du microbiote est lié à l'urbanisation. "on suppose que l'appauvrissement du microbiote est lié à l'urbanisation." La Banque Mondiale du Microbiote : Face à la perte de diversité, une banque suisse conserve des échantillons congelés de matière fécale du monde entier afin de préserver le microbiote de l'humanité : "notre coffre fort contient des communautés microbiennes plus exactement des échantillons congelés de matière fécale provenant du monde entier." Cette initiative est comparée à l'Arche de Noé pour les microbes, visant à préserver des espèces potentiellement importantes pour la santé future. L'Axe Intestin-Cerveau : Le documentaire met en lumière la connexion entre l'intestin et le cerveau, soulignant que l'intestin est le principal producteur de sérotonine, influençant notre humeur : "grâce à son microbiote l'intestin est le principal producteur de sérotonine ce qui influence notre humeur". L'axe intestin-cerveau est décrit comme une route d'information biochimique du ventre à la tête et vice-versa, empruntée par les métabolites produits par les bactéries. Influence de l'Alimentation : L'alimentation joue un rôle crucial dans la composition du microbiote. Les bactéries raffolent des fibres présentes dans les fruits, légumes, fruits à coque et céréales complètes. Une alimentation pauvre en fibres peut conduire les bactéries à grignoter la muqueuse intestinale protectrice. "notre microbiote intestinale dépend de notre alimentation ce qu'on mange nos bactéries le mangent aussi". Colonisation Microbienne : Les trois premières années de vie sont décisives pour l'établissement du microbiote. La naissance par voie basse favorise une meilleure colonisation microbienne que par césarienne. Impact des Antibiotiques : L'utilisation d'antibiotiques, en particulier à large spectre, peut avoir des effets marqués et durables sur le microbiote, surtout s'ils sont administrés dès la petite enfance. Transplantation Fécale : La transplantation fécale est présentée comme une méthode pour restaurer un microbiote sain en réintroduisant les microbes manquants. Reconnexion à l'Environnement : Le documentaire suggère des moyens de stimuler la diversité microbienne, comme avoir des animaux domestiques, des plantes d'intérieur, ouvrir les fenêtres et vivre dans un environnement avec des arbres. Il est recommandé de se frotter au monde extérieur et d'éviter la stérilisation excessive. Idées/Faits Importants :

Le corps humain abrite environ 30 billions de micro-organismes. La diversité microbienne est un indicateur clé de la santé. L'urbanisation et les modes de vie modernes appauvrissent le microbiote. La "banque du microbiote" en Suisse est une tentative de préserver la diversité microbienne. L'alimentation, l'environnement et l'utilisation d'antibiotiques influencent la composition du microbiote. La transplantation fécale est une approche thérapeutique prometteuse. Il est possible de modifier son microbiote par des habitudes de vie saines.

Voici un sommaire minuté de la conférence-débat "Cosmétiques : Lever le voile sur les perturbateurs endocriniens", mettant en évidence les idées forces:

• 0:03-0:31 Introduction de la conférence sur les perturbateurs endocriniens (PE) dans les cosmétiques et annonce d'un bar pour continuer la discussion.

• 0:37-2:01 Définition des perturbateurs endocriniens par l'Organisation Mondiale de la Santé (OMS) comme des substances altérant le système endocrinien, présents dans divers produits du quotidien, y compris les cosmétiques. Le marché mondial des cosmétiques est en essor, avec la France en leader. L'utilisation de PE est courante pour la conservation des produits, malgré leurs effets néfastes potentiels sur la santé et l'environnement.

• 2:06-3:07 Présentation des intervenants : Aurélie Portefaix (pédiatre), Luc Jugla (chimiste), Céline de Laurens (adjointe à la santé de Lyon), et Edouard Raffin (avocat en droit de l'environnement).

• 3:14-8:52 Définition du système endocrinien et explication de son fonctionnement par Aurélie Portefaix. Les PE sont des substances ou mélanges de substances hétérogènes. Certains PE ont une durée de vie courte, tandis que d'autres persistent longtemps dans l'organisme. L'étude Esteban a révélé une exposition généralisée aux parabènes, phtalates et pesticides. Les PE peuvent mimer ou bloquer l'action des hormones, altérer la synthèse des protéines ou provoquer des mutations épigénétiques transmissibles à la descendance.

• 8:58-10:14 Importance de la question des PE dans les cosmétiques en raison de leur utilisation fréquente, de la perméabilité de la peau et de l'interaction contenant-contenu.

• 10:14-11:25 Effets des PE sur l'environnement : le juriste se base sur les analyses scientifiques pour légiférer. Le mécanisme juridique encadre la mise sur le marché des produits contenant des PE, avec identification, restriction ou interdiction.

• 11:25-14:02 Les effets sur l'environnement sont aussi nombreux que les types de PE. Le risque est extrêmement répandu et grave, avec des implications de plus en plus fortes découvertes au fil des ans. Les PE perturbent le système hormonal et sont invisibles et insidieux.

• 14:02-14:42 Effets des PE sur la faune : troubles de la reproduction.

• 15:25-17:39 Raisons de l'utilisation des PE dans les cosmétiques : conservation, résistance, absorption des rayons solaires. Les parabènes sont utilisés comme conservateurs à large spectre et peu coûteux. L'industrie s'adapte en réduisant l'utilisation de certaines familles de PE.

• 17:39-20:55 Enjeux politiques liés aux PE, tant au niveau de la pollution environnementale que de l'exposition individuelle. Les réglementations visent à protéger la santé, mais les autorisations de mise sur le marché reposent souvent sur des estimations négociées avec les industriels.

• 20:55-23:35 Nécessité de recréer un lien avec la science et de renforcer la confiance des citoyens dans le fonctionnement de la société.

• 23:35-25:36 Catégorisation des substances comme perturbateurs endocriniens : PE avérés, présumés ou suspectés. Preuve scientifique d'un effet néfaste, mode d'action compatible et lien de causalité établi sont nécessaires.

• 25:36-29:32 Mécanismes législatifs et réglementaires existants pour encadrer l'utilisation des PE. Le droit est influencé par les lobbyes, nécessitant un équilibre entre réglementation protectrice et compétitivité. Le règlement REACH encadre l'enregistrement, l'évaluation et l'autorisation des substances chimiques.

• 29:32-34:45 Le but est de protéger les consommateurs et leur santé en interdisant ou en restreignant les substances selon leur classification. La France a été pionnière avec les stratégies nationales sur les perturbateurs endocriniens (SNPE). La loi anti-gaspillage et économie circulaire de 2020 impose une meilleure information sur les produits. Un décret de 2023 relatif à l'information sur certains produits de protection intime est un exemple concret.

• 34:45-37:29 Rôle de l'ANSES : Agence de l'État qui répond à une commande, parfois en retard par rapport aux risques. Les avis de l'ANSES sont parfois timorés et la temporalité n'est pas la même que celle des chercheurs académiques. L'ANSES doit tenir compte des réalités économiques et prend des pincettes sur certains sujets.

• 37:29-46:37 Actions de la ville de Lyon : Participation à des instances de coordination, signature de la charte des villes et territoires sans perturbateur endocrinien. Mise en place d'une politique publique locale avec un plan d'action axé sur les écoles et les crèches. Actions concrètes : bio dans les cantines, fin du plastique, nettoyage sans chimie. La ville se concentre sur les 1000 premiers jours et les enfants.

• 46:37-51:33 Choix de ne pas utiliser de PE : Prise de conscience des problématiques environnementales. Dès qu'il y a suspicion, on a décidé de ne pas les utiliser. Transparence dans la composition des produits cosmétiques. Adaptation rapide de la profession aux demandes des clients.

• 51:33-54:03 Progrès scientifiques pour identifier et prouver les effets des PE : Essais randomisés contrôlés difficiles à mettre en place. Études épidémiologiques sur le temps long, biomarqueurs, tests in vitro et in vivo. Recherche sur l'exposome, c'est-à-dire l'ensemble des expositions au cours de la vie.

• 54:03-58:22 Alternatives aux PE : Utilisation d'huiles essentielles (avec prudence), reformulation des conservateurs. Difficultés à remplacer les dérivés fluorés dans le maquillage et les filtres solaires organiques.

• 58:22-1:03:26 Adaptation du consommateur, harmonisation des pratiques, remise à jour de REACH. Libre arbitre et responsabilité individuelle.

• 1:03:26-1:04:46 Les pouvoirs publics et les autorités vont chercher un équilibre avec la compétitivité économique et un caractère raisonnable.

• 1:04:46-1:11:30 Prise de conscience dans la population et des applications comme Yuka.

• 1:04:46-1:17:20 Les risques de non-conformité et les conséquences légales : Les agences européennes ou les agences françaises vont subir à la fois des sanctions administratives et pénales.

• 1:17:20-1:35:27 Les consommateurs qui vont être atteints de symptômes qu' ils ne comprennent pas vont voir leurs médecins et vont ensuite chercher des réponses et vont peut-être intenter un procès administratif ou pénal contre un fabricant ou un distributeur.

• 1:35:27-1:43:23 On manque d'études épidémiologiques, donc il est très difficile à rapporter.

• 1:43:23-1:46:12 Travail sur l'amélioration de cette évaluation scientifique qui passe par une transparence dans les produits qu'on va consommer.

• 1:46:12-1:48:14 Discussion avec le public.

Bien sûr. Voici un résumé de la vidéo avec les idées fortes en gras :

• [00:00:06] Introduction de l'émission et des participants. François Dubet, sociologue et directeur d'études, est l'invité pour discuter de son livre "Tous égaux, tous singuliers", qui traite des inégalités et de la solidarité.

• [00:00:39] Évolution de la notion d'inégalité sociale : Les inégalités sont mesurables, mais la question centrale est de comprendre pourquoi certaines sont acceptées et d'autres suscitent l'indignation. Depuis une trentaine d'années, deux grands changements ont marqué les sociétés : l'explosion des classes sociales et le passage d'une conception de la justice sociale axée sur la réduction des inégalités de conditions à une conception axée sur l'égalité des chances.

• [00:01:44] Justice sociale vs. égalité des chances : La justice sociale est liée à l'affirmation que les hommes naissent libres et égaux, mais vivent dans des sociétés inégalitaires. Deux modèles existent : un modèle européen axé sur la réduction des inégalités de position et un modèle américain axé sur l'égalité des chances. Le second modèle tend à dominer, car les supports sociaux du premier (syndicats, classe ouvrière) s'affaiblissent. L'injustice sociale majeure est perçue comme la discrimination plutôt que l'exploitation.

• [00:02:28] Régime des inégalités vs. inégalités : Les inégalités sont mesurables, tandis que le régime des inégalités est le mode de construction et de justification de ces inégalités. Le premier régime d'inégalités était celui des castes, où les inégalités étaient considérées comme naturelles. Les révolutions démocratiques ont aboli ce régime, mais des traces subsistent, notamment dans les inégalités entre les sexes. Les sociétés industrielles ont ensuite structuré les inégalités autour des fonctions sociales et du marché capitaliste.

• [00:03:15] L'expérience des inégalités : Dans l'ancien régime, les inégalités étaient vécues collectivement, avec une fierté et une culture de classe. Aujourd'hui, les mutations du capitalisme ont individualisé l'expérience des inégalités. Chacun se sent inégal en fonction de multiples facteurs (niveau d'études, lieu de résidence, âge, etc.) plutôt qu'en tant que membre d'une classe sociale.

• [00:03:44] Le mouvement des "gilets jaunes" : Ce mouvement est caractérisé par une colère collective, mais sans revendications claires ni sentiment d'appartenance à une classe. Il y a une hostilité envers les élites et une difficulté à formuler des revendications. Le sentiment dominant est le mépris, où chacun se sent dévalorisé et non reconnu.

• [00:04:18] Déconnexion entre inégalités mesurées et inégalités perçues : L'expérience des inégalités est la rencontre entre les inégalités objectives, la représentation qu'on en a et les conceptions de la justice mobilisées. Les inégalités hommes-femmes ont diminué, mais le sentiment d'injustice est plus fort, car les femmes sont davantage confrontées aux inégalités et ont une plus grande conscience de l'égalité. De même, les inégalités scolaires sont perçues comme plus intolérables, car l'école est devenue une compétition où chacun doit jouer et où les petites inégalités deviennent décisives.

• [00:05:16] L'égalité des chances et ses limites : Les Américains croient en l'égalité des chances, mais la mobilité sociale y est plus faible qu'en France ou en Scandinavie. Il est crucial de comprendre comment les acteurs vivent et se représentent les inégalités pour pouvoir les combattre efficacement. Les inégalités qui créent le moins d'indignation sont les moins importantes à traiter.

• [00:06:05] Repenser la solidarité : L'enjeu est de fabriquer une représentation démocratique où les colères trouvent une expression politique. Il faut rendre lisibles les transferts de solidarité, car le mécanisme actuel est illisible et crée un sentiment d'être lésé. Il est urgent de simplifier les mécanismes de redistribution et de revenir à des politiques universelles plutôt que de cibler des publics particuliers. Il faut également fabriquer des sentiments de solidarité plus actifs, en s'appuyant sur la vie associative locale et en intégrant l'apprentissage de la solidarité dans l'expérience scolaire.

• [00:07:39] Le mécanisme des petites inégalités qui s'agrègent : Autrefois, le parcours scolaire était un destin fixé à la naissance. Aujourd'hui, il est le résultat d'une accumulation de petites différences (notes, choix d'options, établissement fréquenté, etc.). Chacune de ces inégalités n'est pas très forte, mais elle s'accumule et donne l'impression que l'individu est responsable de son propre destin.

• [00:08:28] Les pistes d'action politique : Pour repenser la solidarité, il faut que les colères trouvent une expression politique. Il faut rendre lisibles les transferts de solidarité et simplifier les mécanismes de redistribution. Il faut fabriquer des sentiments de solidarité plus actifs, en s'appuyant sur la vie associative et en intégrant l'apprentissage de la solidarité dans l'école.

• [00:09:16] La sociologie face aux inégalités multiples : La sociologie s'est dispersée dans une multitude d'objets et de théories, perdant sa vision globale de la société. Il faudrait qu'elle refabrique une image de la société et qu'elle s'intéresse à la fois aux acteurs et aux mécanismes qui les structurent. Il faut tenir ensemble les épreuves individuelles et les enjeux collectifs.

• [00:10:13] La sociologie et la stigmatisation : Il y a parfois une posture avantageuse dans la dénonciation des stigmatisations et des discriminations. Il est important d'écouter les acteurs sociaux et de ne pas surinterpréter leurs propos. Les sociologues de la génération actuelle sont peut-être plus techniques et professionnalisés, mais ont peut-être perdu en imagination sociologique. Les laboratoires de recherche devraient favoriser le travail collectif plutôt que de se contenter d'être des plateformes de services individuels.

Voici un sommaire de la vidéo avec les idées fortes, en gras:

• Introduction
  • Présentation des invités : Bruno Humbeeck, psychopédagogue et spécialiste de l'hyper parentalité et de l'éducation positive, et Béatrice Kammerer, journaliste spécialisée en éducation positive.
  • L'émission explore l'éducation positive, un sujet de plus en plus répandu mais controversé, en famille et à l'école.
  • Les invités ne sont ni pour ni contre l'éducation positive, mais proposent une approche mesurée.
• Définition et historique de l'éducation positive
  • L'éducation positive a commencé comme un champ de recherche étudiant les aspects positifs de l'être humain, puis est devenue une forme pédagogique.
  • Le terme est apparu en 2006 avec une recommandation du Conseil de l'Europe définissant une parentalité visant à élever et responsabiliser l'enfant de manière non-violente, en favorisant son plein développement.
  • Cette définition large a permis à divers courants éducatifs de s'y agréger, rendant la notion difficile à cerner précisément.
• Confusion avec les pédagogies nouvelles et critique de l'approche "recette"
  • L'éducation positive est parfois confondue avec les pédagogies nouvelles comme Montessori, issues de l'après-guerre.
  • L'idée de laisser croître les enfants vient de Rousseau, mais il prônait aussi une éducation négative, par exemple en laissant l'enfant subir les conséquences de ses actes.
  • L'éducation positive a parfois le défaut de considérer qu'il n'y a qu'une seule façon d'éduquer, alors que toutes les formes pédagogiques ont une intention bienveillante.
  • L'éducation positive est critiquée pour ses "recettes" simplistes et ses injonctions comme "soyez des parents zen".
• Évolution du regard sur l'enfant et valeurs de l'éducation positive
  • L'éducation positive est une évolution du regard sur l'enfant, notamment la reconnaissance de l'enfant comme personne à part entière.
  • Les châtiments corporels ne sont plus acceptables, et l'on vise l'épanouissement de l'enfant, en tenant compte de ses émotions et de ses besoins.
  • Il faut faire attention à ne pas centrer l'enfant sur lui-même au point de négliger le souci collectif, ce qui pourrait être un danger pour la démocratie.
• Les dérives de l'éducation positive : parents "zen", hyper communicants et hyper tolérants
  • La pédagogie positive peut mener à des enfants tyrans si elle n'est pas modulée par l'empathie.
  • Une des dérives est le parent "zen" qui réprime ses émotions, même quand l'enfant fait une bêtise.
  • Le parent hyper communicant est constamment à la disposition de son enfant, sans sélectionner ce qui est digne d'intérêt.
  • Le parent hyper tolérant ne fixe plus de limites, oubliant tout principe d'autorité, ce qui peut mener à un déni démocratique.
• Principes et pratiques recommandées par l'éducation positive
  • Interdiction de toute forme de violence éducative et de coercition.
  • Pas de punition ni de récompense, car cela nuit à la motivation intrinsèque de l'enfant.
  • Valoriser l'expression des émotions et formuler des demandes mesurées, sans violence.
  • Attention à l'estime de soi de l'enfant : ne pas juger l'enfant, mais ses comportements.
  • Être tourné vers les besoins de l'enfant dès la petite enfance, en s'inspirant de la théorie de l'attachement.
• L'importance de nuancer et de ne pas rejeter en bloc l'éducation positive
  • Il serait dommage de rejeter en bloc l'éducation positive sous prétexte de ses excès.
  • Il faut éviter d'être dans un monde binaire qui dirait l'éducation positive c'est bien ou c'est mal.
  • L'attention portée sur les violences ordinaires est un acquis de la pédagogie positive qu'il faut préserver.
• Critiques de l'école "bienveillante" et nécessité d'un cadre et de règles
  • L'école de la bienveillance est critiquée lorsqu'elle se transforme en école de la complaisance, où les enfants font ce qu'ils veulent sans limite.
  • Il faut exercer une forme d'autorité sur l'enfant, car il a besoin de règles et de lois pour faire société.
  • L'attention portée sur les violences ordinaires est un acquis de la pédagogie positive qu'il faut préserver.
• Les différentes critiques adressées à l'éducation positive : la question de l'autorité
  • Certains parents veulent abolir toute forme de contrainte, considérant que toute imposition est une violence.
  • L'éducation positive a contesté l'autoritarisme, mais a parfois demandé aux parents de ne plus faire preuve d'autorité, ce qui peut mener à d'autres types de comportements critiquables.
• La bienveillance : un concept à nuancer
  • Il faut nuancer le discours autour de la bienveillance éducative, en évitant de transformer les évaluations en "écoles des fans".
  • L'école est un laboratoire permanent, il faut être attentif aux innovations, mais les encadrer.
• Le besoin de cadres et de limites pour l'enfant et l'adulte
  • Les enfants ont besoin d'un cadre et de balises, et une éducation dérégulée ne profite à personne.
  • Il faut des règles et un système de punitions pour que ces règles soient respectées.
• Renoncer à la triple perfection et accepter l'imperfection du monde
  • Il faut renoncer à la triple perfection : ne pas être soi-même parfait, ne pas avoir des enfants parfaits, et accepter que le monde est imparfait.
  • L'inquiétude climatique et le contexte mondial incertain rendent la pédagogie positive difficile à vivre.
  • Il faut accepter l'incertitude et le fait que nos enfants ne seront pas parfaits.
• Les injonctions paradoxales faites aux parents et la difficulté d'être parent aujourd'hui
  • Les parents sont soumis à des injonctions paradoxales : être attentif à son enfant sans l'envahir, répondre à ses besoins tout en lui permettant d'explorer.
  • Il est difficile d'être parent aujourd'hui, car il y a des attentes très diverses de toute la société.
  • Les parents sont tiraillés entre la peur du déclassement social et le désir de voir leur enfant s'épanouir.
• Les inégalités entre pères et mères et la charge mentale des mères
  • L'éducation positive a plutôt tendance à renforcer les inégalités entre pères et mères.
  • Les femmes ont souvent la mission d'être les "chargées de développement" du foyer, et l'éducation positive se dirige énormément vers elles.
  • On demande aux mères de prendre en charge les compétences de communication et l'expression des émotions.
  • Dès la naissance, on adresse aux femmes des injonctions sur l'accouchement, l'allaitement, la disponibilité, etc..
• Le danger du "coaching" parental et l'importance du soutien
  • Le "coaching" est dangereux car il donne l'idée qu'il y aurait une performance à atteindre, alors qu'il faut accepter d'être des parents imparfaits.
  • La parentalité est le mécanisme par lequel chacun de nos enfants transforme l'éducation qu'il reçoit en quelque chose de personnel.
  • La pédagogie positive a sans doute été dans le mur quand elle s'est voulue prescriptive.

• L'enjeu de la coéducation et de la complémentarité des expertises

  • Il faut accepter l'idée qu'on n'éduque pas seul ses enfants et qu'on doit utiliser tous les supports que nos sociétés mettent à notre disposition.
  • La coéducation repose sur la complémentarité des expertises : celles des parents et celles des spécialistes.
  • Il faut redonner de la valeur aux pratiques parentales, même si elles ne suivent pas forcément les manuels d'éducation positive.
  • Les techniques de la coéducation ne sont pas les mêmes que celles du soutien à la parentalité.

• Conclusion

  • Présentation du numéro de Sciences Humaines sur l'éducation positive et d'autres ressources sur le sujet.
  • Remerciements aux invités et aux participants.

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

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Reply to the reviewers

Reply to the Reviewers

I would like to thank the reviewers for their comments and interest in the manuscript and the study.

Referee #1

1. I would assume that there are RNA-seq and/or ChIP-seq data out there produced after knockdown of one or more of these DBPs that show directional positioning.

Response: The directional positioning of CTCF-binding sites at chromatin interaction sites was analyzed by CRISPR experiment (Guo Y et al. Cell 2015). We found that the machine learning and statistical analysis showed the same directional bias of the CTCF-binding motif sequence at chromatin interaction sites as the experimental analysis of Guo Y et al. (lines 229-245, Figure 3b, c, d and Table 1). Since CTCF is involved in different biological functions (Braccioli L et al. Essays Biochem. 2019 ResearchGate webpage), the directional bias of binding sites may be reduced in all binding sites including those at chromatin interaction sites (lines 68-73). In our study, we investigated the DNA-binding sites of proteins using the ChIP-seq data of DNA-binding proteins and DNase-seq data. We also confirmed that the DNA-binding sites of SMC3 and RAD21, which tend to be found in chromatin loops with CTCF, also showed the same directional bias as CTCF by the computational analysis.

1. Figure 6 should be expanded to incorporate analysis of DBPs not overlapping CTCF/cohesin in chromatin interaction data that is important and potentially more interesting than the simple DBPs enrichment reported in the present form of the figure.

Response: Following the reviewer's advice, I performed the same analysis with the DNA-binding sites that do no overlap with the DNA-binding sites of CTCF and cohesin (RAD21 and SMC3) (Fig. 6 and Supplementary Fig. 4). The result showed the same tendency in the distribution of DNA-binding sites. The height of a peak on the graph became lower for some DNA-binding proteins after removing the DNA-binding sites that overlapped with those of CTCF and cohesin. I have added the following sentence on lines 427 and 817: For the insulator-associated DBPs other than CTCF, RAD21, and SMC3, the DNA-binding sites that do not overlap with those of CTCF, RND21, and SMC3 were used to examine their distribution around interaction sites.

1. Critically, I would like to see use of Micro-C/Hi-C data and ChIP-seq from these factors, where insulation scores around their directionally-bound sites show some sort of an effect like that presumed by the authors - and many such datasets are publicly-available and can be put to good use here.

Response: As suggested by the reviewer, I have added the insulator scores and boundary sites from the 4D nucleome data portal as tracks in the UCSC genome browser. The insulator scores seem to correspond to some extent to the H3K27me3 histone marks from ChIP-seq (Fig. 4a and Supplementary Fig. 3). The direction of DNA-binding sites on the genome can be shown with different colors (e.g. red and green), but the directionality of insulator-associated DNA-binding sites is their overall tendency, and it may be difficult to notice the directionality from each binding site because the directionality may be weaker than that of CTCF, RAD21, and SMC3 as shown in Table 1 and Supplementary Table 2.

I found that the CTCF binding sites examined by a wet experiment in the previous study may not always overlap with the boundary sites of chromatin interactions from Micro-C assay (Guo Y et al. Cell 2015). The chromatin interaction data do not include all interactions due to the high sequencing cost of the assay. The number of the boundary sites may be smaller than that of CTCF binding sites acting as insulators and/or some of the CTCF binding sites may not be locate in the boundary sites. It may be difficult for the boundary location algorithm to identify a short boundary location. Due to the limitations of the chromatin interaction data, I planned to search for insulator-associated DNA-binding proteins without using chromatin interaction data in this study. I have added the statistical summary of the analysis in lines 364-387 as follows: Overall, among 20,837 DNA-binding sites of the 97 insulator-associated proteins found at insulator sites identified by H3K27me3 histone modification marks (type 1 insulator sites), 1,315 (6%) overlapped with 264 of 17,126 5kb long boundary sites, and 6,137 (29%) overlapped with 784 of 17,126 25kb long boundary sites in HFF cells. Among 5,205 DNA-binding sites of the 97 insulator-associated DNA-binding proteins found at insulator sites identified by H3K27me3 histone modification marks and transcribed regions (type 2 insulator sites), 383 (7%) overlapped with 74 of 17,126 5-kb long boundary sites, 1,901 (37%) overlapped with 306 of 17,126 25-kb long boundary sites. Although CTCF-binding sites separate active and repressive domains, the limited number of DNA-binding sites of insulator-associated proteins found at type 1 and 2 insulator sites overlapped boundary sites identified by chromatin interaction data. Furthermore, by analyzing the regulatory regions of genes, the DNA-binding sites of the 97 insulator-associated DNA-binding proteins were found (1) at the type 1 insulator sites (based on H3K27me3 marks) in the regulatory regions of 3,170 genes, (2) at the type 2 insulator sites (based on H3K27me3 marks and gene expression levels) in the regulatory regions of 1,044 genes, and (3) at insulator sites as boundary sites identified by chromatin interaction data in the regulatory regions of 6,275 genes. The boundary sites showed the highest number of overlaps with the DNA-binding sites. Comparing the insulator sites identified by (1) and (3), 1,212 (38%) genes have both types of insulator sites. Comparing the insulator sites between (2) and (3), 389 (37%) genes have both types of insulator sites. From the comparison of insulator and boundary sites, we found that (1) or (2) types of insulator sites overlapped or were close to boundary sites identified by chromatin interaction data.

1. The suggested alternative transcripts function, also highlighted in the manuscripts abstract, is only supported by visual inspection of a few cases for several putative DBPs. I believe this is insufficient to support what looks like one of the major claims of the paper when reading the abstract, and a more quantitative and genome-wide analysis must be adopted, although the authors mention it as just an 'observation'.

Response: According to the reviewer's comment, I performed the genome-wide analysis of alternative transcripts where the DNA-binding sites of insulator-associated proteins are located near splicing sites. The DNA-binding sites of insulator-associated DNA-binding proteins were found within 200 bp centered on splice sites more significantly than the other DNA-binding proteins (Fig. 4e and Table 2). I have added the following sentences on lines 397 - 404: We performed the statistical test to estimate the enrichment of insulator-associated DNA-binding sites compared to the other DNA-binding proteins, and found that the insulator-associated DNA-binding sites were significantly more abundant at splice sites than the DNA-binding sites of the other proteins (Fig 4e and Table 2; Mann‒Whitney U test, p value 5. Figure 1 serves no purpose in my opinion and can be removed, while figures can generally be improved (e.g., the browser screenshots in Figs 4 and 5) for interpretability from readers outside the immediate research field.

Response: I believe that the Figure 1 would help researchers in other fields who are not familiar with biological phenomena and functions to understand the study. More explanation has been included in the Figures and legends of Figs. 4 and 5 to help readers outside the immediate research field understand the figures.

1. Similarly, the text is rather convoluted at places and should be re-approached with more clarity for less specialized readers in mind.

Response: Reviewer #2's comments would be related to this comment. I have introduced a more detailed explanation of the method in the Results section, as shown in the responses to Reviewer #2's comments.

Referee #2

1. Introduction, line 95: CTCF appears two times, it seems redundant.

Response: On lines 91-93, I deleted the latter CTCF from the sentence "and examined the directional bias of DNA-binding sites of CTCF and insulator-associated DBPs, including those of known DBPs such as RAD21 and SMC3".

1. Introduction, lines 99-103: Please stress better the novelty of the work. What is the main focus? The new identified DPBs or their binding sites? What are the "novel structural and functional roles of DBPs" mentioned?

Response: Although CTCF is known to be the main insulator protein in vertebrates, we found that 97 DNA-binding proteins including CTCF and cohesin are associated with insulator sites by modifying and developing a machine learning method to search for insulator-associated DNA-binding proteins. Most of the insulator-associated DNA-binding proteins showed the directional bias of DNA-binding motifs, suggesting that the directional bias is associated with the insulator.

I have added the sentence in lines 96-99 as follows: Furthermore, statistical testing the contribution scores between the directional and non-directional DNA-binding sites of insulator-associated DBPs revealed that the directional sites contributed more significantly to the prediction of gene expression levels than the non-directional sites. I have revised the statement in lines 101-110 as follows: To validate these findings, we demonstrate that the DNA-binding sites of the identified insulator-associated DBPs are located within potential insulator sites, and some of the DNA-binding sites in the insulator site are found without the nearby DNA-binding sites of CTCF and cohesin. Homologous and heterologous insulator-insulator pairing interactions are orientation-dependent, as suggested by the insulator-pairing model based on experimental analysis in flies. Our method and analyses contribute to the identification of insulator- and chromatin-associated DNA-binding sites that influence EPIs and reveal novel functional roles and molecular mechanisms of DBPs associated with transcriptional condensation, phase separation and transcriptional regulation.

1. Results, line 111: How do the SNPs come into the procedure? From the figures it seems the input is ChIP-seq peaks of DNBPs around the TSS.

Response: On lines 121-124, to explain the procedure for the SNP of an eQTL, I have added the sentence in the Methods: "If a DNA-binding site was located within a 100-bp region around a single-nucleotide polymorphism (SNP) of an eQTL, we assumed that the DNA-binding proteins regulated the expression of the transcript corresponding to the eQTL".

1. Again, are those SNPs coming from the different cell lines? Or are they from individuals w.r.t some reference genome? I suggest a general restructuring of this part to let the reader understand more easily. One option could be simplifying the details here or alternatively including all the necessary details.

Response: On line 119, I have included the explanation of the eQTL dataset of GTEx v8 as follows: " The eQTL data were derived from the GTEx v8 dataset, after quality control, consisting of 838 donors and 17,382 samples from 52 tissues and two cell lines". On lines 681 and 865, I have added the filename of the eQTL data "(GTEx_Analysis_v8_eQTL.tar)".

1. Figure 1: panel a and b are misleading. Is the matrix in panel a equivalent to the matrix in panel b? If not please clarify why. Maybe in b it is included the info about the SNPs? And if yes, again, what is then difference with a.

Response: The reviewer would mention Figure 2, not Figure 1. If so, the matrices in panels a and b in Figure 2 are equivalent. I have shown it in the figure: The same figure in panel a is rotated 90 degrees to the right. The green boxes in the matrix show the regions with the ChIP-seq peak of a DNA-binding protein overlapping with a SNP of an eQTL. I used eQTL data to associate a gene with a ChIP-seq peak that was more than 2 kb upstream and 1 kb downstream of a transcriptional start site of a gene. For each gene, the matrix was produced and the gene expression levels in cells were learned and predicted using the deep learning method. I have added the following sentences to explain the method in lines 133 - 139: Through the training, the tool learned to select the binding sites of DNA-binding proteins from ChIP-seq assays that were suitable for predicting gene expression levels in the cell types. The binding sites of a DNA-binding protein tend to be observed in common across multiple cell and tissue types. Therefore, ChIP-seq data and eQTL data in different cell and tissue types were used as input data for learning, and then the tool selected the data suitable for predicting gene expression levels in the cell types, even if the data were not obtained from the same cell types.

1. Line 386-388: could the author investigate in more detail this observation? Does it mean that loops driven by other DBPs independent of the known CTCF/Cohesin? Could the author provide examples of chromatin structural data e.g. MicroC?

Response: As suggested by the reviewer, to help readers understand the observation, I have added Supplementary Fig. S4c to show the distribution of DNA-binding sites of "CTCF, RAD21, and SMC3" and "BACH2, FOS, ATF3, NFE2, and MAFK" around chromatin interaction sites. I have modified the following sentence to indicate the figure on line 493: Although a DNA-binding-site distribution pattern around chromatin interaction sites similar to those of CTCF, RAD21, and SMC3 was observed for DBPs such as BACH2, FOS, ATF3, NFE2, and MAFK, less than 1% of the DNA-binding sites of the latter set of DBPs colocalized with CTCF, RAD21, or SMC3 in a single bin (Fig. S4c).

In Aljahani A et al. Nature Communications 2022, we find that depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Together, our data show that loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression. Goel VY et al. Nature Genetics 2023 mentioned in the abstract: Microcompartments frequently connect enhancers and promoters and though loss of loop extrusion and inhibition of transcription disrupts some microcompartments, most are largely unaffected. These results suggested that chromatin loops can be driven by other DBPs independent of the known CTCF/Cohesin.

FOXA1 pioneer factor functions as an initial chromatin-binding and chromatin-remodeling factor and has been reported to form biomolecular condensates (Ji D et al. Molecular Cell 2024). CTCF have also found to form transcriptional condensate and phase separation (Lee R et al. Nucleic acids research 2022). FOS was found to be an insulator-associated DNA-binding protein in this study and is potentially involved in chromatin remodeling, transcription condensation, and phase separation with the other factors such as BACH2, ATF3, NFE2 and MAFK. I have added the following sentence on line 548: FOXA1 pioneer factor functions as an initial chromatin-binding and chromatin-remodeling factor and has been reported to form biomolecular condensates.

1. In general, how the presented results are related to some models of chromatin architecture, e.g. loop extrusion, in which it is integrated convergent CTCF binding sites?

Response: Goel VY et al. Nature Genetics 2023 identified highly nested and focal interactions through region capture Micro-C, which resemble fine-scale compartmental interactions and are termed microcompartments. In the section titled "Most microcompartments are robust to loss of loop extrusion," the researchers noted that a small proportion of interactions between CTCF and cohesin-bound sites exhibited significant reductions in strength when cohesin was depleted. In contrast, the majority of microcompartmental interactions remained largely unchanged under cohesin depletion. Our findings indicate that most P-P and E-P interactions, aside from a few CTCF and cohesin-bound enhancers and promoters, are likely facilitated by a compartmentalization mechanism that differs from loop extrusion. We suggest that nested, multiway, and focal microcompartments correspond to small, discrete A-compartments that arise through a compartmentalization process, potentially influenced by factors upstream of RNA Pol II initiation, such as transcription factors, co-factors, or active chromatin states. It follows that if active chromatin regions at microcompartment anchors exhibit selective "stickiness" with one another, they will tend to co-segregate, leading to the development of nested, focal interactions. This microphase separation, driven by preferential interactions among active loci within a block copolymer, may account for the striking interaction patterns we observe.

The authors of the paper proposed several mechanisms potentially involved in microcompartments. These mechanisms may be involved in looping with insulator function. Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently (Hsieh TS et al. Nature Genetics 2022). Among the identified insulator-associated DNA-binding proteins, Maz and MyoD1 form loops without CTCF (Xiao T et al. Proc Natl Acad Sci USA 2021 ; Ortabozkoyun H et al. Nature genetics 2022 ; Wang R et al. Nature communications 2022). I have added the following sentences on lines 563-567: Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently. I have included the following explanation on lines 574-576: Maz and MyoD1 among the identified insulator-associated DNA-binding proteins form loops without CTCF.

As for the directionality of CTCF, if chromatin loop anchors have some structural conformation, as shown in the paper entitled "The structural basis for cohesin-CTCF-anchored loops" (Li Y et al. Nature 2020), directional DNA binding would occur similarly to CTCF binding sites. Moreover, cohesin complexes that interact with convergent CTCF sites, that is, the N-terminus of CTCF, might be protected from WAPL, but those that interact with divergent CTCF sites, that is, the C-terminus of CTCF, might not be protected from WAPL, which could release cohesin from chromatin and thus disrupt cohesin-mediated chromatin loops (Davidson IF et al. Nature Reviews Molecular Cell Biology 2021). Regarding loop extrusion, the 'loop extrusion' hypothesis is motivated by in vitro observations. The experiment in yeast, in which cohesin variants that are unable to extrude DNA loops but retain the ability to topologically entrap DNA, suggested that in vivo chromatin loops are formed independently of loop extrusion. Instead, transcription promotes loop formation and acts as an extrinsic motor that extends these loops and defines their final positions (Guerin TM et al. EMBO Journal 2024). I have added the following sentences on lines 535-539: Cohesin complexes that interact with convergent CTCF sites, that is, the N-terminus of CTCF, might be protected from WAPL, but those that interact with divergent CTCF sites, that is, the C-terminus of CTCF, might not be protected from WAPL, which could release cohesin from chromatin and thus disrupt cohesin-mediated chromatin loops. I have included the following sentences on lines 569-574: The 'loop extrusion' hypothesis is motivated by in vitro observations. The experiment in yeast, in which cohesin variants that are unable to extrude DNA loops but retain the ability to topologically entrap DNA, suggested that in vivo chromatin loops are formed independently of loop extrusion. Instead, transcription promotes loop formation and acts as an extrinsic motor that extends these loops and defines their final positions.

Another model for the regulation of gene expression by insulators is the boundary-pairing (insulator-pairing) model (Bing X et al. Elife 2024) (Ke W et al. Elife 2024) (Fujioka M et al. PLoS Genetics 2016). Molecules bound to insulators physically pair with their partners, either head-to-head or head-to-tail, with different degrees of specificity at the termini of TADs in flies. Although the experiments do not reveal how partners find each other, the mechanism unlikely requires loop extrusion. Homologous and heterologous insulator-insulator pairing interactions are central to the architectural functions of insulators. The manner of insulator-insulator interactions is orientation-dependent. I have summarized the model on lines 551-559: Other types of chromatin regulation are also expected to be related to the structural interactions of molecules. As the boundary-pairing (insulator-pairing) model, molecules bound to insulators physically pair with their partners, either head-to-head or head-to-tail, with different degrees of specificity at the termini of TADs in flies (Fig. 7). Although the experiments do not reveal how partners find each other, the mechanism unlikely requires loop extrusion. Homologous and heterologous insulator-insulator pairing interactions are central to the architectural functions of insulators. The manner of insulator-insulator interactions is orientation-dependent.

1. Do the authors think that the identified DBPs could work in that way as well?

Response: The boundary-pairing (insulator-pairing) model would be applied to the insulator-associated DNA-binding proteins other than CTCF and cohesin that are involved in the loop extrusion mechanism (Bing X et al. Elife 2024) (Ke W et al. Elife 2024) (Fujioka M et al. PLoS Genetics 2016).

Liquid-liquid phase separation was shown to occur through CTCF-mediated chromatin loops and to act as an insulator (Lee, R et al. Nucleic Acids Research 2022). Among the identified insulator-associated DNA-binding proteins, CEBPA has been found to form hubs that colocalize with transcriptional co-activators in a native cell context, which is associated with transcriptional condensate and phase separation (Christou-Kent M et al. Cell Reports 2023). The proposed microcompartment mechanisms are also associated with phase separation. Thus, the same or similar mechanisms are potentially associated with the insulator function of the identified DNA-binding proteins. I have included the following information on line 546: CEBPA in the identified insulator-associated DNA-binding proteins was also reported to be involved in transcriptional condensates and phase separation.

1. Also, can the authors comment about the mechanisms those newly identified DBPs mediate contacts by active processes or equilibrium processes?

Response: Snead WT et al. Molecular Cell 2019 mentioned that protein post-transcriptional modifications (PTMs) facilitate the control of molecular valency and strength of protein-protein interactions. O-GlcNAcylation as a PTM inhibits CTCF binding to chromatin (Tang X et al. Nature Communications 2024). I found that the identified insulator-associated DNA-binding proteins tend to form a cluster at potential insulator sites (Supplementary Fig. 2d). These proteins may interact and actively regulate chromatin interactions, transcriptional condensation, and phase separation by PTMs. I have added the following explanation on lines 576-582: Furthermore, protein post-transcriptional modifications (PTMs) facilitate control over the molecular valency and strength of protein-protein interactions. O-GlcNAcylation as a PTM inhibits CTCF binding to chromatin. We found that the identified insulator-associated DNA-binding proteins tend to form a cluster at potential insulator sites (Fig. 4f and Supplementary Fig. 3c). These proteins may interact and actively regulate chromatin interactions, transcriptional condensation, and phase separation through PTMs.

1. Can the author provide some real examples along with published structural data (e.g. the mentioned micro-C data) to show the link between protein co-presence, directional bias and contact formation?

Response: Structural molecular model of cohesin-CTCF-anchored loops has been published by Li Y et al. Nature 2020. The structural conformation of CTCF and cohesin in the loops would be the cause of the directional bias of CTCF binding sites, which I mentioned in lines 531 - 535 as follows: These results suggest that the directional bias of DNA-binding sites of insulator-associated DBPs may be involved in insulator function and chromatin regulation through structural interactions among DBPs, other proteins, DNAs, and RNAs. For example, the N-terminal amino acids of CTCF have been shown to interact with RAD21 in chromatin loops. To investigate the principles underlying the architectural functions of insulator-insulator pairing interactions, two insulators, Homie and Nhomie, flanking the Drosophila even skipped locus were analyzed. Pairing interactions between the transgene Homie and the eve locus are directional. The head-to-head pairing between the transgene and endogenous Homie matches the pattern of activation (Fujioka M et al. PLoS Genetics 2016).

Referee #3

1. Some of these TFs do not have specific direct binding to DNA (P300, Cohesin). Since the authors are using binding motifs in their analysis workflow, I would remove those from the analysis.

Response: When a protein complex binds to DNA, one protein of the complex binds to the DNA directory, and the other proteins may not bind to DNA. However, the DNA motif sequence bound by the protein may be registered as the DNA-binding motif of all the proteins in the complex. The molecular structure of the complex of CTCF and Cohesin showed that both CTCF and Cohesin bind to DNA (Li Y et al. Nature 2020). I think there is a possibility that if the molecular structure of a protein complex becomes available, the previous recognition of the DNA-binding ability of a protein may be changed. Therefore, I searched the Pfam database for 99 insulator-associated DNA-binding proteins identified in this study. I found that 97 are registered as DNA-binding proteins and/or have a known DNA-binding domain, and EP300 and SIN3A do not directory bind to DNA, which was also checked by Google search. I have added the following explanation in line 249 to indicate direct and indirect DNA-binding proteins: Among 99 insulator-associated DBPs, EP300 and SIN3A do not directory interact with DNA, and thus 97 insulator-associated DBPs directory bind to DNA. I have updated the sentence in line 20 of the Abstract as follows: We discovered 97 directional and minor nondirectional motifs in human fibroblast cells that corresponded to 23 DBPs related to insulator function, CTCF, and/or other types of chromosomal transcriptional regulation reported in previous studies.

1. I am not sure if I understood correctly, by why do the authors consider enhancers spanning 2Mb (200 bins of 10Kb around eSNPs)? This seems wrong. Enhancers are relatively small regions (100bp to 1Kb) and only a very small subset form super enhancers.

Response: As the reviewer mentioned, I recognize enhancers are relatively small regions. In the paper, I intended to examine further upstream and downstream of promoter regions where enhancers are found. Therefore, I have modified the sentence in lines 917 - 919 of the Fig. 2 legend as follows: Enhancer-gene regulatory interaction regions consist of 200 bins of 10 kbp between -1 Mbp and 1 Mbp region from TSS, not including promoter.

1. I think the H3K27me3 analysis was very good, but I would have liked to see also constitutive heterochromatin as well, so maybe repeat the analysis for H3K9me3.

Response: Following the reviewer's advice, I have added the ChIP-seq data of H3K9me3 as a truck of the UCSC Genome Browser. The distribution of H3K9me3 signal was different from that of H3K27me3 in some regions. I also found the insulator-associated DNA-binding sites close to the edges of H3K9me3 regions and took some screenshots of the UCSC Genome Browser of the regions around the sites in Supplementary Fig. 3b. I have modified the following sentence on lines 962 - 964 in the legend of Fig. 4: a Distribution of histone modification marks H3K27me3 (green color) and H3K9me3 (turquoise color) and transcript levels (pink color) in upstream and downstream regions of a potential insulator site (light orange color). I have also added the following result on lines 348 - 352: The same analysis was performed using H3K9me3 marks, instead of H3K27me3 (Fig. S3b). We found that the distribution of H3K9me3 signal was different from that of H3K27me3 in some regions, and discovered the insulator-associated DNA-binding sites close to the edges of H3K9me3 regions (Fig. S3b).

1. I was not sure I understood the analysis in Figure 6. The binding site is with 500bp of the interaction site, but micro-C interactions are at best at 1Kb resolution. They say they chose the centre of the interaction site, but we don't know exactly where there is the actual interaction. Also, it is not clear what they measure. Is it the number of binding sites of a specific or multiple DBP insulator proteins at a specific distance from this midpoint that they recover in all chromatin loops? Maybe I am missing something. This analysis was not very clear.

Response: The resolution of the Micro-C assay is considered to be 100 bp and above, as the human nucleome core particle contains 145 bp (and 193 bp with linker) of DNA. However, internucleosomal DNA is cleaved by endonuclease into fragments of multiples of 10 nucleotides (Pospelov VA et al. Nucleic Acids Research 1979). Highly nested focal interactions were observed (Goel VY et al. Nature Genetics 2023). Base pair resolution was reported using Micro Capture-C (Hua P et al. Nature 2021). Sub-kilobase (20 bp resolution) chromatin topology was reported using an MNase-based chromosome conformation capture (3C) approach (Aljahani A et al. Nature Communications 2022). On the other hand, Hi-C data was analyzed at 1 kb resolution. (Gu H et al. bioRxiv 2021). If the resolution of Micro-C interactions is at best at 1 kb, the binding sites of a DNA-binding protein will not show a peak around the center of the genomic locations of interaction edges. Each panel shows the number of binding sites of a specific DNA-binding protein at a specific distance from the midpoint of all chromatin interaction edges. I have modified and added the following sentences in lines 585-589: High-resolution chromatin interaction data from a Micro-C assay indicated that most of the predicted insulator-associated DBPs showed DNA-binding-site distribution peaks around chromatin interaction sites, suggesting that these DBPs are involved in chromatin interactions and that the chromatin interaction data has a high degree of resolution. Base pair resolution was reported using Micro Capture-C.

Minor comments:

1. PIQ does not consider TF concentration. Other methods do that and show that TF concentration improves predictions (e.g., https://www.biorxiv.org/content/10.1101/2023.07.15.549134v2 or https://pubmed.ncbi.nlm.nih.gov/37486787/). The authors should discuss how that would impact their results.

Response: The directional bias of CTCF binding sites was identified by ChIA-pet interactions of CTCF binding sites. The analysis of the contribution scores of DNA-binding sites of proteins considering the binding sites of CTCF as an insulator showed the same tendency of directional bias of CTCF binding sites. In the analysis, to remove the false-positive prediction of DNA-binding sites, I used the binding sites that overlapped with a ChIP-seq peak of the DNA-binding protein. This result suggests that the DNA-binding sites of CTCF obtained by the current analysis have sufficient quality. Therefore, if the accuracy of prediction of DNA-binding sites is improved, althought the number of DNA-binding sites may be different, the overall tendency of the directionality of DNA-binding sites will not change and the results of this study will not change significantly.

As for the first reference in the reviewer's comment, chromatin interaction data from Micro-C assay does not include all chromatin interactions in a cell or tissue, because it is expensive to cover all interactions. Therefore, it would be difficult to predict all chromatin interactions based on machine learning. As for the second reference in the reviewer's comment, pioneer factors such as FOXA are known to bind to closed chromatin regions, but transcription factors and DNA-binding proteins involved in chromatin interactions and insulators generally bind to open chromatin regions. The search for the DNA-binding motifs is not required in closed chromatin regions.

1. DeepLIFT is a good approach to interpret complex structures of CNN, but is not truly explainable AI. I think the authors should acknowledge this.

Response: In the DeepLIFT paper, the authors explain that DeepLIFT is a method for decomposing the output prediction of a neural network on a specific input by backpropagating the contributions of all neurons in the network to every feature of the input (Shrikumar A et al. ICML 2017). DeepLIFT compares the activation of each neuron to its 'reference activation' and assigns contribution scores according to the difference. DeepLIFT calculates a metric to measure the difference between an input and the reference of the input.

Truly explainable AI would be able to find cause and reason, and to make choices and decisions like humans. DeepLIFT does not perform causal inferences. I did not use the term "Explainable AI" in our manuscript, but I briefly explained it in Discussion. I have added the following explanation in lines 615-620: AI (Artificial Intelligence) is considered as a black box, since the reason and cause of prediction are difficult to know. To solve this issue, tools and methods have been developed to know the reason and cause. These technologies are called Explainable AI. DeepLIFT is considered to be a tool for Explainable AI. However, DeepLIFT does not answer the reason and cause for a prediction. It calculates scores representing the contribution of the input data to the prediction.

Furthermore, to improve the readability of the manuscript, I have included the following explanation in lines 159-165: we computed DeepLIFT scores of the input data (i.e., each binding site of the ChIP-seq data of DNA-binding proteins) in the deep leaning analysis on gene expression levels. DeepLIFT compares the importance of each input for predicting gene expression levels to its 'reference or background level' and assigns contribution scores according to the difference. DeepLIFT calculates a metric to measure the difference between an input and the reference of the input.

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Reply to the reviewers

I would like to thank the reviewers for their comments and interest in the manuscript and the study.

Reviewer #1

1) I would assume that there are RNA-seq and/or ChIP-seq data out there produced after knockdown of one or more of these DBPs that show directional positioning.

As the reviewer pointed out, a wet experimental validation of the results of this study would give an opportunity for more biological researchers to have an interest in the study. I plan to promote the wet experimental analysis in collaboration with biological experimental researchers as a next step of this study. The same analysis in this study can be performed in immortalized cells for CRISPR experiment (e.g. Guo Y et al. Cell 2015).

2) Figure 6 should be expanded to incorporate analysis of DBPs not overlapping CTCF/cohesin in chromatin interaction data that is important and potentially more interesting than the simple DBPs enrichment reported in the present form of the figure.

Following the reviewer's advice, I performed the same analysis with the DNA-binding sites that do no overlap with the DNA-binding sites of CTCF and cohesin (RAD21 and SMC3) (Fig. 6 and Supplementary Fig. 4). The result showed the same tendency in the distribution of DNA-binding sites. The height of a peak on the graph became lower for some DNA-binding proteins after removing the DNA-binding sites that overlapped with those of CTCF and cohesin. I have added the following sentence on lines 427 and 817: For the insulator-associated DBPs other than CTCF, RAD21, and SMC3, the DNA-binding sites that do not overlap with those of CTCF, RND21, and SMC3 were used to examine their distribution around interaction sites.

3) Critically, I would like to see use of Micro-C/Hi-C data and ChIP-seq from these factors, where insulation scores around their directionally-bound sites show some sort of an effect like that presumed by the authors - and many such datasets are publicly-available and can be put to good use here.

As suggested by the reviewer, I have added the insulator scores and boundary sites from the 4D nucleome data portal as tracks in the UCSC genome browser. The insulator scores seem to correspond to some extent to the H3K27me3 histone marks from ChIP-seq (Fig. 4a and Supplementary Fig. 3). The direction of DNA-binding sites on the genome can be shown with different colors (e.g. red and green), but the directionality is their overall tendency, and it may be difficult to notice the directionality from each binding site.

I found that the CTCF binding sites examined by a wet experiment in the previous study may not always overlap with the boundary sites of chromatin interactions from Micro-C assay (Guo Y et al. Cell 2015). The chromatin interaction data do not include all interactions due to the high sequencing cost of the assay. The number of the boundary sites may be smaller than that of CTCF binding sites acting as insulators and/or some of the CTCF binding sites may not be locate in the boundary sites. It may be difficult for the boundary location algorithm to identify a short boundary location. Due to the limitations of the chromatin interaction data, I planned to search for insulator-associated DNA-binding proteins without using chromatin interaction data in this study. I have added the statistical summary of the analysis in lines 364-387 as follows: Overall, among 20,837 DNA-binding sites of the 97 insulator-associated proteins found at insulator sites identified by H3K27me3 histone modification marks (type 1 insulator sites), 1,315 (6%) overlapped with 264 of 17,126 5kb long boundary sites, and 6,137 (29%) overlapped with 784 of 17,126 25kb long boundary sites in HFF cells. Among 5,205 DNA-binding sites of the 97 insulator-associated DNA-binding proteins found at insulator sites identified by H3K27me3 histone modification marks and transcribed regions (type 2 insulator sites), 383 (7%) overlapped with 74 of 17,126 5-kb long boundary sites, 1,901 (37%) overlapped with 306 of 17,126 25-kb long boundary sites. Although CTCF-binding sites separate active and repressive domains, the limited number of DNA-binding sites of insulator-associated proteins found at type 1 and 2 insulator sites overlapped boundary sites identified by chromatin interaction data. Furthermore, by analyzing the regulatory regions of genes, the DNA-binding sites of the 97 insulator-associated DNA-binding proteins were found (1) at the type 1 insulator sites (based on H3K27me3 marks) in the regulatory regions of 3,170 genes, (2) at the type 2 insulator sites (based on H3K27me3 marks and gene expression levels) in the regulatory regions of 1,044 genes, and (3) at insulator sites as boundary sites identified by chromatin interaction data in the regulatory regions of 6,275 genes. The boundary sites showed the highest number of overlaps with the DNA-binding sites. Comparing the insulator sites identified by (1) and (3), 1,212 (38%) genes have both types of insulator sites. Comparing the insulator sites between (2) and (3), 389 (37%) genes have both types of insulator sites. From the comparison of insulator and boundary sites, we found that (1) or (2) types of insulator sites overlapped or were close to boundary sites identified by chromatin interaction data.

4) The suggested alternative transcripts function, also highlighted in the manuscripts abstract, is only supported by visual inspection of a few cases for several putative DBPs. I believe this is insufficient to support what looks like one of the major claims of the paper when reading the abstract, and a more quantitative and genome-wide analysis must be adopted, although the authors mention it as just an 'observation'.

According to the reviewer's comment, I performed the genome-wide analysis of alternative transcripts where the DNA-binding sites of insulator-associated proteins are located near splicing sites. The DNA-binding sites of insulator-associated DNA-binding proteins were found within 200 bp centered on splice sites more significantly than the other DNA-binding proteins (Fig. 4e and Table 2). I have added the following sentences on lines 397 - 404: We performed the statistical test to estimate the enrichment of insulator-associated DNA-binding sites compared to the other DNA-binding proteins, and found that the insulator-associated DNA-binding sites were significantly more abundant at splice sites than the DNA-binding sites of the other proteins (Fig 4e and Table 2; Mann‒Whitney U test, p value < 0.05). The comparison between the splice sites of both ends of first and last introns and those of other introns showed the similar statistical significance of enrichment and number of splice sites with the insulator-associated DNA-binding proteins (Table 2 and Table S9).

5) Figure 1 serves no purpose in my opinion and can be removed, while figures can generally be improved (e.g., the browser screenshots in Figs 4 and 5) for interpretability from readers outside the immediate research field.

I believe that the Figure 1 would help researchers in other fields who are not familiar with biological phenomena and functions to understand the study. More explanation has been included in the Figures and legends of Figs. 4 and 5 to help readers outside the immediate research field understand the figures.

6) Similarly, the text is rather convoluted at places and should be re-approached with more clarity for less specialized readers in mind.

Reviewer #2's comments would be related to this comment. I have introduced a more detailed explanation of the method in the Results section, as shown in the responses to Reviewer #2’s comments.

Reviewer #2

1) Introduction, line 95: CTCF appears two times, it seems redundant.

On lines 91-93, I deleted the latter CTCF from the sentence "We examine the directional bias of DNA-binding sites of CTCF and insulator-associated DBPs, including those of known DBPs such as RAD21 and SMC3".

2) Introduction, lines 99-103: Please stress better the novelty of the work. What is the main focus? The new identified DPBs or their binding sites? What are the "novel structural and functional roles of DBPs" mentioned?

Although CTCF is known to be the main insulator protein in vertebrates, we found that 97 DNA-binding proteins including CTCF and cohesin are associated with insulator sites by modifying and developing a machine learning method to search for insulator-associated DNA-binding proteins. Most of the insulator-associated DNA-binding proteins showed the directional bias of DNA-binding motifs, suggesting that the directional bias is associated with the insulator.

I have added the sentence in lines 96-99 as follows: Furthermore, statistical testing the contribution scores between the directional and non-directional DNA-binding sites of insulator-associated DBPs revealed that the directional sites contributed more significantly to the prediction of gene expression levels than the non-directional sites. I have revised the statement in lines 101-110 as follows: To validate these findings, we demonstrate that the DNA-binding sites of the identified insulator-associated DBPs are located within potential insulator sites, and some of the DNA-binding sites in the insulator site are found without the nearby DNA-binding sites of CTCF and cohesin. Homologous and heterologous insulator-insulator pairing interactions are orientation-dependent, as suggested by the insulator-pairing model based on experimental analysis in flies. Our method and analyses contribute to the identification of insulator- and chromatin-associated DNA-binding sites that influence EPIs and reveal novel functional roles and molecular mechanisms of DBPs associated with transcriptional condensation, phase separation and transcriptional regulation.

3) Results, line 111: How do the SNPs come into the procedure? From the figures it seems the input is ChIP-seq peaks of DNBPs around the TSS.

On lines 121-124, to explain the procedure for the SNP of an eQTL, I have added the sentence in the Methods: "If a DNA-binding site was located within a 100-bp region around a single-nucleotide polymorphism (SNP) of an eQTL, we assumed that the DNA-binding proteins regulated the expression of the transcript corresponding to the eQTL".

4) Again, are those SNPs coming from the different cell lines? Or are they from individuals w.r.t some reference genome? I suggest a general restructuring of this part to let the reader understand more easily. One option could be simplifying the details here or alternatively including all the necessary details.

On line 119, I have included the explanation of the eQTL dataset of GTEx v8 as follows: " The eQTL data were derived from the GTEx v8 dataset, after quality control, consisting of 838 donors and 17,382 samples from 52 tissues and two cell lines”. On lines 681 and 865, I have added the filename of the eQTL data "(GTEx_Analysis_v8_eQTL.tar)".

5) Figure 1: panel a and b are misleading. Is the matrix in panel a equivalent to the matrix in panel b? If not please clarify why. Maybe in b it is included the info about the SNPs? And if yes, again, what is then difference with a.

The reviewer would mention Figure 2, not Figure 1. If so, the matrices in panels a and b in Figure 2 are equivalent. I have shown it in the figure: The same figure in panel a is rotated 90 degrees to the right. The green boxes in the matrix show the regions with the ChIP-seq peak of a DNA-binding protein overlapping with a SNP of an eQTL. I used eQTL data to associate a gene with a ChIP-seq peak that was more than 2 kb upstream and 1 kb downstream of a transcriptional start site of a gene. For each gene, the matrix was produced and the gene expression levels in cells were learned and predicted using the deep learning method. I have added the following sentences to explain the method in lines 133 - 139: Through the training, the tool learned to select the binding sites of DNA-binding proteins from ChIP-seq assays that were suitable for predicting gene expression levels in the cell types. The binding sites of a DNA-binding protein tend to be observed in common across multiple cell and tissue types. Therefore, ChIP-seq data and eQTL data in different cell and tissue types were used as input data for learning, and then the tool selected the data suitable for predicting gene expression levels in the cell types, even if the data were not obtained from the same cell types.

6) Line 386-388: could the author investigate in more detail this observation? Does it mean that loops driven by other DBPs independent of the known CTCF/Cohesin? Could the author provide examples of chromatin structural data e.g. MicroC?

As suggested by the reviewer, to help readers understand the observation, I have added Supplementary Fig. S4c to show the distribution of DNA-binding sites of "CTCF, RAD21, and SMC3" and "BACH2, FOS, ATF3, NFE2, and MAFK" around chromatin interaction sites. I have modified the following sentence to indicate the figure on line 493: Although a DNA-binding-site distribution pattern around chromatin interaction sites similar to those of CTCF, RAD21, and SMC3 was observed for DBPs such as BACH2, FOS, ATF3, NFE2, and MAFK, less than 1% of the DNA-binding sites of the latter set of DBPs colocalized with CTCF, RAD21, or SMC3 in a single bin (Fig. S4c).

In Aljahani A et al. Nature Communications 2022, we find that depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Together, our data show that loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression. Goel VY et al. Nature Genetics 2023 mentioned in the abstract: Microcompartments frequently connect enhancers and promoters and though loss of loop extrusion and inhibition of transcription disrupts some microcompartments, most are largely unaffected. These results suggested that chromatin loops can be driven by other DBPs independent of the known CTCF/Cohesin.

I added the following sentence on lines 561-569: The depletion of cohesin causes a subtle reduction in longer-range enhancer-promoter interactions and that CTCF depletion can cause rewiring of regulatory contacts. Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently. Furthermore, the loop extrusion is not essential for enhancer-promoter interactions, but contributes to their robustness and specificity and to precise regulation of gene expression.

FOXA1 pioneer factor functions as an initial chromatin-binding and chromatin-remodeling factor and has been reported to form biomolecular condensates (Ji D et al. Molecular Cell 2024). CTCF have also found to form transcriptional condensate and phase separation (Lee R et al. Nucleic acids research 2022). FOS was found to be an insulator-associated DNA-binding protein in this study and is potentially involved in chromatin remodeling, transcription condensation, and phase separation with the other factors such as BACH2, ATF3, NFE2 and MAFK. I have added the following sentence on line 548: FOXA1 pioneer factor functions as an initial chromatin-binding and chromatin-remodeling factor and has been reported to form biomolecular condensates.

7) In general, how the presented results are related to some models of chromatin architecture, e.g. loop extrusion, in which it is integrated convergent CTCF binding sites?

Goel VY et al. Nature Genetics 2023 identified highly nested and focal interactions through region capture Micro-C, which resemble fine-scale compartmental interactions and are termed microcompartments. In the section titled "Most microcompartments are robust to loss of loop extrusion," the researchers noted that a small proportion of interactions between CTCF and cohesin-bound sites exhibited significant reductions in strength when cohesin was depleted. In contrast, the majority of microcompartmental interactions remained largely unchanged under cohesin depletion. Our findings indicate that most P-P and E-P interactions, aside from a few CTCF and cohesin-bound enhancers and promoters, are likely facilitated by a compartmentalization mechanism that differs from loop extrusion. We suggest that nested, multiway, and focal microcompartments correspond to small, discrete A-compartments that arise through a compartmentalization process, potentially influenced by factors upstream of RNA Pol II initiation, such as transcription factors, co-factors, or active chromatin states. It follows that if active chromatin regions at microcompartment anchors exhibit selective "stickiness" with one another, they will tend to co-segregate, leading to the development of nested, focal interactions. This microphase separation, driven by preferential interactions among active loci within a block copolymer, may account for the striking interaction patterns we observe.

The authors of the paper proposed several mechanisms potentially involved in microcompartments. These mechanisms may be involved in looping with insulator function. Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently (Hsieh TS et al. Nature Genetics 2022). Among the identified insulator-associated DNA-binding proteins, Maz and MyoD1 form loops without CTCF (Xiao T et al. Proc Natl Acad Sci USA 2021 ; Ortabozkoyun H et al. Nature genetics 2022 ; Wang R et al. Nature communications 2022). I have added the following sentences on lines 563-567: Another group reported that enhancer-promoter interactions and transcription are largely maintained upon depletion of CTCF, cohesin, WAPL or YY1. Instead, cohesin depletion decreased transcription factor binding to chromatin. Thus, cohesin may allow transcription factors to find and bind their targets more efficiently. I have included the following explanation on lines 574-576: Maz and MyoD1 among the identified insulator-associated DNA-binding proteins form loops without CTCF.

As for the directionality of CTCF, if chromatin loop anchors have some structural conformation, as shown in the paper entitled "The structural basis for cohesin-CTCF-anchored loops" (Li Y et al. Nature 2020), directional DNA binding would occur similarly to CTCF binding sites. Moreover, cohesin complexes that interact with convergent CTCF sites, that is, the N-terminus of CTCF, might be protected from WAPL, but those that interact with divergent CTCF sites, that is, the C-terminus of CTCF, might not be protected from WAPL, which could release cohesin from chromatin and thus disrupt cohesin-mediated chromatin loops (Davidson IF et al. Nature Reviews Molecular Cell Biology 2021). Regarding loop extrusion, the ‘loop extrusion’ hypothesis is motivated by in vitro observations. The experiment in yeast, in which cohesin variants that are unable to extrude DNA loops but retain the ability to topologically entrap DNA, suggested that in vivo chromatin loops are formed independently of loop extrusion. Instead, transcription promotes loop formation and acts as an extrinsic motor that extends these loops and defines their final positions (Guerin TM et al. EMBO Journal 2024). I have added the following sentences on lines 535-539: Cohesin complexes that interact with convergent CTCF sites, that is, the N-terminus of CTCF, might be protected from WAPL, but those that interact with divergent CTCF sites, that is, the C-terminus of CTCF, might not be protected from WAPL, which could release cohesin from chromatin and thus disrupt cohesin-mediated chromatin loops. I have included the following sentences on lines 569-574: The ‘loop extrusion’ hypothesis is motivated by in vitro observations. The experiment in yeast, in which cohesin variants that are unable to extrude DNA loops but retain the ability to topologically entrap DNA, suggested that in vivo chromatin loops are formed independently of loop extrusion. Instead, transcription promotes loop formation and acts as an extrinsic motor that extends these loops and defines their final positions.

Another model for the regulation of gene expression by insulators is the boundary-pairing (insulator-pairing) model (Bing X et al. Elife 2024) (Ke W et al. Elife 2024) (Fujioka M et al. PLoS Genetics 2016). Molecules bound to insulators physically pair with their partners, either head-to-head or head-to-tail, with different degrees of specificity at the termini of TADs in flies. Although the experiments do not reveal how partners find each other, the mechanism unlikely requires loop extrusion. Homologous and heterologous insulator-insulator pairing interactions are central to the architectural functions of insulators. The manner of insulator-insulator interactions is orientation-dependent. I have summarized the model on lines 551-559: Other types of chromatin regulation are also expected to be related to the structural interactions of molecules. As the boundary-pairing (insulator-pairing) model, molecules bound to insulators physically pair with their partners, either head-to-head or head-to-tail, with different degrees of specificity at the termini of TADs in flies (Fig. 7). Although the experiments do not reveal how partners find each other, the mechanism unlikely requires loop extrusion. Homologous and heterologous insulator-insulator pairing interactions are central to the architectural functions of insulators. The manner of insulator-insulator interactions is orientation-dependent.

8) Do the authors think that the identified DBPs could work in that way as well?

The boundary-pairing (insulator-pairing) model would be applied to the insulator-associated DNA-binding proteins other than CTCF and cohesin that are involved in the loop extrusion mechanism (Bing X et al. Elife 2024) (Ke W et al. Elife 2024) (Fujioka M et al. PLoS Genetics 2016).

Liquid-liquid phase separation was shown to occur through CTCF-mediated chromatin loops and to act as an insulator (Lee, R et al. Nucleic Acids Research 2022). Among the identified insulator-associated DNA-binding proteins, CEBPA has been found to form hubs that colocalize with transcriptional co-activators in a native cell context, which is associated with transcriptional condensate and phase separation (Christou-Kent M et al. Cell Reports 2023). The proposed microcompartment mechanisms are also associated with phase separation. Thus, the same or similar mechanisms are potentially associated with the insulator function of the identified DNA-binding proteins. I have included the following information on line 546: CEBPA in the identified insulator-associated DNA-binding proteins was also reported to be involved in transcriptional condensates and phase separation.

9) Also, can the authors comment about the mechanisms those newly identified DBPs mediate contacts by active processes or equilibrium processes?

Snead WT et al. Molecular Cell 2019 mentioned that protein post-transcriptional modifications (PTMs) facilitate the control of molecular valency and strength of protein-protein interactions. O-GlcNAcylation as a PTM inhibits CTCF binding to chromatin (Tang X et al. Nature Communications 2024). I found that the identified insulator-associated DNA-binding proteins tend to form a cluster at potential insulator sites (Supplementary Fig. 2d). These proteins may interact and actively regulate chromatin interactions, transcriptional condensation, and phase separation by PTMs. I have added the following explanation on lines 576-582: Furthermore, protein post-transcriptional modifications (PTMs) facilitate control over the molecular valency and strength of protein-protein interactions. O-GlcNAcylation as a PTM inhibits CTCF binding to chromatin. We found that the identified insulator-associated DNA-binding proteins tend to form a cluster at potential insulator sites (Fig. 4f and Supplementary Fig. 3c). These proteins may interact and actively regulate chromatin interactions, transcriptional condensation, and phase separation through PTMs.

10) Can the author provide some real examples along with published structural data (e.g. the mentioned micro-C data) to show the link between protein co-presence, directional bias and contact formation?

Structural molecular model of cohesin-CTCF-anchored loops has been published by Li Y et al. Nature 2020. The structural conformation of CTCF and cohesin in the loops would be the cause of the directional bias of CTCF binding sites, which I mentioned in lines 531 – 535 as follows: These results suggest that the directional bias of DNA-binding sites of insulator-associated DBPs may be involved in insulator function and chromatin regulation through structural interactions among DBPs, other proteins, DNAs, and RNAs. For example, the N-terminal amino acids of CTCF have been shown to interact with RAD21 in chromatin loops.

To investigate the principles underlying the architectural functions of insulator-insulator pairing interactions, two insulators, Homie and Nhomie, flanking the Drosophila even skipped locus were analyzed. Pairing interactions between the transgene Homie and the eve locus are directional. The head-to-head pairing between the transgene and endogenous Homie matches the pattern of activation (Fujioka M et al. PLoS Genetics 2016).

Reviewer #3

1. Some of these TFs do not have specific direct binding to DNA (P300, Cohesin). Since the authors are using binding motifs in their analysis workflow, I would remove those from the analysis.

When a protein complex binds to DNA, one protein of the complex binds to the DNA directory, and the other proteins may not bind to DNA. However, the DNA motif sequence bound by the protein may be registered as the DNA-binding motif of all the proteins in the complex. The molecular structure of the complex of CTCF and Cohesin showed that both CTCF and Cohesin bind to DNA (Li Y et al. Nature 2020). I think there is a possibility that if the molecular structure of a protein complex becomes available, the previous recognition of the DNA-binding ability of a protein may be changed. Therefore, I searched the Pfam database for 99 insulator-associated DNA-binding proteins identified in this study. I found that 97 are registered as DNA-binding proteins and/or have a known DNA-binding domain, and EP300 and SIN3A do not directory bind to DNA, which was also checked by Google search. I have added the following explanation in line 249 to indicate direct and indirect DNA-binding proteins: Among 99 insulator-associated DBPs, EP300 and SIN3A do not directory interact with DNA, and thus 97 insulator-associated DBPs directory bind to DNA. I have updated the sentence in line 22 of the Abstract as follows: We discovered 97 directional and minor nondirectional motifs in human fibroblast cells that corresponded to 23 DBPs related to insulator function, CTCF, and/or other types of chromosomal transcriptional regulation reported in previous studies.

2. I am not sure if I understood correctly, by why do the authors consider enhancers spanning 2Mb (200 bins of 10Kb around eSNPs)? This seems wrong. Enhancers are relatively small regions (100bp to 1Kb) and only a very small subset form super enhancers.

As the reviewer mentioned, I recognize enhancers are relatively small regions. In the paper, I intended to examine further upstream and downstream of promoter regions where enhancers are found. Therefore, I have modified the sentence in lines 917 – 919 of the Fig. 2 legend as follows: Enhancer-gene regulatory interaction regions consist of 200 bins of 10 kbp between -1 Mbp and 1 Mbp region from TSS, not including promoter.

3. I think the H3K27me3 analysis was very good, but I would have liked to see also constitutive heterochromatin as well, so maybe repeat the analysis for H3K9me3.

Following the reviewer's advice, I have added the ChIP-seq data of H3K9me3 as a truck of the UCSC Genome Browser. The distribution of H3K9me3 signal was different from that of H3K27me3 in some regions. I also found the insulator-associated DNA-binding sites close to the edges of H3K9me3 regions and took some screenshots of the UCSC Genome Browser of the regions around the sites in Supplementary Fig. 3b. I have modified the following sentence on lines 962 – 964 in the legend of Fig. 4: a Distribution of histone modification marks H3K27me3 (green color) and H3K9me3 (turquoise color) and transcript levels (pink color) in upstream and downstream regions of a potential insulator site (light orange color). I have also added the following result on lines 348 – 352: The same analysis was performed using H3K9me3 marks, instead of H3K27me3 (Fig. S3b). We found that the distribution of H3K9me3 signal was different from that of H3K27me3 in some regions, and discovered the insulator-associated DNA-binding sites close to the edges of H3K9me3 regions (Fig. S3b).

4. I was not sure I understood the analysis in Figure 6. The binding site is with 500bp of the interaction site, but micro-C interactions are at best at 1Kb resolution. They say they chose the centre of the interaction site, but we don't know exactly where there is the actual interaction. Also, it is not clear what they measure. Is it the number of binding sites of a specific or multiple DBP insulator proteins at a specific distance from this midpoint that they recover in all chromatin loops? Maybe I am missing something. This analysis was not very clear.

The resolution of the Micro-C assay is considered to be 100 bp and above, as the human nucleome core particle contains 145 bp (and 193 bp with linker) of DNA. However, internucleosomal DNA is cleaved by endonuclease into fragments of multiples of 10 nucleotides (Pospelov VA et al. Nucleic Acids Research 1979). Highly nested focal interactions were observed (Goel VY et al. Nature Genetics 2023). Base pair resolution was reported using Micro Capture-C (Hua P et al. Nature 2021). Sub-kilobase (20 bp resolution) chromatin topology was reported using an MNase-based chromosome conformation capture (3C) approach (Aljahani A et al. Nature Communications 2022). On the other hand, Hi-C data was analyzed at 1 kb resolution. (Gu H et al. bioRxiv 2021). If the resolution of Micro-C interactions is at best at 1 kb, the binding sites of a DNA-binding protein will not show a peak around the center of the genomic locations of interaction edges. Each panel shows the number of binding sites of a specific DNA-binding protein at a specific distance from the midpoint of all chromatin interaction edges. I have modified and added the following sentences in lines 585-589: High-resolution chromatin interaction data from a Micro-C assay indicated that most of the predicted insulator-associated DBPs showed DNA-binding-site distribution peaks around chromatin interaction sites, suggesting that these DBPs are involved in chromatin interactions and that the chromatin interaction data has a high degree of resolution. Base pair resolution was reported using Micro Capture-C.

1.PIQ does not consider TF concentration. Other methods do that and show that TF concentration improves predictions (e.g.,https://www.biorxiv.org/content/10.1101/2023.07.15.549134v2 or https://pubmed.ncbi.nlm.nih.gov/37486787/). The authors should discuss how that would impact their results.

The directional bias of CTCF binding sites was identified by ChIA-pet interactions of CTCF binding sites. The analysis of the contribution scores of DNA-binding sites of proteins considering the binding sites of CTCF as an insulator showed the same tendency of directional bias of CTCF binding sites. In the analysis, to remove the false-positive prediction of DNA-binding sites, I used the binding sites that overlapped with a ChIP-seq peak of the DNA-binding protein. This result suggests that the DNA-binding sites of CTCF obtained by the current analysis have sufficient quality. Therefore, if the accuracy of prediction of DNA-binding sites is improved, althought the number of DNA-binding sites may be different, the overall tendency of the directionality of DNA-binding sites will not change and the results of this study will not change significantly.

As for the first reference in the reviewer's comment, chromatin interaction data from Micro-C assay does not include all chromatin interactions in a cell or tissue, because it is expensive to cover all interactions. Therefore, it would be difficult to predict all chromatin interactions based on machine learning. As for the second reference in the reviewer's comment, pioneer factors such as FOXA are known to bind to closed chromatin regions, but transcription factors and DNA-binding proteins involved in chromatin interactions and insulators generally bind to open chromatin regions. The search for the DNA-binding motifs is not required in closed chromatin regions.

2. DeepLIFT is a good approach to interpret complex structures of CNN, but is not truly explainable AI. I think the authors should acknowledge this.

In the DeepLIFT paper, the authors explain that DeepLIFT is a method for decomposing the output prediction of a neural network on a specific input by backpropagating the contributions of all neurons in the network to every feature of the input (Shrikumar A et al. ICML 2017). DeepLIFT compares the activation of each neuron to its 'reference activation' and assigns contribution scores according to the difference. DeepLIFT calculates a metric to measure the difference between an input and the reference of the input.

Truly explainable AI would be able to find cause and reason, and to make choices and decisions like humans. DeepLIFT does not perform causal inferences. I did not use the term "Explainable AI" in our manuscript, but I briefly explained it in Discussion. I have added the following explanation in lines 615-620: AI (Artificial Intelligence) is considered as a black box, since the reason and cause of prediction are difficult to know. To solve this issue, tools and methods have been developed to know the reason and cause. These technologies are called Explainable AI. DeepLIFT is considered to be a tool for Explainable AI. However, DeepLIFT does not answer the reason and cause for a prediction. It calculates scores representing the contribution of the input data to the prediction.

Furthermore, to improve the readability of the manuscript, I have included the following explanation in lines 159-165: we computed DeepLIFT scores of the input data (i.e., each binding site of the ChIP-seq data of DNA-binding proteins) in the deep leaning analysis on gene expression levels. DeepLIFT compares the importance of each input for predicting gene expression levels to its 'reference or background level' and assigns contribution scores according to the difference. DeepLIFT calculates a metric to measure the difference between an input and the reference of the input.

make sure to use strong action statements here (accomplished X through Y resulting in Z). instead of saying 'i used', you could rephrase to something along the lines of 'Developed the front-end of a JSON compiler, implementing lexical, syntactic, and semantic analysis, ensuring proper error handling and recovery'.

Voici des documents de synthèse pour un briefing sur le Cadre d'action et de coopération pour la transformation écologique (CACTÉ), en tirant parti des sources et de notre conversation précédente :

Titre : Briefing sur le Cadre d'action et de coopération pour la transformation écologique (CACTÉ)

Introduction (0:00-1:31):

• Le CACTÉ est un cadre d'action et de coopération pour la transformation écologique.
• Il a été élaboré par la direction générale de la création artistique (DGCA) du ministère de la Culture.
• Objectif principal : intégrer les enjeux écologiques dans le secteur de la création artistique.
• Les intervenants clés sont Frédérique Sarre, responsable de la mission Transformation écologique de la création au Ministère de la Culture, et Maxime Gueudet, Chargé de mission transition écologique de la création au Ministère de la Culture.

Contexte et Stratégie Globale (1:43-3:22):

• Le CACTÉ est une mesure du plan d'action de la DGCA pour la transition écologique du secteur de la création.
• Il s'inscrit dans la stratégie "mieux produire, mieux diffuser".
• Il est aligné avec les accords internationaux et la feuille de route ministérielle.
• Il constitue le volet écologique de "mieux produire, mieux diffuser".

Élaboration du Plan d'Action (3:29-4:26):

• Le plan d'action a été élaboré à partir de 2020.
• Il a impliqué une approche transversale avec les services de la DGCA et les DRAC.
• Une mission dédiée à la transformation écologique a été créée.

Objectifs du Plan d'Action et du CACTÉ (4:32-5:22):

• "Éviter l'ingérable et gérer l'inévitable".
• Contribuer à la réduction des impacts environnementaux du secteur de la création.
• Assurer la robustesse du secteur face aux crises écologiques.
• Respecter les valeurs du secteur, notamment la liberté de création.

Principes Clés du CACTÉ (6:28-7:26):

• Souplesse : Adaptation aux réalités et priorités locales.
• Structuration : Engagement obligatoire et méthodologie imposée.
• Pédagogie : Présentation sous forme de guide.

Application du CACTÉ (7:32-8:22):

• Obligatoire pour les structures ayant une convention pluriannuelle d'objectifs (CPO) de 3 ans ou plus avec la DGCA.
• Recommandé pour les structures financées de manière régulière depuis plus de 3 ans.
• Utilisable par toute autre structure du secteur public ou privé.

Calendrier (8:29-9:06):

• Expérimentation en 2024 dans cinq régions.
• Publication de la version révisée.
• Généralisation prévue en 2025-2026 lors des renouvellements de CPO.

Bilan de l'Expérimentation (9:11-10:13):

• Réception positive et adaptabilité aux réalités locales.
• Association des partenaires financiers.
• Nécessité d'accompagnement et crainte d'un manque de disponibilité des équipes.

Réponses aux Craintes (10:13-11:25):

• Accompagnement par les DRAC et la DGCA.
• Webinaires et outils de soutien.

Documents du CACTÉ (11:31-12:21):

• Document présentant le dispositif.
• Guide thématique pour l'action avec des fiches action.
• Volet ressources (réglementation, sites internet, guides, rapports).

Fonctionnement Détaillé du CACTÉ (12:26-13:53):

• Deux types d'engagements :
  • Engagement méthodologique obligatoire.
  • Engagements thématiques au choix.
• Engagement méthodologique obligatoire :
  • Définir une stratégie basée sur des données objectives (évaluation des impacts environnementaux).
  • Formation (minimum une journée pour toute l'équipe).
  • Coopération en interne et en externe.

Importance de la Formation (13:59-14:23):

• La formation est essentielle pour développer les questions de transition écologique.
• Elle doit inclure les enjeux de la transition écologique et leur application au secteur de la création.

Nécessité de Coopération (14:23-15:18):

• La coopération interne et externe est cruciale pour une démarche écoresponsable efficace.
• Impliquer l'ensemble de l'équipe dans l'élaboration et la mise en œuvre du plan d'action.
• S'associer avec des acteurs locaux, y compris ceux hors du secteur culturel.

Engagements Thématiques (15:25-17:06):

• Dix engagements thématiques au choix.
• Les structures choisissent les engagements qu'elles souhaitent mettre en œuvre.
• Les engagements sont choisis en fonction des impacts estimés, de la réalité de la structure, de son contexte local, et en concertation avec les partenaires financiers.
• Le nombre d'engagements minimum varie selon le type et la taille de la structure.
• Exemples d'engagements thématiques: mobilité, réduction de la consommation des fluides, alimentation, écoconception des œuvres, numérique, communication, gestion des déchets, bâti et sites, biodiversité.

Leviers d'Action (17:31-19:57):

• Pour chaque engagement, des leviers d'action sont proposés pour assurer que l'engagement est rempli dans son intégralité.

Fiches Action (20:02-20:46):

• Les fiches action sont des guides thématiques et des supports d'auto-évaluation.
• Elles permettent de visualiser rapidement les actions mises en œuvre.
• Elles développent des leviers et exemples d'actions pour chaque engagement.

Évaluation et Certification (20:46-21:47):

• L'évaluation donne lieu à une certification (niveaux 1, 2, 3, 3+) correspondant au nombre d'engagements thématiques mis en œuvre.
• Le niveau 3+ inclut l'intégration des questions écologiques dans la programmation ou les actions d'EAC.

Absence d'Indicateurs Chiffrés Obligatoires (21:47-23:53):

• L'évaluation ne repose pas sur des indicateurs chiffrés obligatoires, mais sur une obligation de moyens.
• Cela est dû à la difficulté de définir des indicateurs pertinents et à la capacité limitée des structures à les renseigner.

Processus d'Évaluation (23:53-26:22):

• L'évaluation associe les collectivités territoriales et permet un suivi régional.
• Auto-évaluation par la structure.
• Réunion d'un groupe régional d'évaluation coopérative.

Groupe Régional d'Évaluation Coopérative (26:22-27:31):

• Le groupe donne un avis sur le niveau de certification.
• Il recommande des actions aux structures et à la puissance publique.

Instances de Suivi et Attribution de la Certification (27:31-28:27):

• La discussion revient aux instances de suivi (comité de suivi, conseil d'administration).
• La DRAC attribue la certification.

Questions Fréquentes et Réponses :

• Rôle des collectivités : Application par les DRAC en lien avec les collectivités, dialogue au sein des comités de suivi.
• Exemples de déploiement : Diversité des structures concernées (centres nationaux, FRAC, scènes dramatiques, équipes indépendantes).
• Calendrier pour les festivals : Se rapprocher de la DRAC, Coprog comme outil possible, pas de "carotte" ou de "bâton", mais écoconditionnalité douce.
• Possibilité pour une compagnie non conventionnée d'être certifiée : Oui, en adressant une demande à la DRAC.
• Accompagnement : privilégier un accompagnement collectif, en s'appuyant sur des écoconseillers, des dispositifs existants (Avdas), ou les DLA.
• Empilement des dispositifs : le CACTÉ doit s'adapter aux priorités politiques locales et aux démarches déjà en place.

Ces documents devraient fournir une base solide pour un briefing complet sur le CACTÉ.

Voici un sommaire minuté des idées fortes du webinaire sur le Cadre d’action et de coopération pour la transformation écologique (CACTÉ) :

• 0:00-1:05: Introduction par Antoine Dunan de la FNADAC, remerciant les intervenants et soulignant l'importance du CACTÉ pour les associations.
• 1:12-1:31: Présentation de Frédérique Sarre et Maxime Gueudet du Ministère de la Culture, exprimant leur enthousiasme à partager les résultats de leurs travaux sur le CACTÉ.
• 1:43-3:22: Contexte et stratégie globale du CACTÉ, le positionnant comme une mesure du plan d'action de la DGCA pour la transition écologique du secteur de la création, intégré à la stratégie "mieux produire, mieux diffuser" et aligné avec les accords internationaux et la feuille de route ministérielle.
• 3:29-4:26: Élaboration du plan d'action de la DGCA depuis 2020, soulignant la transversalité, la formation aux enjeux écologiques, et la création d'une mission dédiée à la transformation écologique.
• 4:32-5:22: Objectifs du plan d'action et du CACTÉ : éviter l'ingérable et gérer l'inévitable, assurer la robustesse du secteur face aux crises écologiques, tout en respectant les valeurs du secteur, notamment la liberté de création.
• 5:28-6:21: Les mesures du plan d'action sont fondées sur une enquête préalable, des auditions, et un repérage des initiatives existantes, avec une approche itérative et évolutive.
• 6:28-7:26: Le CACTÉ est construit sur trois piliers : souplesse (adaptation aux réalités et priorités locales), structuration (obligation d'engagement et méthodologie imposée), et pédagogie (présentation sous forme de guide).
• 7:32-8:22: Application du CACTÉ : obligatoire pour les structures ayant un document de contractualisation de 3 ans ou plus avec la DGCA, recommandé pour les autres, et utilisable par toute structure.
• 8:29-9:06: Calendrier : expérimentation en 2024 dans cinq régions, révision, et publication de la nouvelle version, généralisation prévue en 2025-2026 lors des renouvellements de CPO.
• 9:11-10:13: Bilan de l'expérimentation : réception positive, adaptabilité aux réalités locales, association des partenaires financiers, mais nécessité d'accompagnement et crainte d'un manque de disponibilité des équipes.
• 10:13-11:25: Réponses apportées aux craintes : accompagnement par les DRAC et la DGCA, webinaires, etc.
• 11:31-12:21: Présentation des trois documents du CACTÉ : un document présentant le dispositif, un guide thématique pour l'action avec des fiches action, et un volet ressources.
• 12:26-13:53: Détail du fonctionnement du CACTÉ : deux types d'engagements, un engagement méthodologique obligatoire (définir une stratégie basée sur des données objectives, formation, coopération en interne et externe), et des engagements thématiques.
• 13:59-14:23: Importance de la formation pour développer les questions de transition écologique, avec un minimum d'une journée pour toute l'équipe.
• 14:23-15:18: Nécessité de coopération interne et externe pour une démarche écoresponsable efficace.
• 15:25-17:06: Présentation des 10 engagements thématiques au choix, à sélectionner en fonction des impacts estimés, de la réalité de la structure, de son projet, de son contexte local, et en dialogue avec les partenaires financiers.
• 17:06-17:31: Le nombre d'engagements minimum varie selon le type et la taille de la structure.
• 17:31-19:57: Pour chaque engagement, des leviers d'action sont proposés pour assurer que l'engagement est rempli dans son intégralité.
• 20:02-20:46: Les fiches action sont des guides thématiques et des supports d'auto-évaluation, permettant de visualiser rapidement les actions mises en œuvre.
• 20:46-21:47: L'évaluation donne lieu à une certification (niveaux 1, 2, 3, 3+) correspondant au nombre d'engagements thématiques mis en œuvre, le niveau 3+ incluant l'introduction des questions écologiques dans la programmation ou les actions d'EAC.
• 21:47-23:53: L'évaluation ne repose pas sur des indicateurs chiffrés obligatoires, mais sur une obligation de moyens, en raison de la difficulté de définir des indicateurs pertinents et de la capacité limitée des structures à les renseigner.
• 23:53-26:22: L'évaluation associe les collectivités territoriales et permet un suivi régional, avec une auto-évaluation par la structure et la réunion d'un groupe régional d'évaluation coopérative.
• 26:22-27:31: Le groupe régional d'évaluation coopérative donne un avis sur le niveau de certification et recommande des actions aux structures et à la puissance publique.
• 27:31-28:27: La discussion revient ensuite aux instances de suivi (comité de suivi, conseil d'administration) pour confirmer le niveau de certification et envisager des solutions aux problèmes rencontrés, puis attribution de la certification par la DRAC.
• 28:46-32:30: Questions sur le rôle des collectivités et la déclinaison du CACTÉ au niveau régional (PACA), réponse sur l'application par les DRAC en lien avec les collectivités, et sur le dialogue au sein des comités de suivi et conseils d'administration.
• 32:30-34:55: Questions sur les récits d'expérience de la phase d'expérimentation, réponse sur l'observation de la mise en œuvre et l'enclenchement de la démarche, et sur les principaux engagements choisis (biodiversité moins choisie, mobilité des professionnels et des œuvres plus fréquente).
• 34:55-38:41: Questions sur le travail avec les référentiels carbone (FRAC, centres d'art), réponse sur le lien entre ces référentiels et le CACTÉ, et sur la création d'un outil pour estimer facilement le profil d'émission des structures.
• 38:41-40:03: Questions sur des exemples de déploiement du CACTÉ dans des structures labellisées et des festivals, réponse sur la diversité des structures concernées et la possibilité d'obtenir des détails par mail.
• 40:03-45:35: Questions sur le calendrier de mise en place du CACTÉ pour un festival de cirque, réponse sur l'absence d'obligation dans ce cas, mais l'invitation à se rapprocher de la DRAC, sur l'intégration de Coprog comme outil possible, et sur l'absence de carotte ou de bâton, mais une écoconditionnalité douce et une évaluation qui fait partie des critères de poursuite de la subvention.
• 45:35-47:31: Questions sur la mise en place du groupe régional d'évaluation coopérative et les autres lieux de coopération, réponse sur une mise en place réelle en 2027, une préfiguration possible en 2026, et sur les discussions au sein des CLTC et COREPS.
• 47:31-50:19: Questions sur la possibilité pour une compagnie non conventionnée d'être certifiée, réponse positive avec une demande à adresser à la DRAC, et sur une communication en direction de l'ensemble des structures du secteur, réponse sur un webinaire prévu et des présentations par les DRAC.
• 50:19-54:27: Questions sur l'empilement des strates d'implication des collectivités, réponse sur l'adaptabilité du CACTÉ et l'importance de choisir les engagements correspondant aux priorités politiques locales, et sur la synchronisation progressive avec les conseillers DRAC.
• 54:27-59:10: Questions d'Olivier, il représente une compagnie musicale. Il demande si le conseiller référent des DRAC pour le CACTE serait le conseiller musique. La réponse est "oui".
• 59:10-1:03:27: Il n'y aura pas de formations obligatoires imposées par le ministère, mais que l'AVDAS a intégré le CACTÉ dans son programme de formation.
• 1:03:27-1:05:53: Demande sur le volet lié à l'intégration des questions de transition dans les programmations et projets EAC, la réponse est que le principe du CACTÉ est avant tout de permettre de contraindre les structures de s'engager dans une démarche d'écoresponsabilité de leurs activités..
• 1:05:53-1:07:20: Une structure d'accompagnement demande si elle peut être référencée pour délivrer les formations obligatoires dans le cadre du CACTÉ, la réponse est que pour le moment, toute structure est agrée en quelque sorte.
• 1:07:20-1:08:46: Une question par rapport aux engagements thématiques les plus et moins populaires, la réponse est que l'engagement sur la biodiversité est assez peu choisi, mais la prudence reste de mise.
• 1:09:05-1:12:09: Antoine Dunan a expérimenté en PACA et trouve qu'il y a des montagnes à déplacer pour traiter certains objets et demande comment financer et mettre en place des nouvelles infrastructures. La fnadaac a mis en place un groupe de travail qui s'appelle crisealide.
• 1:12:16-1:16:41: Y a-t-il des écoconseillers ? La réponse est l'appropriation du sujet par chacun est essentiel, et ils peuvent accompagner un certain nombre de structures.
• 1:16:54-1:18:12: Les interlocuteurs remercient les participants et se tiennent à disposition.

Voici un sommaire minuté des idées fortes de la conférence de Gérald Bronner :

• 0:09-1:36 : Introduction et remerciements. Gérald Bronner se présente et remercie la Sorbonne et la Fondation Descartes pour leur soutien, soulignant la gratuité de l'événement grâce à leur contribution. Il met en évidence l'importance de la Fondation Descartes, unique en France à s'intéresser à la qualité de l'information et à la désinformation.

• 1:41-3:30 : Présentation du séminaire et contrat moral. Bronner explique le déroulement du séminaire en quatre parties, encourageant les participants à reproduire ce séminaire auprès de leurs proches pour diffuser l'esprit critique. Il annonce que les supports de cours seront disponibles en ligne.

• 3:36-6:01 : Pourquoi un séminaire sur l'esprit critique ?. Bronner justifie la nécessité de ce séminaire face à la désinformation et aux fausses nouvelles, un problème reconnu par de nombreuses institutions internationales.

• 6:08-8:05 : Le Forum économique mondial et les risques pour l'humanité. Il cite le Forum économique mondial qui identifie la mésinformation et la désinformation comme les principaux risques à court terme pour l'humanité, car ils empêchent de s'accorder sur la réalité des problèmes. Le risque est la fracture du socle épistémique commun et la polarisation des sociétés.

• 8:10-9:36 : La démocratie des crédules et les conditions métacognitives. Bronner évoque ses travaux antérieurs, notamment "La démocratie des crédules", et souligne que nous vivons aujourd'hui dans cette réalité. Il explique que les prochaines séances porteront sur les conditions métacognitives pour développer l'esprit critique.

• 9:42-10:31 : Exercices d'esprit critique et commune humanité. Les dernières séances seront consacrées à des exercices d'esprit critique sur des problèmes amusants et neutres, encourageant à se rappeler notre commune humanité face aux divergences d'interprétation.

• 10:31-12:29 : Disponibilité de l'information et pression concurrentielle. Bronner aborde l'explosion de la disponibilité de l'information grâce à Internet, qui a créé une pression concurrentielle sur le marché des idées. Contrairement aux espoirs initiaux, la rationalité ne s'impose pas naturellement.

• 12:29-14:05 : Paradoxe de l'information et biais de confirmation. Il explique le paradoxe de l'information : plus il y a d'informations, plus il est facile de trouver des informations qui confirment nos croyances préalables, renforçant le biais de confirmation.

• 14:13-16:11 : Intervention de tous et super-diffuseurs. Tout le monde peut intervenir sur le marché de l'information, mais certains individus (super-diffuseurs) parlent plus fort et plus que les autres. 1 % des comptes produisent 33 % de l'information.

• 16:11-17:44 : Radicalité et antivaccins. Les plus actifs sont souvent porteurs d'une forme de radicalité. L'exemple des antivaccins montre comment une minorité suractive peut essaimer ses arguments dans l'espace public.

• 17:44-19:38 : Prosélytisme et apathie. Les antivaccins font plus de prosélytisme que les provaccins. Bronner souligne que "le mal n'a besoin de rien d'autre pour s'imposer que de l'apathie des gens de bien", appelant à ne pas se laisser faire.

• 19:38-21:33 : Rapidité de diffusion et effet de primauté. Les théories du complot se diffusent très rapidement, exploitant les "Data void". L'effet de primauté fait que la première information rencontrée imprime une impression durable, même en cas de démenti.

• 21:33-23:01 : Stratégies des théoriciens du complot et exploitation des Data void. Les plus radicaux exploitent stratégiquement les vides de données (Data void) pour diffuser leurs théories alternatives.

• 23:01-25:56 : Puissance argumentative et millefeuille argumentatif. Il ne faut pas sous-estimer la puissance argumentative de ces raisonnements, qui forment un "millefeuille argumentatif" impressionnant grâce au travail en essaim permis par le numérique. Il ne faut pas prendre les gens qui croient ces théories pour des imbéciles.

• 25:56-28:18 : Variables sociales et intimidation intellectuelle. Les gens qui se sentent déclassés sont plus sensibles aux théories du complot. L'intimidation intellectuelle et l'asymétrie de visibilité des points de vue sont des conséquences de ce phénomène.

• 28:18-31:01 : Loi de Brandolini. Bronner explique la loi de Brandolini : il faut moins de temps pour diffuser une ânerie que pour la défaire. Il donne l'exemple d'une théorie du complot autour du film Captain America et de la bière Corona.

• 31:01-33:51 : Leviers possibles et modération. Un article de Science montre que les fausses histoires se diffusent plus vite et plus profondément que les vraies. Bronner évoque la modération des réseaux sociaux, mais souligne que leur intérêt économique n'est pas toujours convergent avec les intérêts de la démocratie.

• 33:51-35:36 : Le cerveau comme meilleur régulateur et illusions mentales. Les meilleurs régulateurs sont nos cerveaux. Il faut apprendre à dompter certains mécanismes cognitifs. Bronner parle d'illusions mentales, qu'il préfère qualifier de tentations mentales auxquelles on peut résister.

• 35:36-38:12 : Lazy thinking et avarie cognitive. La "lazy thinking" (pensée paresseuse) ou "avarice cognitive" est ce qui prédit le plus la diffusion de fausses informations. Il faut apprendre à reconnaître les situations où l'on risque de se tromper.

• 38:12-40:22 : Biais cognitifs et exemple de la COVID-19 et la 5G. La littérature a dénombré environ 150 biais cognitifs. Bronner donne l'exemple de la corrélation fallacieuse entre la COVID-19 et les antennes 5G.

• 40:22-43:02 : Corrélation n'est pas causalité. Il rappelle que corrélation n'est pas causalité, citant l'exemple des cigognes et des bébés.

• 43:02-47:21 : Interrogations métaphysiques et paradoxe de Fermi. Bronner exprime son inquiétude quant à l'avenir de notre civilisation. Il évoque le paradoxe de Fermi (où sont les extraterrestres ?) et les interprétations pessimistes sur la durée des civilisations.

• 47:21-50:11 : Croyance aux extraterrestres et soucoupes volantes. Il aborde le regain de la croyance aux extraterrestres et l'histoire de Kenneth Arnold, à l'origine du terme "soucoupe volante".

• 50:11-52:51 : Modes et observations. Il explique comment les modes et les prédictions peuvent susciter des observations et des mésinterprétations.

• 52:51-54:40 : Arguments rationnels et platistes. Bronner souligne l'importance de présenter des arguments rationnels et cite l'exemple des platistes pour illustrer l'ambivalence de la croyance.

Voici un résumé minuté de la transcription concernant l'impact du sel sur la santé, basé sur les informations des sources fournies:

• 0:08-0:20: Le sel est présenté comme essentiel et indispensable à la cuisine de qualité.
• 0:27-0:34: Le sel est potentiellement dangereux pour la santé, voire mortel, en cas de surconsommation.
• 0:41-0:47: Il est avancé que ces craintes pourraient être infondées.
• 0:47-0:53: Le sodium est indispensable au fonctionnement normal du corps humain.
• 1:52-2:06: Importance du sel en cuisine, notamment pour l'assaisonnement du poisson.
• 2:19-2:25: Importance d'un assaisonnement équilibré, ni trop discret, ni trop présent.
• 2:45-2:52: Nécessité de saler généreusement l'eau pour blanchir les légumes verts.
• 3:47-3:55: Estimation de l'utilisation de plusieurs centaines de grammes de sel pour 50 convives, ce qui peut sembler impressionnant comparé aux recommandations nutritionnelles.
• 4:00-4:25: Le sel a toujours été diabolisé, notamment dans les ouvrages destinés au grand public, et fait l'objet de campagnes de sensibilisation sur la consommation excessive.
• 5:01-5:18: Le sel de table est du chlorure de sodium, essentiel pour la rétention de liquide, le fonctionnement du cœur, des nerfs, des muscles et la tension artérielle.
• 5:23-5:46: Le sel est un électrolyte qui transporte une charge électrique, permettant au cœur de battre et alimentant le cerveau, les muscles et le système nerveux.
• 5:46-6:05: Le sel maintient l'équilibre des fluides et l'hydratation du corps.
• 6:13-6:19: Une personne de 70 kg doit avoir environ 62 cuillères à café de sel dans l'organisme.
• 6:19-6:33: Le sel est filtré et éliminé par les reins, évitant l'accumulation de toxines.
• 6:33-6:38: Le sel est indispensable à notre organisme.
• 6:52-7:16: Le sel est tombé en disgrâce aux États-Unis en 1977, conduisant à une recommandation de 3g de sel par jour.
• 7:16-8:15: Cette recommandation est basée sur des études contestables, notamment une étude sur des rats ayant ingéré d'énormes quantités de sel et une étude comparant la consommation de sel et la tension artérielle de populations isolées.
• 8:15-8:35: Le sel est devenu le grand ennemi, accusé d'entraîner la rétention de liquide, l'hypertension artérielle et les infarctus.
• 8:35-9:00: Ces conclusions sont le résultat d'hypothèses incertaines et ne tiennent pas compte du fait que la consommation de sel n'est pas la seule cause de l'hypertension.
• 9:05-9:13: Parmi les facteurs de risque de l'hypertension figurent le tabac, l'alcool, le stress, l'obésité et le sel.
• 9:13-9:32: Le sel est l'élément le plus facile à changer dans notre alimentation, ce qui en fait le coupable idéal de l'hypertension.
• 9:45-10:02: La consommation de sel n'augmente la tension artérielle que chez certaines personnes.
• 10:02-10:10: Contrairement au tabac et à l'alcool, le sel est indispensable à notre organisme.
• 10:10-10:16: Théorie selon laquelle nous raffolons du sel parce que nous descendons de créatures marines.
• 10:35-10:56: Comparaison entre la composition de l'eau de mer et de notre milieu intérieur, expliquant notre besoin de sel.
• 11:08-11:13: Le rein est l'organe le plus important du corps pour la gestion du sel.
• 11:18-11:40: Les reins filtrent et réinjectent le sel dans notre organisme, permettant de maintenir un environnement interne équilibré.
• 11:40-11:46: Nous avons besoin du sel pour vivre.
• 11:53-12:19: Une consommation importante de sel pourrait être mauvaise pour la santé en augmentant la tension artérielle chez les personnes sensibles au sel (environ 1/4 de la population).
• 13:31-13:39: La dose nécessaire de sel est estimée à 1,5 g par jour, mais la plupart des britanniques en consomment environ 20 fois plus.
• 13:53-14:17: La Finlande a réussi à réduire sa consommation de sel grâce à des campagnes de sensibilisation.
• 14:50-15:02: L'American Heart Association recommande de ne pas dépasser 1500 mg de sodium par jour.
• 15:14-15:20: Le lien entre la consommation de sodium et les maladies cardiovasculaires n'a jamais été remis en question et s'est imposé comme une vérité.
• 15:56-16:02: Rien n'indique que la restriction en sel serait bonne pour l'organisme selon certaines études.
• 16:12-16:25: Il ne faut pas exiger systématiquement des malades souffrant d'insuffisance cardiaque qu'ils réduisent leur consommation de sodium sans éléments concrets.
• 17:11-17:17: Il faut tenir compte des difficultés que vont rencontrer certains patients comme les personnes âgées, celles qui ont des revenus limités ou qui font partie de minorités.
• 17:36-17:43: Il faut des éléments tangibles qui prouvent que le changement améliorera son état avant de demander à un patient atteint d'une maladie cardiovasculaire de changer son alimentation.
• 19:01-19:07: L'être humain est programmé pour aimer le sel.
• 20:54-21:07: La majeure partie du sel que l'on mange vient des produits industriels.
• 21:40-21:47: Certains nutritionnistes conseillent à leurs clients d'en consommer davantage.
• 21:53-22:10: Exemple de Miguel, qui avait des symptômes indiquant une carence en sel et à qui on a donné des boissons riches en électrolytes.
• 22:22-22:34: Une carence en sel peut être mortelle, comme dans le cas d'une coureuse de marathon ayant bu trop d'eau et perdu trop de sel (hyponatrémie).
• 23:26-23:44: Plusieurs organisations préconisent un apport très faible en sodium, mais aucun élément probant n'a été apporté pour justifier ce chiffre.
• 23:44-24:11: De plus en plus d'études prouvent que le contrôle de l'apport en sodium n'apporte rien et pourrait même augmenter les risques, sauf en Chine où la consommation est très élevée.
• 24:49-25:08: Une faible consommation de sodium est associée à une augmentation du niveau de certaines hormones, avec des effets délétères sur le système vasculaire.
• 25:13-25:26: Chaque nutriment essentiel doit être consommé dans une certaine quantité : au-delà la dose est toxique, en dessous on a des carences.
• 26:52-27:06: Les usages du sel sont multiples : il ne sert pas seulement à assaisonner, mais aussi à contrôler l'activité de la levure dans le pain et à tempérer la puissance du sucre dans les desserts.
• 30:28-30:44: Dans les années 20, on a ajouté de l'iode au sel pour compenser une carence chez les consommateurs américains.
• 32:09-32:16: D'un point de vue chimique, le sel de mer et le sel extrait d'une mine contiennent la même quantité de sodium pour un poids donné.
• 32:58-33:11: C'est à cause de cette diversité que les études sur le sel se contredisent autant.
• 33:11-33:40: Il est très complexe de mener des études sur la nutrition humaine, car il est difficile de contrôler l'alimentation des participants.
• 33:40-34:32: Présentation du programme Mars 500, une expérience d'isolement permettant d'étudier les effets du sel dans des conditions contrôlées.
• 34:32-35:28: Découverte surprenante : la quantité de sodium stockée ou éliminée dans l'organisme ne dépend pas de l'alimentation du sujet.
• 35:47-36:00: Le sodium disparu s'est dispersé dans l'organisme.
• 36:00-36:10: Si on ne sait pas où se trouve ce sel, il y a de quoi se demander s'il est vraiment nécessaire de réduire notre consommation.
• 36:28-36:34: On a créé la sauce soja pour diluer le sel qui était rare à l'époque.
• 37:44-37:50: Umami est un mot japonais qui désigne la 5e saveur de base après l'acide, l'amer, le sucré et le salé.
• 38:30-38:38: Partout dans le monde, les êtres humains sont fous de sel, car cette envie découle d'un véritable besoin.
• 38:38-39:09: On nous sert souvent des biscuits apéritifs salés dans les bars, car le sel donne envie de boire.
• 39:15-39:26: Le sel donne soif à court terme, mais pendant le programme Mars 500, les cosmonautes qui ont mangé une plus grande quantité de sel ont aussi eu plus d'appétit.
• 39:26-39:58: Selon une étude menée sur des cosmonautes, une augmentation de l'apport en sel ferait baisser la soif et augmenterait la faim.
• 39:58-40:09: Les réserves d'eau sont stockées dans des dépôts de sel.
• 40:09-40:17: Il faut continuer à creuser pour en savoir plus sur les stocks de sodium.
• 40:24-40:32: Utilisation de l'IRM pour détecter le sodium dans le corps.
• 40:37-40:51: Le sel apparaît en blanc sur l'écran, plus la zone est blanche, plus la concentration est importante.
• 41:10-41:17: Notre cœur envoie 4,5 g de sel par minute dans notre corps.
• 41:23-41:42: Plus le patient est âgé, plus la quantité de sodium stockée dans les muscles est importante.
• 41:42-42:07: Plus on vieillit, plus notre corps stocke le sel, ce qui pourrait être associé aux problèmes de santé liés à l'âge.
• 42:18-42:25: Le sel stocké aiderait le corps à conserver l'eau, comme une crème hydratante.
• 42:30-42:44: S'hydrater, ce n'est pas seulement boire de l'eau, il faut que cette eau reste dans le corps, et cette étape est gérée par notre métabolisme.
• 42:50-42:56: Ces mécanismes surpassent la fonction rénale.
• 43:20-43:32: Avec le temps, la peau est de plus en plus perméable et perd en élasticité.
• 43:32-43:46: On cherche à savoir si les patients dont la peau laisse passer plus d'eau stockent davantage de sodium au niveau cutané.
• 44:17-44:23: Il est trop tôt pour affirmer que le stockage de sel joue un rôle dans notre organisme, mais ces observations soulèvent des questions.
• 44:23-44:35: Si une quantité indéterminée de sel ingéré est stockée dans notre corps, a-t-on besoin de contrôler notre consommation au milligram près ?.
• 45:44-45:55: Le sel est à la fois un conservateur essentiel et une substance très corrosive, présente dans l'eau mais pouvant entraîner déshydratation.
• 45:55-46:02: Il est essentiel à la vie animale et humaine, mais a été qualifié d'aliment mortel.
• 46:02-46:09: C'est le condiment de toutes les contradictions.
• 46:09-46:40: On aime les modèles simples, mais notre biologie est un peu plus compliquée que ça.
• 47:53-47:58: Tâchons d'épargner nos reins et de les aider à faire le travail dans des conditions normales.
• 48:28-48:40: Les gens doivent avoir conscience de ce qu'ils mangent, et les agences de santé doivent recommander un régime équilibré plutôt que de se concentrer sur le sel.
• 48:55-49:01: Une alimentation équilibrée à base de fruits et légumes est bénéfique.
• 49:01-49:08: La modération est une bonne ligne de conduite.
• 49:08-49:14: On ne comprend pas encore tous les effets que le sel a sur notre corps.

DOI: 10.1038/s44321-024-00187-y

Resource: RRID:Addgene_8454

Curator: @olekpark

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DOI: 10.1038/s44321-024-00187-y

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DOI: 10.1038/s44161-024-00593-y

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DOI: 10.1038/s41598-025-89908-y

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DOI: 10.1038/s41467-025-56696-y

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DOI: 10.1038/s41467-025-56151-y

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

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Reply to the reviewers

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

Authors has provided a mechanism by which how presence of truncated P53 can inactivate function of full length P53 protein. Authors proposed this happens by sequestration of full length P53 by truncated P53.

In the study, performed experiments are well described.

My area of expertise is molecular biology/gene expression, and I have tried to provide suggestions on my area of expertise. The study has been done mainly with overexpression system and I have included few comments which I can think can be helpful to understand effect of truncated P53 on endogenous wild type full length protein. Performing experiments on these lines will add value to the observation according to this reviewer.

Major comments:

1. What happens to endogenous wild type full length P53 in the context of mutant/truncated isoforms, that is not clear. Using a P53 antibody which can detect endogenous wild type P53, can authors check if endogenous full length P53 protein is also aggregated as well? It is hard to differentiate if aggregation of full length P53 happens only in overexpression scenario, where lot more both of such proteins are expressed. In normal physiological condition P53 expression is usually low, tightly controlled and its expression get induced in altered cellular condition such as during DNA damage. So, it is important to understand the physiological relevance of such aggregation, which could be possible if authors could investigate effect on endogenous full length P53 following overexpression of mutant isoforms. Response: Thank you very much for your insightful comments. 1) To address "what happens to endogenous wild-type full-length P53 in the context of mutant/truncated isoforms," we employed a human A549 cell line expressing endogenous wild-type p53 under DNA damage conditions such as an etoposide treatment1. We choose the A549 cell line since similar to H1299, it is a lung cancer cell line (www.atcc.org). For comparison, we also transfected the cells with 2 μg of V5-tagged plasmids encoding FLp53 and its isoforms Δ133p53 and Δ160p53. As shown in Figure R1A, lanes 1 and 2, endogenous p53 expression, remained undetectable in A549 cells despite etoposide treatment, which limits our ability to assess the effects of the isoforms on the endogenous wild-type FLp53. We could, however, detect the V5-tagged FLp53 expressed from the plasmid using anti-V5 (rabbit) as well as with anti-DO-1 (mouse) antibody (Figure R1). The latter detects both endogenous wild-type p53 and the V5-tagged FLp53 since the antibody epitope is within the N-terminus (aa 20-25). This result supports the reviewer's comment regarding the low level of expression of endogenous p53 that is insufficient for detection in our experiments. (Figure R1 is included in the file "RC-2024-02608 Figures of Response to Reviewer.)__

In summary, in line with the reviewer's comment that 'under normal physiological conditions p53 expression is usually low,' we could not detect p53 with an anti-DO-1 antibody. Thus, we proceeded with V5/FLAG-tagged p53 for detection of the effects of the isoforms on p53 stability and function. We also found that protein expression in H1299 cells was more easily detectable than in A549 cells (Compare Figures R1A and B). Thus, we decided to continue with the H1299 cells (p53-null), which would serve as a more suitable model system for this study.

2) We agree with the reviewer that 'It is hard to differentiate if aggregation of full-length p53 happens only in overexpression scenario'. However, it is not impossible to imagine that such aggregation of FLp53 happens under conditions when p53 and its isoforms are over-expressed in the cell. Although the exact physiological context is not known and beyond the scope of the current work, our results indicate that at higher expression, p53 isoforms drive aggregation of FLp53. Given the challenges of detecting endogenous FLp53, we had to rely on the results obtained with plasmid mediated expression of p53 and its isoforms in p53-null cells.

Can presence of mutant P53 isoforms can cause functional impairment of wild type full length endogenous P53? That could be tested as well using similar ChIP assay authors has performed, but instead of antibody against the Tagged protein if the authors could check endogenous P53 enrichment in the gene promoter such as P21 following overexpression of mutant isoforms. May be introducing a condition such as DNA damage in such experiment might help where endogenous P53 is induced and more prone to bind to P53 target such as P21.

Response: Thank you very much for your valuable comments and suggestions. To investigate the potential functional impairment of endogenous wild-type p53 by p53 isoforms, we initially utilized A549 cells (p53 wild-type), aiming to monitor endogenous wild-type p53 expression following DNA damage. However, as mentioned and demonstrated in Figure R1, endogenous p53 expression was too low to be detected under these conditions, making the ChIP assay for analyzing endogenous p53 activity unfeasible. Thus, we decided to utilize plasmid-based expression of FLp53 and focus on the potential functional impairment induced by the isoforms.

3. On similar lines, authors described:

"To test this hypothesis, we escalated the ratio of FLp53 to isoforms to 1:10. As expected, the activity of all four promoters decreased significantly at this ratio (Figure 4A-D). Notably, Δ160p53 showed a more potent inhibitory effect than Δ133p53 at the 1:5 ratio on all promoters except for the p21 promoter, where their impacts were similar (Figure 4E-H). However, at the 1:10 ratio, Δ133p53 and Δ160p53 had similar effects on all transactivation except for the MDM2 promoter (Figure 4E-H)."

Again, in such assay authors used ratio 1:5 to 1:10 full length vs mutant. How authors justify this result in context (which is more relevant context) where one allele is Wild type (functional P53) and another allele is mutated (truncated, can induce aggregation). In this case one would except 1:1 ratio of full-length vs mutant protein, unless other regulation is going which induces expression of mutant isoforms more than wild type full length protein. Probably discussing on these lines might provide more physiological relevance to the observed data.

Response: Thank you for raising this point regarding the physiological relevance of the ratios used in our study. 1) In the revised manuscript (lines 193-195), we added in this direction that "The elevated Δ133p53 protein modulates p53 target genes such as miR‑34a and p21, facilitating cancer development2, 3. To mimic conditions where isoforms are upregulated relative to FLp53, we increased the ratios to 1:5 and 1:10." This approach aims to simulate scenarios where isoforms accumulate at higher levels than FLp53, which may be relevant in specific contexts, as also elaborated above.

2) Regarding the issue of protein expression, where one allele is wild-type and the other is isoform, this assumption is not valid in most contexts. First, human cells have two copies of TPp53 gene (one from each parent). Second, the TP53 gene has two distinct promoters: the proximal promoter (P1) primarily regulates FLp53 and ∆40p53, whereas the second promoter (P2) regulates ∆133p53 and ∆160p534, 5. Additionally, ∆133TP53 is a p53 target gene6, 7 and the expression of Δ133p53 and FLp53 is dynamic in response to various stimuli. Third, the expression of p53 isoforms is regulated at multiple levels, including transcriptional, post-transcriptional, translational, and post-translational processing8. Moreover, different degradation mechanisms modify the protein level of p53 isoforms and FLp538. These differential regulation mechanisms are regulated by various stimuli, and therefore, the 1:1 ratio of FLp53 to ∆133p53 or ∆160p53 may be valid only under certain physiological conditions. In line with this, varied expression levels of FLp53 and its isoforms, including ∆133p53 and ∆160p53, have been reported in several studies3, 4, 9, 10.

3) In our study, using the pcDNA 3.1 vector under the human cytomegalovirus (CMV) promoter, we observed moderately higher expression levels of ∆133p53 and ∆160p53 relative to FLp53 (Figure R1B). This overexpression scenario provides a model for studying conditions where isoform accumulation might surpass physiological levels, impacting FLp53 function. By employing elevated ratios of these isoforms to FLp53, we aim to investigate the potential effects of isoform accumulation on FLp53.

4. Finally does this altered function of full length P53 (preferably endogenous one) in presence of truncated P53 has any phenotypic consequence on the cells (if authors choose a cell type which is having wild type functional P53). Doing assay such as apoptosis/cell cycle could help us to get this visualization.

Response: Thank you for your insightful comments. In the experiment with A549 cells (p53 wild-type), endogenous p53 levels were too low to be detected, even after DNA damage induction. The evaluation of the function of endogenous p53 in the presence of isoforms is hindered, as mentioned above. In the revised manuscript, we utilized H1299 cells with overexpressed proteins for apoptosis studies using the Caspase-Glo® 3/7 assay (Figure 7). This has been shown in the Results section (lines 254-269). "The Δ133p53 and Δ160p53 proteins block pro-apoptotic function of FLp53.

One of the physiological read-outs of FLp53 is its ability to induce apoptotic cell death11. To investigate the effects of p53 isoforms Δ133p53 and Δ160p53 on FLp53-induced apoptosis, we measured caspase-3 and -7 activities in H1299 cells expressing different p53 isoforms (Figure 7). Caspase activation is a key biochemical event in apoptosis, with the activation of effector caspases (caspase-3 and -7) ultimately leading to apoptosis12. The caspase-3 and -7 activities induced by FLp53 expression was approximately 2.5 times higher than that of the control vector (Figure 7). Co-expression of FLp53 and the isoforms Δ133p53 or Δ160p53 at a ratio of 1: 5 significantly diminished the apoptotic activity of FLp53 (Figure 7). This result aligns well with our reporter gene assay, which demonstrated that elevated expression of Δ133p53 and Δ160p53 impaired the expression of apoptosis-inducing genes BAX and PUMA (Figure 4G and H). Moreover, a reduction in the apoptotic activity of FLp53 was observed irrespective of whether Δ133p53 or Δ160p53 protein was expressed with or without a FLAG tag (Figure 7). This result, therefore, also suggests that the FLAG tag does not affect the apoptotic activity or other physiological functions of FLp53 and its isoforms. Overall, the overexpression of p53 isoforms Δ133p53 and Δ160p53 significantly attenuates FLp53-induced apoptosis, independent of the protein tagging with the FLAG antibody epitope."

**Referees cross-commenting**

I think the comments from the other reviewers are very much reasonable and logical.

Especially all 3 reviewers have indicated, a better way to visualize the aggregation of full-length wild type P53 by truncated P53 (such as looking at endogenous P53# by reviewer 1, having fluorescent tag #by reviewer 2 and reviewer 3 raised concern on the FLAG tag) would add more value to the observation.

Response: Thank you for these comments. The endogenous p53 protein was undetectable in A549 cells induced by etoposide (Figure R1A). Therefore, we conducted experiments using FLAG/V5-tagged FLp53. To avoid any potential side effects of the FLAG tag on p53 aggregation, we introduced untagged p53 isoforms in the H1299 cells and performed subcellular fractionation. Our revised results, consistent with previous FLAG-tagged p53 isoforms findings, demonstrate that co-expression of untagged isoforms with FLAG-tagged FLp53 significantly induced the aggregation of FLAG-FLp53, while no aggregation was observed when FLAG-tagged FLp53 was expressed alone (Supplementary Figure 6). These results clearly indicate that the FLAG tag itself does not contribute to protein aggregation.

Additionally, we utilized the A11 antibody to detect protein aggregation, providing additional validation (Figure R3). Given that the fluorescent proteins (~30 kDa) are substantially bigger than the tags used here (~1 kDa) and may influence oligomerization (especially GFP), stability, localization, and function of p53 and its isoforms, we avoided conducting these vital experiments with such artificial large fusions.

Reviewer #1 (Significance (Required)):

The work in significant, since it points out more mechanistic insight how wild type full length P53 could be inactivated in the presence of truncated isoforms, this might offer new opportunity to recover P53 function as treatment strategies against cancer.

Response: Thank you for your insightful comments. We appreciate your recognition of the significance of our work in providing mechanistic insights into how wild-type FLp53 can be inactivated by truncated isoforms. We agree that these findings have potential for exploring new strategies to restore p53 function as a therapeutic approach against cancer.

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

The manuscript by Zhao and colleagues presents a novel and compelling study on the p53 isoforms, Δ133p53 and Δ160p53, which are associated with aggressive cancer types. The main objective of the study was to understand how these isoforms exert a dominant negative effect on full-length p53 (FLp53). The authors discovered that the Δ133p53 and Δ160p53 proteins exhibit impaired binding to p53-regulated promoters. The data suggest that the predominant mechanism driving the dominant-negative effect is the co-aggregation of FLp53 with Δ133p53 and Δ160p53.

This study is innovative, well-executed, and supported by thorough data analysis. However, the authors should address the following points:

1. • Introduction on Aggregation and Co-aggregation: Given that the focus of the study is on the aggregation and co-aggregation of the isoforms, the introduction should include a dedicated paragraph discussing this issue. There are several original research articles and reviews that could be cited to provide context.* Response: Thank you very much for the valuable comments. We have added the following paragraph in the revised manuscript (lines 74-82): "Protein aggregation has become a central focus of modern biology research and has documented implications in various diseases, including cancer13, 14, 15. Protein aggregates can be of different types ranging from amorphous aggregates to highly structured amyloid or fibrillar aggregates, each with different physiological implications. In the case of p53, whether protein aggregation, and in particular, co-aggregation with large N-terminal deletion isoforms, plays a mechanistic role in its inactivation is yet underexplored. Interestingly, the Δ133p53β isoform has been shown to aggregate in several human cancer cell lines16. Additionally, the Δ40p53α isoform exhibits a high aggregation tendency in endometrial cancer cells17. Although no direct evidence exists for Δ160p53 yet, these findings imply that p53 isoform aggregation may play a major role in their mechanisms of actions."

2. Antibody Use for Aggregation: To strengthen the evidence for aggregation, the authors should consider using antibodies that specifically bind to aggregates.

Response: Thank you for your insightful suggestion. We addressed protein aggregation using the A11 antibody which specifically recognizes amyloid-like protein aggregates. We analyzed insoluble nuclear pellet samples prepared under identical conditions as described in Figure 6B. To confirm the presence of p53 proteins, we employed the anti-p53 M19 antibody (Santa Cruz, Cat No. sc-1312) to detect bands corresponding to FLp53 and its isoforms Δ133p53 and Δ160p53. The monomer FLp53 was not detected (Figure R3, lower panel), which may be attributed to the lower binding affinity of the anti-p53 M19 antibody to it. These samples were also immunoprecipitated using the A11 antibody (Thermo Fischer Scientific, Cat No. AHB0052) to detect aggregated proteins. Interestingly, FLp53 and its isoforms, Δ133p53 and Δ160p53, were clearly visible with Anti-A11 antibody when co-expressed at a 1:5 ratio suggesting that they underwent co-aggregation__.__ However, no FLp53 aggregates were observed when it was expressed alone (Figure R2). These results support the conclusion in our manuscript that Δ133p53 and Δ160p53 drive FLp53 aggregation.

(Figure R2 is included in the file "RC-2024-02608 Figures of Response to Reviewer.)__

3. Fluorescence Microscopy: Live-cell fluorescence microscopy could be employed to enhance visualization by labeling FLp53 and the isoforms with different fluorescent markers (e.g., EGFP and mCherry tags).

Response: We appreciate the suggestion to use live-cell fluorescence microscopy with EGFP and mCherry tags for the visualization FLp53 and its isoforms. While we understand the advantages of live-cell imaging with EGFP / mCherry tags, we restrained us from doing such fusions as the GFP or corresponding protein tags are very big (~30 kDa) with respect to the p53 isoform variants (~30 kDa). Other studies have shown that EGFP and mCherry fusions can alter protein oligomerization, solubility and aggregation18, 19. Moreover, most fluorescence proteins are prone to dimerization (i.e. EGFP) or form obligate tetramers (DsRed)20, 21, 22, potentially interfering with the oligomerization and aggregation properties of p53 isoforms, particularly Δ133p53 and Δ160p53.

Instead, we utilized FLAG- or V5-tag-based immunofluorescence microscopy, a well-established and widely accepted method for visualizing p53 proteins. This method provided precise localization and reliable quantitative data, which we believe meet the needs of the current study. We believe our chosen method is both appropriate and sufficient for addressing the research question.

Reviewer #2 (Significance (Required)):

The manuscript by Zhao and colleagues presents a novel and compelling study on the p53 isoforms, Δ133p53 and Δ160p53, which are associated with aggressive cancer types. The main objective of the study was to understand how these isoforms exert a dominant negative effect on full-length p53 (FLp53). The authors discovered that the Δ133p53 and Δ160p53 proteins exhibit impaired binding to p53-regulated promoters. The data suggest that the predominant mechanism driving the dominant-negative effect is the co-aggregation of FLp53 with Δ133p53 and Δ160p53.

Response: We sincerely thank the reviewer for the thoughtful and positive comments on our manuscript and for highlighting the significance of our findings on the p53 isoforms, Δ133p53 and Δ160p53.

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

In this manuscript entitled "Δ133p53 and Δ160p53 isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation", the authors suggest that the Δ133p53 and Δ160p53 isoforms have high aggregation propensity and that by co-aggregating with canonical p53 (FLp53), they sequestrate it away from DNA thus exerting a dominant-negative effect over it.

First, the authors should make it clear throughout the manuscript, including the title, that they are investigating Δ133p53α and Δ160p53α since there are 3 Δ133p53 isoforms (α, β, γ), and 3 Δ160p53 isoforms (α, β, γ).

Response: Thank you for your suggestion. We understand the importance of clearly specifying the isoforms under study. Following your suggestion, we have added α in the title, abstract, and introduction and added the following statement in the Introduction (lines 57-59): "For convenience and simplicity, we have written Δ133p53 and Δ160p53 to represent the α isoforms (Δ133p53α and Δ160p53α) throughout this manuscript."

One concern is that the authors only consider and explore Δ133p53α and Δ160p53α isoforms as exclusively oncogenic and FLp53 dominant-negative while not discussing evidences of different activities. Indeed, other manuscripts have also shown that Δ133p53α is non-oncogenic and non-mutagenic, do not antagonize every single FLp53 functions and are sometimes associated with good prognosis. To cite a few examples:

• Hofstetter G. et al. D133p53 is an independent prognostic marker in p53 mutant advanced serous ovarian cancer. Br. J. Cancer 2011, 105, 1593-1599.
• Bischof, K. et al. Influence of p53 Isoform Expression on Survival in High-Grade Serous Ovarian Cancers. Sci. Rep. 2019, 9,5244.
• Knezovi´c F. et al. The role of p53 isoforms' expression and p53 mutation status in renal cell cancer prognosis. Urol. Oncol. 2019, 37, 578.e1-578.e10.
• Gong, L. et al. p53 isoform D113p53/D133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage. Cell Res. 2015, 25, 351-369.
• Gong, L. et al. p53 isoform D133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming. Sci. Rep. 2016, 6, 37281.
• Horikawa, I. et al. D133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells. Cell Death Differ. 2017, 24, 1017-1028.
• Gong, L. p53 coordinates with D133p53 isoform to promote cell survival under low-level oxidative stress. J. Mol. Cell Biol. 2016, 8, 88-90. Response: Thank you very much for your comment and for highlighting these important studies.

We agree that Δ133p53 isoforms exhibit complex biological functions, with both oncogenic and non-oncogenic potentials. However, our mission here was primarily to reveal the molecular mechanism for the dominant-negative effects exerted by the Δ133p53α and Δ160p53α isoforms on FLp53 for which the Δ133p53α and Δ160p53α isoforms are suitable model systems. Exploring the oncogenic potential of the isoforms is beyond the scope of the current study and we have not claimed anywhere that we are reporting that. We have carefully revised the manuscript and replaced the respective terms e.g. 'pro-oncogenic activity' with 'dominant-negative effect' in relevant places (e.g. line 90). We have now also added a paragraph with suitable references that introduces the oncogenic and non-oncogenic roles of the p53 isoforms.

After reviewing the papers you cited, we are not sure that they reflect on oncogenic /non-oncogenic role of the Δ133p53α isoform in different cancer cases. Although our study is not about the oncogenic potential of the isoforms, we have summarized the key findings below:

• Hofstetter et al., 2011: Demonstrated that Δ133p53α expression improved recurrence-free and overall survival (in a p53 mutant induced advanced serous ovarian cancer, suggesting a potential protective role in this context.
• Bischof et al., 2019: Found that Δ133p53 mRNA can improve overall survival in high-grade serous ovarian cancers. However, out of 31 patients, only 5 belong to the TP53 wild-type group, while the others carry TP53 mutations.
• Knezović et al., 2019: Reported downregulation of Δ133p53 in renal cell carcinoma tissues with wild-type p53 compared to normal adjacent tissue, indicating a potential non-oncogenic role, but not conclusively demonstrating it.
• Gong et al., 2015: Showed that Δ133p53 antagonizes p53-mediated apoptosis and promotes DNA double-strand break repair by upregulating RAD51, LIG4, and RAD52 independently of FLp53.
• Gong et al., 2016: Demonstrated that overexpression of Δ133p53 promotes efficiency of cell reprogramming by its anti-apoptotic function and promoting DNA DSB repair. The authors hypotheses that this mechanism is involved in increasing RAD51 foci formation and decrease γH2AX foci formation and chromosome aberrations in induced pluripotent stem (iPS) cells, independent of FL p53.
• Horikawa et al., 2017: Indicated that induced pluripotent stem cells derived from fibroblasts that overexpress Δ133p53 formed non-cancerous tumors in mice compared to induced pluripotent stem cells derived from fibroblasts with complete p53 inhibition. Thus, Δ133p53 overexpression is "non- or less oncogenic and mutagenic" compared to complete p53 inhibition, but it still compromises certain p53-mediated tumor-suppressing pathways. "Overexpressed Δ133p53 prevented FL-p53 from binding to the regulatory regions of p21WAF1 and miR-34a promoters, providing a mechanistic basis for its dominant-negative inhibition of a subset of p53 target genes."
• Gong, 2016: Suggested that Δ133p53 promotes cell survival under low-level oxidative stress, but its role under different stress conditions remains uncertain. We have revised the Introduction to provide a more balanced discussion of Δ133p53's dule role (lines 62-73):

"The Δ133p53 isoform exhibit complex biological functions, with both oncogenic and non-oncogenic potentials. Recent studies demonstrate the non-oncogenic yet context-dependent role of the Δ133p53 isoform in cancer development. Δ133p53 expression has been reported to correlate with improved survival in patients with TP53 mutations23, 24, where it promotes cell survival in a non-oncogenic manner25, 26, especially under low oxidative stress27. Alternatively, other recent evidences emphasize the notable oncogenic functions of Δ133p53 as it can inhibit p53-dependent apoptosis by directly interacting with the FLp53 4, 6. The oncogenic function of the newly identified Δ160p53 isoform is less known, although it is associated with p53 mutation-driven tumorigenesis28 and in melanoma cells' aggressiveness10. Whether or not the Δ160p53 isoform also impedes FLp53 function in a similar way as Δ133p53 is an open question. However, these p53 isoforms can certainly compromise p53-mediated tumor suppression by interfering with FLp53 binding to target genes such as p21 and miR-34a2, 29 by dominant-negative effect, the exact mechanism is not known."

On the figures presented in this manuscript, I have three major concerns:

*1- Most results in the manuscript rely on the overexpression of the FLAG-tagged or V5-tagged isoforms. The validation of these construct entirely depends on Supplementary figure 3 which the authors claim "rules out the possibility that the FLAG epitope might contribute to this aggregation. However, I am not entirely convinced by that conclusion. Indeed, the ratio between the "regular" isoform and the aggregates is much higher in the FLAG-tagged constructs than in the V5-tagged constructs. We can visualize the aggregates easily in the FLAG-tagged experiment, but the imaging clearly had to be overexposed (given the white coloring demonstrating saturation of the main bands) to visualize them in the V5-tagged experiments. Therefore, I am not convinced that an effect of the FLAG-tag can be ruled out and more convincing data should be added. *

Response: Thank you for raising this important concern. We have carefully considered your comments and have made several revisions to clarify and strengthen our conclusions.

First, to address the potential influence of the FLAG and V5 tags on p53 isoform aggregation, we have revised Figure 2 and removed the previous Supplementary Figure 3, where non-specific antibody bindings and higher molecular weight aggregates were not clearly interpretable. In the revised Figure 2, we have removed these potential aggregates, improving the clarity and accuracy of the data.

To further rule out any tag-related artifacts, we conducted a co-immunoprecipitation assay with FLAG-tagged FLp53 and untagged Δ133p53 and Δ160p53 isoforms. The results (now shown in the new Supplementary Figure 3) completely agree with our previous result with FLAG-tagged and V5-tagged Δ133p53 and Δ160p53 isoforms and show interaction between the partners. This indicates that the FLAG / V5-tags do not influence / interfere with the interaction between FLp53 and the isoforms. We have still used FLAG-tagged FLp53 as the endogenous p53 was undetectable and the FLAG-tagged FLp53 did not aggregate alone.

In the revised paper, we added the following sentences (Lines 146-152): "To rule out the possibility that the observed interactions between FLp53 and its isoforms Δ133p53 and Δ160p53 were artifacts caused by the FLAG and V5 antibody epitope tags, we co-expressed FLAG-tagged FLp53 with untagged Δ133p53 and Δ160p53. Immunoprecipitation assays demonstrated that FLAG-tagged FLp53 could indeed interact with the untagged Δ133p53 and Δ160p53 isoforms (Supplementary Figure 3, lanes 3 and 4), confirming formation of hetero-oligomers between FLp53 and its isoforms. These findings demonstrate that Δ133p53 and Δ160p53 can oligomerize with FLp53 and with each other."

Additionally, we performed subcellular fractionation experiments to compare the aggregation and localization of FLAG-tagged FLp53 when co-expressed either with V5-tagged or untagged Δ133p53/Δ160p53. In these experiments, the untagged isoforms also induced FLp53 aggregation, mirroring our previous results with the tagged isoforms (Supplementary Figure 5). We've added this result in the revised manuscript (lines 236-245): "To exclude the possibility that FLAG or V5 tags contribute to protein aggregation, we also conducted subcellular fractionation of H1299 cells expressing FLAG-tagged FLp53 along with untagged Δ133p53 or Δ160p53 at a 1:5 ratio. The results showed (Supplementary Figure 6) a similar distribution of FLp53 across cytoplasmic, nuclear, and insoluble nuclear fractions as in the case of tagged Δ133p53 or Δ160p53 (Figure 6A to D). Notably, the aggregation of untagged Δ133p53 or Δ160p53 markedly promoted the aggregation of FLAG-tagged FLp53 (Supplementary Figure 6B and D), demonstrating that the antibody epitope tags themselves do not contribute to protein aggregation."

We've also discussed this in the Discussion section (lines 349-356): "In our study, we primarily utilized an overexpression strategy involving FLAG/V5-tagged proteins to investigate the effects of p53 isoforms Δ133p53 and Δ160p53 on the function of FLp53. To address concerns regarding potential overexpression artifacts, we performed the co-immunoprecipitation (Supplementary Figure 6) and caspase-3 and -7 activity (Figure 7) experiments with untagged Δ133p53 and Δ160p53. In both experimental systems, the untagged proteins behaved very similarly to the FLAG/V5 antibody epitope-containing proteins (Figures 6 and 7 and Supplementary Figure 6). Hence, the C-terminal tagging of FLp53 or its isoforms does not alter the biochemical and physiological functions of these proteins."

In summary, the revised data set and newly added experiments provide strong evidence that neither the FLAG nor the V5 tag contributes to the observed p53 isoform aggregation.

2- The authors demonstrate that to visualize the dominant-negative effect, Δ133p53α and Δ160p53α must be "present in a higher proportion than FLp53 in the tetramer" and the need at least a transfection ratio 1:5 since the 1:1 ration shows no effect. However, in almost every single cell type, FLp53 is far more expressed than the isoforms which make it very unlikely to reach such stoichiometry in physiological conditions and make me wonder if this mechanism naturally occurs at endogenous level. This limitation should be at least discussed.

Response: Thank you for your insightful comment. However, evidence suggests that the expression levels of these isoforms such as Δ133p53, can be significantly elevated relative to FLp53 in certain physiological conditions3, 4, 9. For example, in some breast tumors, with Δ133p53 mRNA is expressed at a much levels than FLp53, suggesting a distinct expression profile of p53 isoforms compared to normal breast tissue4. Similarly, in non-small cell lung cancer and the A549 lung cancer cell line, the expression level of Δ133p53 transcript is significantly elevated compared to non-cancerous cells3. Moreover, in specific cholangiocarcinoma cell lines, the Δ133p53 /TAp53 expression ratio has been reported to increase to as high as 3:19. These observations indicate that the dominant-negative effect of isoform Δ133p53 on FLp53 can occur under certain pathological conditions where the relative amounts of the FLp53 and the isoforms would largely vary. Since data on the Δ160p53 isoform are scarce, we infer that the long N-terminal truncated isoforms may share a similar mechanism.

Figure 5C: I am concerned by the subcellular location of the Δ133p53α and Δ160p53α as they are commonly considered nuclear and not cytoplasmic as shown here, particularly since they retain the 3 nuclear localization sequences like the FLp53 (Bourdon JC et al. 2005; Mondal A et al. 2018; Horikawa I et al, 2017; Joruiz S. et al, 2024). However, Δ133p53α can form cytoplasmic speckles (Horikawa I et al, 2017) when it colocalizes with autophagy markers for its degradation.

3-The authors should discuss this issue. Could this discrepancy be due to the high overexpression level of these isoforms? A co-staining with autophagy markers (p62, LC3B) would rule out (or confirm) activation of autophagy due to the overwhelming expression of the isoform.

Response: Thank you for your thoughtful comments. We have thoroughly reviewed all the papers you recommended (Bourdon JC et al., 2005; Mondal A et al., 2018; Horikawa I et al., 2017; Joruiz S. et al., 2024)4, 29, 30, 31. Among these, only the study by Bourdon JC et al. (2005) provided data regarding the localization of Δ133p534. Interestingly, their findings align with our observations, indicating that the protein does not exhibit predominantly nuclear localization in the Figure below. The discrepancy may be caused by a potentially confusing statement in that paper4

(The Figure from Bourdon JC et al. (2005) is included in the file "RC-2024-02608 Figures of Response to Reviewer.)__

The localization of p53 is governed by multiple factors, including its nuclear import and export32. The isoforms Δ133p53 and Δ160p53 contain three nuclear localization sequences (NLS)4 . However, the isoforms Δ133p53 and Δ160p53 were potentially trapped in the cytoplasm by aggregation and masking the NLS. This mechanism would prevent nuclear import.

Further, we acknowledge that Δ133p53 co-aggregates with autophagy substrate p62/SQSTM1 and autophagosome component LC3B in cytoplasm by autophagic degradation during replicative senescence33. We agree that high overexpression of these aggregation-prone proteins may induce endoplasmic reticulum (ER) stress and activates autophagy34. This could explain the cytoplasmic localization in our experiments. However, it is also critical to consider that we observed aggregates in both the cytoplasm and the nucleus (Figures 6B and E and Supplementary Figure 6B). While cytoplasmic localization may involve autophagy-related mechanisms, the nuclear aggregates likely arise from intrinsic isoform properties, such as altered protein folding, independent of autophagy. These dual localizations reflect the complex behavior of Δ133p53 and Δ160p53 isoforms under our experimental conditions.

In the revised manuscript, we discussed this in Discussion (lines 328-335): "Moreover, the observed cytoplasmic isoform aggregates may reflect autophagy-related degradation, as suggested by the co-localization of Δ133p53 with autophagy substrate p62/SQSTM1 and autophagosome component LC3B33. High overexpression of these aggregation-prone proteins could induce endoplasmic reticulum stress and activate autophagy34. Interestingly, we also observed nuclear aggregation of these isoforms (Figure 6B and E and Supplementary Figure 6B), suggesting that distinct mechanisms, such as intrinsic properties of the isoforms, may govern their localization and behavior within the nucleus. This dual localization underscores the complexity of Δ133p53 and Δ160p53 behavior in cellular systems."

Minor concerns:

- Figure 1A: the initiation of the "Δ140p53" is shown instead of "Δ40p53"

Response: Thank you! The revised Figure 1A has been created in the revised paper.

• Figure 2A: I would like to see the images cropped a bit higher, so the cut does not happen just above the aggregate bands

Response: Thank you for this suggestion. We've changed the image and the new Figure 2 has been shown in the revised paper.

• Figure 3C: what ratio of FLp53/Delta isoform was used?

Response: We have added the ratio in the figure legend of Figure 3C (lines 845-846) "Relative DNA-binding of the FLp53-FLAG protein to the p53-target gene promoters in the presence of the V5-tagged protein Δ133p53 or Δ160p53 at a 1: 1 ratio."

• Figure 3C suggests that the "dominant-negative" effect is mostly senescence-specific as it does not affect apoptosis target genes, which is consistent with Horikawa et al, 2017 and Gong et al, 2016 cited above. Furthermore, since these two references and the others from Gong et al. show that Δ133p53α increases DNA repair genes, it would be interesting to look at RAD51, RAD52 or Lig4, and maybe also induce stress.

Response: Thank you for your thoughtful comments and suggestions. In Figure 3C, the presence of Δ133p53 or Δ160p53 only significantly reduced the binding of FLp53 to the p21 promoter. However, isoforms Δ133p53 and Δ160p53 demonstrated a significant loss of DNA-binding activity at all four promoters: p21, MDM2, and apoptosis target genes BAX and PUMA (Figure 3B). This result suggests that Δ133p53 and Δ160p53 have the potential to influence FLp53 function due to their ability to form hetero-oligomers with FLp53 or their intrinsic tendency to aggregate. To further investigate this, we increased the isoform to FLp53 ratio in Figure 4, which demonstrate that the isoforms Δ133p53 and Δ160p53 exert dominant-negative effects on the function of FLp53.

These results demonstrate that the isoforms can compromise p53-mediated pathways, consistent with Horikawa et al. (2017), which showed that Δ133p53α overexpression is "non- or less oncogenic and mutagenic" compared to complete p53 inhibition, but still affects specific tumor-suppressing pathways. Furthermore, as noted by Gong et al. (2016), Δ133p53's anti-apoptotic function under certain conditions is independent of FLp53 and unrelated to its dominant-negative effects.

We appreciate your suggestion to investigate DNA repair genes such as RAD51, RAD52, or Lig4, especially under stress conditions. While these targets are intriguing and relevant, we believe that our current investigation of p53 targets in this manuscript sufficiently supports our conclusions regarding the dominant-negative effect. Further exploration of additional p53 target genes, including those involved in DNA repair, will be an important focus of our future studies.

• Figure 5A and B: directly comparing the level of FLp53 expressed in cytoplasm or nucleus to the level of Δ133p53α and Δ160p53α expressed in cytoplasm or nucleus does not mean much since these are overexpressed proteins and therefore depend on the level of expression. The authors should rather compare the ratio of cytoplasmic/nuclear FLp53 to the ratio of cytoplasmic/nuclear Δ133p53α and Δ160p53α.

Response: Thank you very much for this valuable suggestion. In the revised paper, Figure 5B has been recreated. Changes have been made in lines 214-215: "The cytoplasm-to-nucleus ratio of Δ133p53 and Δ160p53 was approximately 1.5-fold higher than that of FLp53 (Figure 5B)."

**Referees cross-commenting**

I agree that the system needs to be improved to be more physiological.

Just to precise, the D133 and D160 isoforms are not truncated mutants, they are naturally occurring isoforms expressed in almost every normal human cell type from an internal promoter within the TP53 gene.

Using overexpression always raises concerns, but in this case, I am even more careful because the isoforms are almost always less expressed than the FLp53, and here they have to push it 5 to 10 times more expressed than the FLp53 to see the effect which make me fear an artifact effect due to the overwhelming overexpression (which even seems to change the normal localization of the protein).

To visualize the endogenous proteins, they will have to change cell line as the H1299 they used are p53 null.

Response: Thank you for these comments. We've addressed the motivation of overexpression in the above responses. We needed to use the plasmid constructs in the p53-null cells to detect the proteins but the expression level was certainly not 'overwhelmingly high'.

First, we tried the A549 cells (p53 wild-type) under DNA damage conditions, but the endogenous p53 protein was undetectable. Second, several studies reported increased Δ133p53 level compared to wild-type p53 and that it has implications in tumor development2, 3, 4, 9. Third, the apoptosis activity of H1299 cells overexpressing p53 proteins was analyzed in the revised manuscript (Figure 7). The apoptotic activity induced by FLp53 expression was approximately 2.5 times higher than that of the control vector under identical plasmid DNA transfection conditions (Figure 7). These results rule out the possibility that the plasmid-based expression of p53 and its isoforms introduced artifacts in the results. We've discussed this in the Results section (lines 254-269).

Reviewer #3 (Significance (Required)):

Overall, the paper is interesting particularly considering the range of techniques used which is the main strength.

The main limitation to me is the lack of contradictory discussion as all argumentation presents Δ133p53α and Δ160p53α exclusively as oncogenic and strictly FLp53 dominant-negative when, particularly for Δ133p53α, a quite extensive literature suggests a not so clear-cut activity.

The aggregation mechanism is reported for the first time for Δ133p53α and Δ160p53α, although it was already published for Δ40p53α, Δ133p53β or in mutant p53.

This manuscript would be a good basic research addition to the p53 field to provide insight in the mechanism for some activities of some p53 isoforms.

My field of expertise is the p53 isoforms which I have been working on for 11 years in cancer and neuro-degenerative diseases

Response: Thank you very much for your positive and critical comments. We've included a fair discussion on the oncogenic and non-oncogenic function of Δ133p53 in the Introduction following your suggestion (lines 62-73).

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I don't know if I agree with this; it does look like there are some calibration issues. I also imagine the plot would look worse if the x and y axes had the same scales.

One question - is this run for ALL batter/seasons in the training set? Would be interested in what this looks like if we restrict the population to player/seasons with some sample size threshold (maybe \(PA > 50\). Don't know if that's the right way to evaluate the model, but just something I'm curious about. My prior is that it would make the calibration look even worse, since the model will be more confident about their true talent and their sample size makes the expected noise drop.

Regardless - we need to think more about what this is telling us. In my mind, it's saying that the model is overconfident. It's estimating true talent too close to the observed values in some cases (too much coverage of low probabilities), and that's likely what's hurting the top end as well (not enough coverage of high probabilities).

When I read this it makes me think of illusions, obsession, and a lingering presence of something/someone powerful. The steps in the sentence go from being deeply captivated by someone--the story coming to an end--to it being a dream? Similar to Moby dick regarding the presense of the whale and Ahabs obsession with finding it. Tangled up with Ishmael and following Ahab through the journey, but when the book ended, the "spell" was over and everything was gone, but Ishmael.

Why utilize the description of "Earth's Holocaust"? Might this seem insensitive to connect literary elements with traumatic historic events. When I read this passage, I found this quite inappropriate to be honest.

It has no romance and the main character has no experience as a harpooneer. The books is not a personal experience either. He is trying to make it sound good that he can get is published.

I knew Millville was patriotic of American but not to this extent.

La revue Recherches en éducation, numéro 49, explore l'autorité et le pouvoir dans le contexte éducatif contemporain. Plusieurs articles de ce numéro thématique mettent en lumière le rôle des élèves, des parents et des familles dans le système éducatif, que ce soit directement ou indirectement.

Références aux élèves:

• Plusieurs articles traitent directement des élèves, notamment en ce qui concerne leurs expériences et leurs apprentissages.
• L'article de Sylvain Fabre examine l'accompagnement des élèves dans leurs expériences artistiques, en explorant la notion de milieux rythmiques. L'auteur met en évidence l'importance de considérer l'élève dans son dialogue avec les environnements culturels et scolaires, soulignant comment le sujet s'inscrit dans des milieux et y détermine les conditions de son activité.
• Emmanuel Sander, Géry Marcoux, et al. présentent les résultats d'une étude sur les conceptions intuitives des notions de justice et de liberté chez des collégiens dans le cadre du cours d'enseignement moral et civique. Cette étude vise à identifier les conceptions prédominantes d’élèves de collège en réseau d’éducation prioritaire en France sur les notions de justice sociale et de liberté individuelle ainsi qu’à évaluer leur flexibilité cognitive relativement à ces notions.

Références aux parents et aux familles:

• Bien que les parents ne soient pas toujours le sujet central, ils sont mentionnés dans le contexte des relations entre l'école et la société.
• Dans l'article de Cécile Roaux, il est souligné que les directeurs d'école primaire doivent organiser l'école autour de principes managériaux tout en restant soumis à une culture professionnelle de l'égalité entre pairs portée par les enseignants. Dans ce contexte, les directeurs doivent négocier en permanence pour qu'émerge un réel collectif de professionnels, ce qui implique également une prise en compte des relations avec les familles.
• L'étude d'Emmanuel Sander, Géry Marcoux, et al. prend en compte l'influence de la famille dans la construction de l'identité politique des élèves. L'étude de Wilfried Lignier et Julie Pagis (2017) tend à démontrer que la construction de l’identité politique se fait en lien entre la famille, les groupes des pairs et l’école.

En résumé, la revue aborde les thématiques du pouvoir et de l'autorité en éducation, en considérant les élèves comme des acteurs centraux de l'apprentissage et en reconnaissant l'influence des parents et des familles dans le contexte éducatif. Les articles mettent en évidence la complexité des relations entre les différents acteurs du système éducatif et soulignent l'importance de prendre en compte les conceptions et les expériences de chacun.

Le numéro 49 de la revue Recherches en éducation (2022) est un dossier thématique qui aborde les questions d'autorité et de pouvoir des personnels de direction, d’encadrement et de formation dans les politiques publiques contemporaines d’éducation. Ce dossier examine la recomposition des rapports de pouvoir et d'autorité au sein du système scolaire, en se penchant sur les relations entre les différents acteurs chargés de définir, d'encadrer et de mettre en œuvre les politiques publiques. Les articles de ce numéro se concentrent sur le personnel de direction, d'encadrement ou de formation, et mettent en évidence la multi-référentialité des sciences de l'éducation et de la formation. L'objectif de ce dossier est d'analyser les implications à l'échelle globale et les incidences professionnelles concrètes.

Voici un aperçu des articles inclus dans ce numéro thématique:

• Édito par Camille Roelens et Stéphan Mierzejewski, qui introduit les enjeux de l'autorité et du pouvoir éducatifs à l'épreuve des politiques d'éducation.
• "Rapports de force et crise de l’autorité dans le mouvement Freinet entre 1945 et 1968 : quand l’horizontalité questionne la verticalité": Xavier Riondet analyse les rapports de force et la crise de l'autorité dans le mouvement Freinet entre 1945 et 1968, en examinant comment l'horizontalité remet en question la verticalité. L'article de Xavier Riondet cherche à penser les enjeux de l'école actuelle à partir des crises qui ont marqué l'histoire de l'Éducation Nouvelle entre 1945 et 1967. Les rapports de force et d'autorité y ont été précocement bouleversés, en particulier en ce qui concerne les questions de direction et de formation. L'auteur questionne et explicite certaines influences des expériences d'Éducation Nouvelle sur l'Éducation nationale, en revenant sur la puissance transformatrice de la dynamique d'égalisation et d'individualisation au cœur des collectifs éducatifs.
• "L’évolution des rapports hiérarchiques entre directeurs et adjoints dans le champ de l’enseignement primaire de la Seine sous la IIIe République : aux origines d’une autonomie professionnelle": Jérôme Krop étudie l'évolution des rapports hiérarchiques entre directeurs et adjoints dans l'enseignement primaire de la Seine sous la IIIe République, explorant les origines d'une autonomie professionnelle. L'article de Jérôme Krop se propose de remonter aux origines historiques de la condition des directeurs d'école, sous la IIIe République, une période de reconfiguration du champ de l'enseignement primaire et des rapports de pouvoir entre ces derniers et les instituteurs adjoints. Son étude repose sur les données issues de l’étude exhaustive d’un corpus représentatif composé de dossiers d’instituteurs et d’institutrices appartenant à la première génération des enseignants des écoles publiques entrés dans l’enseignement primaire de la Seine entre 1870 et 1886. Son analyse de la conflictualité entre les instituteurs et les directeurs d’écoles montre comment l’histoire des relations sociales constitutives de ce champ de l’enseignement primaire a produit les schèmes de perception et les dispositions pratiques hostiles aux rapports d’autorité fondés sur la subordination hiérarchique.
• "De l’engagement dans la fonction d’adjoint d’établissement scolaire à l’exercice partagé du pouvoir et de l’autorité": Simon Mallard, Gwénola Réto et Rozenn Décret-Rouillard s'intéressent à l'engagement dans la fonction d'adjoint d'établissement scolaire et à l'exercice partagé du pouvoir et de l'autorité. Cet article s’intéresse à l’engagement de professionnels de l’éducation et de l’enseignement dans la fonction d’adjoint d’établissement scolaire. L'analyse des entretiens semi-directifs menés auprès d'adjoints de l’enseignement public, a fait émerger trois axes saillants : s’engager pour prendre des responsabilités, traverser des épreuves et enfin tenir le rôle pour devenir le chef. Ces axes permettent de qualifier l’engagement dans la fonction d’adjoint et de comprendre ce qui apparaît comme la colonne vertébrale de leur fonction : l’exercice partagé du pouvoir et de l’autorité avec le chef d’établissement.
• "La direction d’école primaire, une question de pouvoir ?": Cécile Roaux questionne la notion de pouvoir dans la direction d'école primaire.
• "Le leadership : la fin d’un tabou et le début d’un mythe. Évolution des métiers de l’encadrement scolaire et de leur formation en Suisse romande": Laetitia Progin examine l'évolution des métiers de l'encadrement scolaire et de leur formation en Suisse romande, en mettant en perspective le leadership entre attentes, mythe et réalité. Cet article présente l’évolution des métiers de l’encadrement et de leur formation en Suisse romande en particulier. Il analyse, d’une manière empirique, comment le positionnement des cadres scolaires vis-à-vis de l’appel au leadership a évolué (entre tabou et émergence d’un mythe) sous l’effet notamment de leur formation et propose, pour conclure, quelques éléments de synthèse.
• "Pression temporelle et situation de porte-à-faux. Regard socio-didactique sur les positionnements professionnels des conseillers pédagogiques de circonscription - « tuteurs terrain »": Stéphan Mierzejewski et Abdelkarim Zaid analysent la pression temporelle et les situations de porte-à-faux vécues par les conseillers pédagogiques de circonscription, en adoptant une approche socio-didactique. Cet article aborde la problématique générale du dossier, en l’envisageant du double point de vue de la pression temporelle et des contradictions structurelles qui caractérisent l’évolution des missions de conseiller pédagogique de circonscription.

La section "Varia" propose également des articles qui élargissent le champ de la réflexion:

• "Rythmes scolaires et éducation artistique : l’expérience de la danse à l’école": Sylvain Fabre propose une réflexion sur l'accompagnement des élèves dans leurs expériences artistiques, en explorant la notion de milieux rythmiques.
• "Valeurs de l’éducation et capitalisme contemporain : l’exemple de l’idéal d’autonomie": Renaud Hétier examine comment le capitalisme transforme les valeurs de l'éducation.
• "Conceptions intuitives des notions de justice et de liberté : résultats d'une étude au collège dans le cours d'enseignement moral et civique": Emmanuel Sander, Géry Marcoux, et al. présentent les résultats d'une étude sur les conceptions intuitives des notions de justice et de liberté chez des collégiens dans le cadre du cours d'enseignement moral et civique. Cette étude vise à identifier les conceptions prédominantes d’élèves de collège en réseau d’éducation prioritaire en France sur les notions de justice sociale et de liberté individuelle ainsi qu’à évaluer leur flexibilité cognitive relativement à ces notions.

Author response:

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

Public Reviews:

Reviewer 1 (Public Review):

O’Neill et al. have developed a software analysis application, miniML, that enables the quantification of electrophysiological events. They utilize a supervised deep learned-based method to optimize the software. miniML is able to quantify and standardize the analyses of miniature events, using both voltage and current clamp electrophysiology, as well as optically driven events using iGluSnFR3, in a variety of preparations, including in the cerebellum, calyx of held, Golgi cell, human iPSC cultures, zebrafish, and Drosophila. The software appears to be flexible, in that users are able to hone and adapt the software to new preparations and events. Importantly, miniML is an open-source software free for researchers to use and enables users to adapt new features using Python.

Overall this new software has the potential to become widely used in the field and an asset to researchers. However, the authors fail to discuss or even cite a similar analysis tool recently developed (SimplyFire), and determine how miniML performs relative to this platform. There are a handful of additional suggestions to make miniML more user-friendly, and of broad utility to a variety of researchers, as well as some suggestions to further validate and strengthen areas of the manuscript:

(1) miniML relative to existing analysis methods: There is a major omission in this study, in that a similar open source, Python-based software package for event detection of synaptic events appears to be completely ignored. Earlier this year, another group published SimplyFire in eNeuro (Mori et al., 2024; doi: 10.1523/eneuro.0326-23.2023). Obviously, this previous study needs to be discussed and ideally compared to miniML to determine if SimplyFire is superior or similar in utility, and to underscore differences in approach and accuracy.

We thank the reviewer for bringing this interesting publication to our attention. We have included SimplyFire in our benchmarking for comprehensive comparison with miniML. The approach taken by SimplyFire differs from miniML in a number of ways. Our results show that miniML provides higher recall and precision than SimplyFire (revised Figure 3). We appreciate that SimplyFire provides a user-interface similar to the commonly used MiniAnalysis software. In addition, the peak-finding-based approach of SimplyFire makes it relatively robust to event shape, which facilitates analysis of diverse data. However, we noted a strong threshold-dependence and long run time of SimplyFire (revised Figure 3 and Figure 3—figure supplement 1). In addition, SimplyFire is not robust against various types of noise typically encountered in electrophysiological recordings. Our extended benchmark analysis thus indicates that AI-based event detection is superior to existing algorithmic approaches, including SimplyFire.

(2) The manuscript should comment on whether miniML works equally well to quantify current clamp events (voltage; e.g. EPSP/mEPSPs) compared to voltage clamp (currents, EPSC/mEPSCs), which the manuscript highlights. Are rise and decay time constants calculated for each event similarly?

miniML works equally well for current- and voltage events (Figure 5, Figure 9). In general, events of opposite polarity can be analyzed by simply inverting the data. Transfer learning models may further improve the detection.

For each detected event, independent of data/recording type, rise times are calculated as 10–90% times (baseline–peak), and decay times are calculated as time to 50% of the peak. In addition, event decay time constants are calculated from a fit to the event average. With miniML being open-source, researchers can adapt the calculations of event statistics to their needs, if desired. In the revised manuscript, we have expanded the Methods section that describes the quantification of event statistics (Methods, Quantification).

(3) The interface and capabilities of miniML appear quite similar to Mini Analysis, the free software that many in the field currently use. While the ability and flexibility for users to adapt and adjust miniML for their own uses/needs using Python programming is a clear potential advantage, can the authors comment, or better yet, demonstrate, whether there is any advantage for researchers to use miniML over Mini Analysis or SimplyFire if they just need the standard analyses?

Following the reviewer’s suggestion, we developed a graphical user interface (GUI) for miniML to enhance its usability (Figure 2—figure supplement 2), which is provided on the GitHub repository. Our comprehensive benchmark analysis demonstrated that miniML outperforms existing tools such as MiniAnalysis and SimplyFire. The main advantages are (i) increased reliability of results, which eliminates the need for visual inspection; (ii) fast runtime and easy automation; (iii) superior detection performance as demonstrated by higher recall in both synthetic and real data; (iv) open-source Python-based design. We believe that these advantages make miniML a valuable tool for researchers recording various types of synaptic events, offering a more efficient and reliable solution compared to existing methods.

(4) Additional utilities for miniML: The authors show miniML can quantify miniature electrophysiological events both current and voltage clamp, as well as optical glutamate transients using iGluSnFR. As the authors mention in the discussion, the same approach could, in principle, be used to quantify evoked (EPSC/EPSP) events using electrophysiology, Ca2+ events (using GCaMP), and AP waveforms using voltage indicators like ASAP4. While I don’t think it is reasonable to ask the authors to generate any new experimental data, it would be great to see how miniML performs when analysing data from these approaches, particularly to quantify evoked synaptic events and/or Ca2+ (ideally postsynaptic Ca2+ signals from miniature events, as the Drosophila NMJ have developed nice approaches).

In the revised manuscript, we have extended the application examples of miniML. We applied miniML to detect mEPSPs recorded with the novel voltage-sensitive indicator ASAP5 (Figure 9 and Figure 9—figure supplement 1). We performed simultaneous recordings of membrane voltage through electrophysiology and ASAP5 voltage imaging in rat cultured neurons at physiological temperature. Data were analyzed using miniML, with electrophysiology data being used as ground-truth for assessing detection performance in imaging data. Our results demonstrate that miniML robustly detects mEPSPs in current-clamp, and can localize corresponding transients in imaging data. Furthermore, we observed that miniML performs better than template matching and deconvolution on ASAP5 imaging data (Figure 9 and Figure 9—figure supplement 2).

Reviewer 2 (Public Review):

This paper presents miniML as a supervised method for the detection of spontaneous synaptic events. Recordings of such events are typically of low SNR, where state-of-the-art methods are prone to high false positive rates. Unlike current methods, training miniML requires neither prior knowledge of the kinetics of events nor the tuning of parameters/thresholds.

The proposed method comprises four convolutional networks, followed by a bi-directional LSTM and a final fully connected layer which outputs a decision event/no event per time window. A sliding window is used when applying miniML to a temporal signal, followed by an additional estimation of events’ time stamps. miniML outperforms current methods for simulated events superimposed on real data (with no events) and presents compelling results for real data across experimental paradigms and species. Strengths:

The authors present a pipeline for benchmarking based on simulated events superimposed on real data (with no events). Compared to five other state-of-the-art methods, miniML leads to the highest detection rates and is most robust to specific choices of threshold values for fast or slow kinetics. A major strength of miniML is the ability to use it for different datasets. For this purpose, the CNN part of the model is held fixed and the subsequent networks are trained to adapt to the new data. This Transfer Learning (TL) strategy reduces computation time significantly and more importantly, it allows for using a substantially smaller data set (compared to training a full model) which is crucial as training is supervised (i.e. uses labeled examples).

Weaknesses:

The authors do not indicate how the specific configuration of miniML was set, i.e. number of CNNs, units, LSTM, etc. Please provide further information regarding these design choices, whether they were based on similar models or if chosen based on performance.

The data for the benchmark system was augmented with equal amounts of segments with/without events. Data augmentation was undoubtedly crucial for successful training.

(1) Does a balanced dataset reflect the natural occurrence of events in real data? Could the authors provide more information regarding this matter?

In a given recording, the event frequency determines the ratio of event-containing vs. nonevent-containing data segments. Whereas many synapses have a skew towards non-events, high event frequencies as observed, e.g., in pyramidal cells or Purkinje neurons, can shift the ratio towards event-containing data.

For model training, we extracted data segments from mEPSC recordings in cerebellar granule cells, which have a low mEPSC frequency (about 0.2 Hz, Delvendahl et al. 2019). Unbalanced training data may complicate model training (Drummond and Holte 2003; Prati et al. 2009; Tyagi and Mittal 2020). We therefore decided to balance the training dataset for miniML by down-sampling the majority class (i.e., non-event segments), so that the final datasets for model training contained roughly equal amounts of events and non-events.

(2) Please provide a more detailed description of this process as it would serve users aiming to use this method for other sub-fields.

We thank the reviewer for raising this point. In the revised manuscript, we present a systematic analysis of the impact of imbalanced training data on model training (Figure 1—figure supplement 2). In addition, we have revised the description of model training and data augmentation in the Methods section (Methods, Training data and annotation).

The benchmarking pipeline is indeed valuable and the results are compelling. However, the authors do not provide comparative results for miniML for real data (Figures 4-8). TL does not apply to the other methods. In my opinion, presenting the performance of other methods, trained using the smaller dataset would be convincing of the modularity and applicability of the proposed approach.

Quantitative comparison of synaptic detection methods on real-world data is challenging because the lack of ground-truth data prevents robust, quantitative analyses. Nevertheless, we compared miniML to common template-based and finite-threshold based methods on four different types of synapses. We noted that miniML generally detects more events, whereas other methods are susceptible to false-positives (Figure 4—figure supplement 1). In addition, we analyzed the performance of miniML on voltage imaging data (Figure 9). Simultaneous recordings of electrophysiological and imaging data allowed a quantitative comparison of detection methods in this dataset. Our results demonstrate that miniML provides higher recall for optical minis recorded using ASAP5 (Figure 9 and Figure 9—figure supplement 2; F1 score, Cohen’s d 1.35 vs. template matching and 5.1 vs. deconvolution).

Impact:

Accurate detection of synaptic events is crucial for the study of neural function. miniML has a great potential to become a valuable tool for this purpose as it yields highly accurate detection rates, it is robust, and is relatively easily adaptable to different experimental setups.

Additional comments:

Line 73: the authors describe miniML as "parameter-free". Indeed, miniML does not require the selection of pulse shape, rise/fall time, or tuning of a threshold value. Still, I would not call it "parameter-free" as there are many parameters to tune, starting with the number of CNNs, and number of units through the parameters of the NNs. A more accurate description would be that as an AI-based method, the parameters of miniML are learned via training rather than tuned by the user.

We agree that a deep learning model is not parameter-free, and this term may be misleading. We have therefore changed this sentence in the introduction as follows: "The method is fast, robust to threshold choice, and generalizable across diverse data types [...]"

Line 302: the authors describe miniML as "threshold-independent". The output trace of the model has an extremely high SNR so a threshold of 0.5 typically works. Since a threshold is needed to determine the time stamps of events, I think a better description would be "robust to threshold choice".

To detect event localizations, a peak search is performed on the model output, which uses a minimum peak height parameter (or threshold). Extreme values for this parameter do indeed have a small impact on detection performance (Figure 3J). We have changed the description in the introduction and discussion according to the reviewer’s suggestion.

Reviewer 3 (Public Review):

miniML as a novel supervised deep learning-based method for detecting and analyzing spontaneous synaptic events. The authors demonstrate the advantages of using their methods in comparison with previous approaches. The possibility to train the architecture on different tasks using transfer learning approaches is also an added value of the work. There are some technical aspects that would be worth clarifying in the manuscript:

(1) LSTM Layer Justification: Please provide a detailed explanation for the inclusion of the LSTM layer in the miniML architecture. What specific benefits does the LSTM layer offer in the context of synaptic event detection?

Our model design choice was inspired by similar approaches in the literature (Donahue et al. 2017; Islam et al. 2020; Passricha and Aggarwal 2019; Tasdelen and Sen 2021; Wang et al. 2020). Convolutional and recurrent neural networks are often combined for time-series classification problems as they allow learning spatial and temporal features, respectively. Combining the strengths of both network architectures can thus help improve the classification performance. Indeed, a CNN-LSTM architecture proved to be superior in both training accuracy and detection performance (Figure 1—figure supplement 2). Further, this architecture requires fewer free parameters than comparable model designs using fully connected layers instead. The revised manuscript shows a comparison of different model architectures (Figure 1—figure supplement 2), and we added the following description to the text (Methods, Deep learning model architecture):

"The combination of convolutional and recurrent neural network layers helps to improve the classification performance for time-series data. In particular, LSTM layers allow learning temporal features."

(2) Temporal Resolution: Can you elaborate on the reasons behind the lower temporal resolution of the output? Understanding whether this is due to specific design choices in the model, data preprocessing, or post-processing will clarify the nature of this limitation and its impact on the analysis.

When running inference on a continuous recording, we choose to use a sliding window approach with stride. Therefore, the model output has a lower temporal resolution than the raw data, which is determined by the stride length (i.e., how many samples to advance the sliding window). While using a stride is not required, it significantly reduces inference time (cf. Figure 2—figure supplement 1). We recommend a stride of 20 samples, which does not impact the detection of events. Any subsequent quantification of events (amplitude, area, risetimes, etc.) is performed on raw data. Based on the reviewer’s comment, we have adapted the code to resample the prediction trace to the sampling rate of the original data. This maintains temporal precision and avoids confusion.

The Methods now include the following statement:

"To maintain temporal precision, the prediction trace is resampled to the sampling frequency of the raw data."

(3) Architecture optimization: how was the architecture CNN+LSTM optimized in terms of a number of CNN layers and size?

We performed a Bayesian optimization over a defined range of hyperparameters in combination with empirical hyperparameter tuning. We now describe this in the Methods section as follows:

"To optimise the model architecture, we performed a Bayesian optimisation of hyperparameters. Hyperparameter ranges were chosen for the free parameters of all layers. Optimisation was then performed with a maximum number of trials of 50. Models were evaluated using the validation dataset. Because higher number of free parameters tended to increase inference times, we then empirically tuned the chosen hyperparameter combination to achieve a trade-off between number of free parameters and accuracy."

Recommendations For The Authors

Reviewing Editor (Recommendations For The Authors):

Overall suggestions to the authors:

(1) Directly compare miniML with SimplyFire (which was not cited or discussed in the original manuscript), with both idealized and actual data. Discuss the pros/cons of each software.

We have conducted an extensive comparison between miniML and SimplyFire using both simulated and actual experimental data. This analysis is now presented in the revised Figure 3, Figure 3—figure supplement 1, and Figure 4—figure supplement 1. In addition, we have included relevant citations for SimplyFire in our manuscript. These additions provide a more comprehensive and balanced view of the available tools in the field, positioning our work within the broader context of existing solutions.

(2) Generate a better user interface akin to MiniAnalysis or SimplyFire.

We thank the editor and reviewers for the suggestion to improve the user interface. We have created a user-friendly graphical user interface (GUI) for miniML that is available on our GitHub repository. This GUI is now showcased in Figure 2—figure supplement 2 of the manuscript. The new interface allows users to load and analyze data through an intuitive point-and-click system, visualize results in real-time, and adjust parameters easily without coding knowledge. We have incorporated user feedback to refine the interface and improve user experience. These improvements significantly enhance the accessibility of miniML, making it more user-friendly for researchers with varying levels of programming expertise.

Reviewer 1 (Recommendations For The Authors):

Related to point (1) of the Public Review, we have taken the liberty to compare electrophysiological data using miniAnalysis, SimiplyFire, and miniML. In our comparison, we note the following in our experience:

(1.1) In contrast to both SimplyFire and miniAnalysis, miniML does not currently have a user-friendly interface where the user can directly control or change the parameters of interest, nor does miniML have a user control center, so the user cannot simply type or select the mini manually. Rather, if any parameter needs to be changed, the user needs to read, understand, and change the original source code to generate the preferred change. This level of "activation energy" and required user coding expertise in computer science, which many researchers do not have, renders miniML much less accessible when directly compared to SimplyFire and miniAnalysis. Hence, unless miniML’s interface can be made more user-friendly, this is a major disadvantage, especially when compared to SimplyFire, which has many of the same features as miniML but with a much easier interface and user controls.

As suggested by the reviewer, we have created a graphical user interface (GUI) for miniML. The GUI allows easy data loading, filtering, analysis, event inspection, and saving of results without the need for writing Python code. Figure 2—figure supplement 2 illustrates the typical workflow for event analysis with miniML using the GUI and a screenshot of the user interface. Code to use miniML via the GUI is now included in the project’s GitHub repository. The GUI provides a simple and intuitive way to analyze synaptic events, whereas running miniML as Python script allows for more customization and a high degree of automatization.

(1.2) We compared electrophysiological miniature events between miniML, SimplyFire, and miniAnalysis. All three achieved similar mean amplitudes in "wild type" conditions, and conditions in which mini events were enhanced and diminished, so the overall means and utilities are similar, with miniML and SimplyFire being preferred given the flexibility and much faster analysis. We did note a few differences, however. SimplyFire tends to capture a high number of mini-events over miniML, especially in conditions of diminished mini amplitude (e.g., miniML found 76 events, while SimplyFire 587). The mean amplitudes, however, were similar. It seems that in data with low SNR, SimplyFire captures many more events as real minis that are probably noise, while miniML is more selective, which might be an advantage in miniML. That being said, we found SimplyFire to be superior in many respects, not least of which the user interface and experience.

We appreciate the reviewer’s thorough comparison of miniML, SimplyFire, and MiniAnalysis. While we acknowledge SimplyFire’s user-friendly interface, our study highlights several advantages of AI-based event analysis over conventional algorithmic approaches. Our updated benchmark analysis revealed better detection performance of miniML compared with SimplyFire (revised Figure 3), which had similar performance to deconvolution. As already noted by the reviewer, high false positive rates are a major issue of the SimplyFire approach. Although a minimum amplitude cutoff can partially resolve this problem, detection performance is highly sensitive to threshold setting (revised Figure 3). Another apparent disadvantage of SimplyFire is its relatively slow runtime (Figure 3—figure supplement 1). Finally, we have enhanced miniML’s accessibility by providing a graphical user interface that is easy to use and provides additional functionality.

Some technical comments:

(1) Improvements to the dependence version of miniML: There is a need to clarify the dependence version of the python and tensor flow used in this study and in the GitHub. We used Python version 3.8.19 to load the miniML model. However, if Python versions >=3.9, as described on the GitHub provided, it is difficult to have a matched h5py version installed. It is also inaccurate to say using Python >=3.9, because tensor flow version for this framework needs to be around 2.13. However, if using Python >=3.10, it will only allow 2.16 version tensor flow to be the download choice. Therefore, as a Python framework, the dependency version needs to be specified on GitHub to allow researchers to access the model using the entire work.

Thank you for highlighting this issue. We have now included specific version numbers in the requirements to avoid version conflicts and to ensure proper functioning of the code.

(2) Due to the intrinsic characteristics of the trained model, every model is only suitable for analyzing data with similar attributes. It is hard for researchers without a strong computer science background to train a new model themselves for their specific data. Therefore, it would be preferred if there were more available transfer learning models on GitHub accessible for researchers to adapt to their data.

We would like to thank the reviewer for this feedback. Trained models (such as the default model) can often be used on different data (see, e.g., Figure 4, where data from four distinct synaptic preparations were analyzed with the base model, and Figure 5—figure supplement 1). However, changes in event waveform and/or noise characteristics may necessitate transfer learning to obtain optimal results with miniML. We have revised the description and tutorial for model training on the project’s GitHub repository to provide more guidance in this process. In addition, we now provide a tutorial on how to use existing models on out-of-sample data with distinct kinetics, using resampling. We hope these updates to the miniML GitHub repository will facilitate the use of the method.

Following the suggestion by the reviewer, we have provided the transfer learning models used for the manuscript on the project’s GitHub repository to increase the number of available machine learning models for event detection. In addition, users of miniML are encouraged to supply their custom models. We hope that this will facilitate model exchange between laboratories in the future.

Reviewer 3:

I congratulate all authors for the convincing demonstration of their methodology, I do not have additional recommendations.

We would like to thank the reviewer for the positive assessment of our manuscript.

References

Delvendahl, I., Kita, K., & Müller, M. (2019). Rapid and sustained homeostatic control of presynaptic exocytosis at a central synapse. Proceedings of the National Academy of Sciences, 116(47), 23783–23789. https://doi.org/10.1073/pnas.1909675116

Donahue, J., Hendricks, L. A., Rohrbach, M., Venugopalan, S., Guadarrama, S., Saenko, K., & Darrell, T. (2017). Long-term recurrent convolutional networks for visual recognition and description. IEEE Transactions on Pattern Analysis and Machine Intelligence, 39(4), 677–691. https://doi.org/10.1109/tpami.2016.2599174

Drummond, C., & Holte, R. C. (2003). C4.5, class imbalance, and cost sensitivity: Why under-sampling beats over-sampling. https: //api.semanticscholar.org/CorpusID:204083391

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Passricha, V., & Aggarwal, R. K. (2019). A hybrid of deep CNN and bidirectional LSTM for automatic speech recognition. Journal of Intelligent Systems, 29(1), 1261–1274. https://doi.org/10.1515/jisys-2018-0372

Prati, R. C., Batista, G. E. A. P. A., & Monard, M. C. (2009). Data mining with imbalanced class distributions: Concepts and methods. Indian International Conference on Artificial Intelligence. https://api.semanticscholar.org/CorpusID:16651273

Tasdelen, A., & Sen, B. (2021). A hybrid CNN-LSTM model for pre-miRNA classification. Scientific Reports, 11(1). https://doi.org/10. 1038/s41598-021-93656-0

Tyagi, S., & Mittal, S. (2020). Sampling approaches for imbalanced data classification problem in machine learning. In P. K. Singh, A. K. Kar, Y. Singh, M. H. Kolekar, & S. Tanwar (Eds.), Proceedings of icric 2019 (pp. 209–221). Springer International Publishing.

Wang, H., Zhao, J., Li, J., Tian, L., Tu, P., Cao, T., An, Y., Wang, K., & Li, S. (2020). Wearable sensor-based human activity recognition using hybrid deep learning techniques. Security and Communication Networks, 2020, 1–12. https://doi.org/10.1155/2020/ 2132138

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What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_50594

Curator: @scibot

SciCrunch record: RRID:BDSC_50594

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_26297

Curator: @scibot

SciCrunch record: RRID:BDSC_26297

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_55275

Curator: @scibot

SciCrunch record: RRID:BDSC_55275

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_29306

Curator: @scibot

SciCrunch record: RRID:BDSC_29306

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_82181

Curator: @scibot

SciCrunch record: RRID:BDSC_82181

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_58993

Curator: @scibot

SciCrunch record: RRID:BDSC_58993

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_62142

Curator: @scibot

SciCrunch record: RRID:BDSC_62142

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_52265

Curator: @scibot

SciCrunch record: RRID:BDSC_52265

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_52268

Curator: @scibot

SciCrunch record: RRID:BDSC_52268

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_32198

Curator: @scibot

SciCrunch record: RRID:BDSC_32198

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_44222

Curator: @scibot

SciCrunch record: RRID:BDSC_44222

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_32184

Curator: @scibot

SciCrunch record: RRID:BDSC_32184

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_30005

Curator: @scibot

SciCrunch record: RRID:BDSC_30005

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_26206

Curator: @scibot

SciCrunch record: RRID:BDSC_26206

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_30118

Curator: @scibot

SciCrunch record: RRID:BDSC_30118

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_25856

Curator: @scibot

SciCrunch record: RRID:BDSC_25856

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_52262

Curator: @scibot

SciCrunch record: RRID:BDSC_52262

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_66562

Curator: @scibot

SciCrunch record: RRID:BDSC_66562

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_6874

Curator: @scibot

SciCrunch record: RRID:BDSC_6874

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_79471

Curator: @scibot

SciCrunch record: RRID:BDSC_79471

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_25927

Curator: @scibot

SciCrunch record: RRID:BDSC_25927

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_66542

Curator: @scibot

SciCrunch record: RRID:BDSC_66542

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_7018

Curator: @scibot

SciCrunch record: RRID:BDSC_7018

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_71993

Curator: @scibot

SciCrunch record: RRID:BDSC_71993

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_30142

Curator: @scibot

SciCrunch record: RRID:BDSC_30142

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_38689

Curator: @scibot

SciCrunch record: RRID:BDSC_38689

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_75312

Curator: @scibot

SciCrunch record: RRID:BDSC_75312

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_79030

Curator: @scibot

SciCrunch record: RRID:BDSC_79030

What is this?

DOI: 10.1038/s41586-023-06627-y

Resource: RRID:BDSC_60321

Curator: @scibot

SciCrunch record: RRID:BDSC_60321

What is this?

DOI: 10.1016/j.devcel.2025.01.016

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2145180

What is this?